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Accurate chromosome segregation in meiosis is essential for production of euploid gametes. The error correction and the spindle assembly checkpoint (SAC) pathways ensure homolog segregation to opposite poles during meiosis I, but how each component of the SAC contributes to chromosome segregation remains unclear. My work as well as previous studies report that the SAC components Mad1 and Mad2 promote accurate homolog segregation, but the contribution of SAC protein Mad3 is dispensable. Instead, I identify previously unreported non-canonical interactions of Mad3 with microtubule regulators Stu1 and Slk19, and with protein phosphatase PP2C subunit Ptc7 during meiosis in S. cerevisiae. Domain analysis revealed that the TOGL1 domain on Stu1 is necessary for Mad3-Stu1 and Mad3-Slk19 interaction. Mad3 and Stu1 appear to act together in a secondary pathway that is crucial for homolog segregation in the absence of Mad2, and the Mad3-Stu1 pathway of chromosome segregation becomes crucial for the segregation of achiasmate chromosomes- chromosomes that lack crossovers. While Mad3-Stu1 interact since meiotic prophase, Slk19 joins the Mad3-Stu1 complex only after exit from prophase I. Slk19 and Stu1 are known to promote kinetochore capture by microtubules in prometaphase, so Slk19 might also promote achiasmate chromosome segregation. Strikingly, Mad3 arrests cells in prophase I in the absence of its other interactor Ptc7, suggesting a checkpoint-like function of Mad3 in regulating the duration of prophase I. SAC-independent roles of Mad3 homolog BubR1 have been identified in higher eukaryotes. This work introduces novel interactors of SAC component Mad3, and explores interesting non-canonical roles of these interactions in the context of meiotic cell cycle and chromosome segregation in budding yeast.

Meiosis is a highly conserved specialized cell division unique to eukaryotes that functions to increase genetic diversity in offspring. Crossover formation is an outcome of the process of meiotic recombination, a cascading series of events during which homologous chromosomes undergo pairing and reciprocal exchange of genetic information. During meiotic recombination, meiotic DSB formation and crossover localization is negative correlated in plant species with high levels of heterochromatic features such as H3K9me2, DNA methylation, and nucleosome density. In plant species, maintenance of heterochromatin is controlled via the concerted efforts CHROMOMETHYLASE 3 (CMT3) and KRYPTONITE/SUVH5/SUVH6 (KSS), and mutation of these features can remodel recombination events to previously suppressed regions of the genome. Remodelling of crossovers to cold regions is of particular interest to crop breeders, who seek to unlock genomic regions to crossovers to identify novel, beneficial allelic combinations. To further characterize the meiotic recombination landscape in bread wheat, I have analyzed the genome wide distribution of the meiotic axis protein, ASY1, and the meiotic recombinase, DMC1 via Chromatin-Immunoprecipitation and sequencing (ChIP-seq). Consistent with crossover distribution, I have observed enrichment of both factors in the distal, highly recombining regions of the genome, correlating with facultative heterochromatin, signatures of adaption, and defense response genes (i.e. NLR containing gene families). I have also generated homozygous mutant lines for kryptonite and suvh5/6 orthologues in wheat, and also characterized a CMT3 related, cmt6S-ε mutant. Here, I have observed reduction of heterochromatin, resulting in reduced fertility and high prevalence of homoeologous recombination events in metaphase I chromosomes in cmt6S-ε, similar to Ph1 locus mutant phenotypes. This confirms an intimate relationship with early meiotic recombination events and crossovers in bread wheat and presents a novel function for epigenetic regulation of homoeologous recombination. In addition, I present the ChIP-seq analysis of DMC1 in Arabidopsis thaliana in wildtype versus cmt3 and kss mutants. DMC1 in wildtype is enriched strikingly in regions flanking meiotic DSB sites, however this pattern is remodelled to the DSB sites themselves in cmt3 and kss mutants. This presents a novel function for DNA methylation in determining the fine-scale distributions of recombinases in Arabidopsis meiosis. Together, these results advance the understanding of epigenetic regulation of meiotic processes in plants.

The accurate transmission of genetic material in the germline is of vital importance to the offspring since the entire organism is affected. It is therefore critical that chromosomes are faithfully distributed during the formation of gametes in a specialised form of cell division known as meiosis. Errors in meiotic cell divisions are a frequent cause of infertility, miscarriages, and birth defects in humans. The meiotic chromatin undergoes dynamic rearrangements during these divisions that are poorly understood at the molecular level. In prophase of the first meiotic division, the chromatin of Drosophila melanogaster oocytes detaches from the nuclear envelope to form a compact spherical cluster known as karyosome. It was previously shown that the karyosome is required for faithful chromosome segregation, but knowledge about its formation and maintenance is limited. I wish to understand how karyosome formation is regulated and identified genes important for karyosome formation in a genome-wide cytological screen of Drosophila melanogaster oocytes. The screen comprised 3,916 candidate genes expressed in ovaries, of which 209 genes showed karyosome defects upon knockdown. I found that genes encoding mitochondrial proteins, including electron transport chain components, are overrepresented amongst genes whose knockdown results in severe and reproducible karyosome defects. Interestingly, mitochondrial dysfunction induced a distinct karyosome defect characterised by three individual chromatin clusters in proximity to the nuclear envelope. Furthermore, my studies revealed that mitochondrial dysfunction not only impairs karyosome formation, but also karyosome maintenance throughout mid-oogenesis and synaptonemal complex dynamics. I asked how mitochondrial dysfunction triggers karyosome defects and aimed at further mechanistical insights. I found that mitochondrial dysfunction forces a low percentage of oocytes into apoptotic cell death, but karyosome defects occur independent of apoptosis. The knockdown of ATP synthase subunits induced the distinct karyosome defects observed upon mitochondrial dysfunction, suggesting a direct link between the karyosome phenotype and reduced levels of cellular ATP. I further determined the dependence of observed karyosome abnormalities on meiotic checkpoint activation. My work thus identified a set of genes with reproducible and checkpoint-independent karyosome defects upon knockdown. The uncharacterised function of these genes in karyosome formation and maintenance remains to be investigated and future research will pave the way for a better understanding of the karyosome at a molecular level. Furthermore, I established a link between mitochondrial dysfunction and a karyosome phenotype characterised by chromatin attachment to the nuclear envelope. The functional role of mitochondria is an increasingly important consideration in both male and female fertility. My study therefore provides a novel insight, considering that mechanistical details on how mitochondrial diseases link to infertility are sparse.

Meiosis is a type of cell division that leads to the creation of gametes. Unlike mitosis, which produces exact copies of the mother cell, the aim of meiosis is to halve the cell's chromosomal content. To achieve this, the mechanisms governing the division processes have been heavily modified. This applies particularly to the first of two divisions, when genome reduction happens. At the beginning of meiosis I, the homologous chromosomes pair and create linked bivalents, which generates tension between the homologues and leads them to be segregated towards the opposite spindle poles. This type of segregation, which is referred to as "reductional", is further biased by the fusion of the sister kinetochores. In budding yeast, the fused kinetochore allows the attachment of only one microtubule, leading to kinetochore mono-orientation. Kinetochore mono-orientation, in cooperation with the pericentromeric cohesin, precludes precocious segregation of sister chromatids. It has been proposed that kinetochore fusion depends on the monopolin complex, which provides a bridge between the sister kinetochores. In turn, the recruitment of monopolin complex depends on Spo13 and the polo-like kinase Cdc5. While the current model of the Spo13 role indicates that it functions as a platform for kinetochore localisation of Cdc5, the exact functions of both proteins in sister-kinetochore mono-orientation remain obscure. During my project, I tried to learn the precise localisation of Spo13. Using mass spectrometry, I have shown that Spo13 is recruited to the inner kinetochore and that this localisation depends on Spo13 interaction with Cdc5. However, the abundance of dispersed monopolin complex precluded studying it by the same method. Cdc5 is a versatile kinase with plenty of possible substrates. To discover the phosphorylation sites that may play role in the mono-orientation establishment, I immunoprecipitated the kinetochore and submitted it to be analysed by mass spectrometry. I utilised a Cdc5 kinetochore-tethered (Cdc5-kt) system which allows Cdc5 to localise on kinetochores independently of Spo13. Because CDC5-kt cells co-segregate the sister chromatids in the absence of Spo13 and the monopolin complex, I hoped to understand why Cdc5-kt helps rescue the phenotype. I found that several components of the spindle assessment and error correction pathways are enriched in spo13Δ cells, while Cdc5-kt lowers their kinetochore occupancy to wild type levels. Therefore, Spo13 likely participates in two parallel pathways to establish sister kinetochore mono-orientation: firstly, it recruits monopolin to provide the kinetochore fusion and secondly, it modulates the mechanisms involved in microtubule-kinetochore attachment tension sensing.

The synaptonemal complex is a tripartite proteinaceous structure consisting of two filamentous lateral elements, joined by zipper-like transverse filaments and central elements that assemble to join homologous chromosomes and ensure recombination by formation of crossovers during meiotic prophase I. In polyploid wheat, components of the synaptonemal complex and its installation process are still elusive. The application of several distinct methodologies, such as Sanger sequencing, cloning and bioinformatics was conducted to analyse gene expression contribution and transcript variants for the Triticum aestivum ASYNAPTIC 1 (ASY1) and ZYPPER-LIKE 1 (ZYP1) genes. Mutant TILLING lines provided a valid strategy to functionally analyse asy1 and zyp1 and to characterize putative meiotic genes in polyploid wheat, identified via a computational screening. Molecular analysis demonstrated a balanced expression level among the ASY1 and ZYP1 sub-genomes of hexaploid wheat, being also expressed in non-meiotic tissues, and uncovered the presence of de-novo transcript isoforms within their 3' UTRs. Cytological analysis revealed that ASY1 is involved in chromosome homology search, since asy1 hypomorphic mutants showed multivalent associations, and is required for formation of the obligate crossover, mirroring the ph1 phenotype. Preliminary data of zyp1 single knockouts suggested a conserved role of ZYP1 in mantaining obligate crossovers, and in controlling the timing of synaptonemal complex assembly in tetraploid wheat. Furthermore, a wheat SWITCH1 (SWI1) orthologue was identified using co-expression bioinformatics tools. Lastly, a method to implement FISH-based single copy oligo-probes to discriminate individual wheat chromosomes was developed. This work provides advanced directions for studying meiosis in polyploid wheat, expanding the opportunities to manipulate crossovers and introgress agronomically important genes.

Fundamental to meiosis is the programmed formation of DNA double-strand breaks (DSBs), which are catalysed by the meiosis-specific transesterase Spo11. These DSBs are tightly regulated, occurring non-randomly and with a higher frequency at meiotic DSB hot spots. Apart from Spo11, break formation also requires a network of co-factors, as well as components of the chromosome axis, which intricately regulate the recruitment and activity of Spo11 within a complex meiosis-specific chromatin environment. Once formed, meiotic breaks are preferentially repaired using the homologous chromosome as the template. Repair can result in various outcomes. However, only crossover events establish physical connections between the parental chromosomes which are essential for correct chromosome segregation during the first meiotic division. To better understand meiotic recombination and its regulation, this project sought to develop a meiotically expressed CRISPR/Cas9 system that works in combination with an ade6-based genetic reporter system, to investigate meiotic repair mechanisms in the model organism Schizosaccharomyces pombe. Using this experimental approach, I show that Cas9 induces recombination at ade6 heteroalleles with different DSB forming activities and that the repair outcome of these breaksis different to that of Spo11-DSBs. I also present a systematic analysis of Cas9-DSB repair from which the main findings were: (I) many early meiotic factors critical in coordinating DSB formation do not influence the repair outcome of Cas9 breaks; (II) surprisingly, the axis-associated protein Hop1 may act without being recruited to the meiotic chromosome axis, at least for Cas9 breaks; and (III) repair pathway choice is altered in favour of unbiased Mus81-Eme1 resolution and synthesis-dependent strand annealing during the repair of meiotic Cas9 breaks. The CRISPR/Cas9-based experimental approach developed in this thesis provides a novel strategy for exploring the genetic requirements of DSB formation and the mechanistics of DSB repair in Schizosaccharomyces pombe. As a tool, this has the potential to provide more control over the meiotic recombination landscape, which could be useful in other applications.

In early meiosis, programmed-DNA double strand breaks occur throughout the genome which are predominantly repaired via homologous recombination. During this process, the homologous chromosomes pair and reciprocally exchange genetic information, also called crossovers. Crossovers break the linkage between genes and create new combinations of alleles which are inherited in the offspring population. Meiotic crossover events can be beneficial to plant breeding programmes, because they facilitate the introgression of alleles from wild crop relatives to elite cultivars. However, crossover distributions are non-random and with recombination preferentially enriched in distal regions of the chromosomes, and low numbers of crossovers in the interstitial and centromere-proximal regions. This is particularly true in plant species with large, repetitive genome, for example major crops such as maize and wheat. This considerably limits the potential to introduce allelic diversity throughout the genome during crop improvement. My study aims to provide novel insights in the control of meiotic recombination and to identify new factors involved in crossover repression across the centromere-proximal heterochromatic regions. Heterochromatin is compacted with a high DNA density and is enriched in nucleosome occupancy and specific chromatin modifications, including H3K9me2 and DNA methylation. Analysis of histone H2A variants via ChIP-sequencing has revealed that H2A.W is enriched in the heterochromatin. By combining cytogenetic, genetic and genomic assays, I have demonstrated that HTA7 is specifically involved in crossover repression within the pericentromeric and centromeric heterochromatin. The meiotic recombination phenotype of hta7 was not further increased in hta7 cmt3, where CHG DNA methylation and H3K9me2 are reduced, revealing an epistatic interaction between these epigenetic components. In the absence of all three H2A.W proteins, the elevation of recombination observed in hta7 was decreased and the organisation of the heterochromatin was changed, likely in response to the replacement of H2A.W by other canonical and variant histones. Overall, my study identified a new factor repressing meiotic recombination within Arabidopsis heterochromatin and provides new insights in the coordination between chromatin organisation and meiotic recombination.

Meiosis creates genetic diversity by recombination and segregation of chromosomes. Meiotic errors can lead to chromosome missegregation and consequently infertility or birth defects. In Drosophila oocytes chromosomes undergo crucial reorganisation events during meiotic prophase to prepare for proper recombination and chromosome segregation. However, the molecular mechanisms regulating chromatin reorganisation remain poorly understood. The importance of post-translational modifications during chromatin rearrangements has been shown across many species. Here, I report the role of the SCF complex, an E3 ubiquitin ligase, in synaptonemal complex assembly and karyosome formation in Drosophila oocytes. The SCF complex is constituted by SkpA, Cul-1, Roc and a substrate recognising protein (F-box). I have identified two specific F-box proteins, Slmb and CG6758, to be required for the karyosome formation and synaptonemal complex assembly. Moreover, I identified PP2A as an interactor of the F-box protein CG6758 and described the role of CG6758 in regulating the amount of PP2A-B56 subunit. Thus, in this work, I have demonstrated that the SCF-CG6758 protein complex downregulates the phosphatase subunit PP2A-B56, which is required for proper synaptonemal complex and karyosome formation. To further determine the role of ubiquitination during meiotic prophase, I carried out an RNAi screen for ubiquitin-associated proteins. I found that multiple other ubiquitin ligases have an important role in karyosome formation. However, only the components of the neddylation cycle, an important regulator of the SC, showed to be required for synaptonemal complex assembly and karyosome formation. Overall, my findings uncover a novel role for the ubiquitin ligase SCF in meiotic prophase. Moreover, it opens a new line of inquiry about the importance of post-translational modifications in synaptonemal complex assembly and karyosome formation.

Meiosis is a specialised cell division producing the haploid gametes required for sexual reproduction. A key function of this process is to generate genetic diversity through recombination and independent assortment of homologous chromosomes. Delineation of gene expression during this process has been challenging due to the heterogeneity of testis tissue and the lack of faithful in vitro models. Here we jointly analyse a single-cell resolution transcriptomic dataset of over 20,000 cells from both wild type and mutant mice. We use dimensionality reduction methods to infer latent components of variation that represent transcriptional programmes, mutant specific pathological processes, and technical effects. This approach simultaneously soft clusters cells, infers corresponding groups of co-expressed marker genes, and imputes sparse, noisy gene expression. We were also able to infer, de novo, transcription factor binding motifs for each component, revealing a general switch at the meiotic divisions. We facilitate access to this resource by providing an interactive website testisatlas.ml. The high-resolution delineation of gene expression during the spermatogenic programme provides high-resolution clues to the function of understudied genes. One such gene, Zcwpw1, is highly co-expressed with Prdm9, and has domains capable of recognising the unique combination of histone marks that PRDM9 deposits (H3K4me3 and H3K36me3). By using a human in vitro system of Zcwpw1 co-transfection with either human or chimp Prdm9, we show that PRDM9 causes the recruitment of ZCWPW1 to its binding sites. This recruitment is stronger than for sites with H3K4me3 alone and is CpG dependent. Male Zcwpw1-/- mice have completely normal double strand break positioning, but severe repair and synapsis defects leading to complete testicular azoospermia. Although PRDM9’s effect of DSB positioning remains intact, PRDM9’s effect of aiding synapsis appears to be abolished, with persistent DMC1 signal - most dramatically at the most strongly PRDM9 bound hotspots.

Meiosis is a conserved eukaryotic cell division that increases genetic diversity in progeny. During meiosis, homologous chromosomes pair and undergo reciprocal exchange, called crossover. Meiotic recombination initiates from DNA double strand breaks, which are repaired using either sister or homologous chromatids as templates. When meiosis occurs in heterozygous (hybrid) organisms, interactions between homologous chromosomes have the potential to generate mismatched DNA structures. Several proteins with roles in DNA mismatch repair (MMR) are known to influence meiotic recombination in several eukaryotes. In Arabidopsis thaliana this includes three MSH heterodimers (MSH2-MSH3, MSH2-MSH6 and MSH2-MSH7) that recognise mismatched nucleotides and have demonstrated roles in repressing meiotic crossovers in hybrid plants. To further investigate the meiotic function of MMR genome-wide, I generated a series of msh2 mutant introgression lines in three different genetic backgrounds – Ct-1, Ler-0 and CLC – which have varying patterns and levels of polymorphism. Consistent with the function of MSH2 as a hybrid-specific anti-recombinase, I observed significant crossover increases in the chromosome arms in all three hybrid msh2 mutants. However, I also found evidence for accession and region specific effects of msh2 on crossover frequency. For example, crossovers appeared to decrease over centromere proximal regions in msh2 compared to wild type. A genotyping-by-sequencing experiment was performed to generate genome-wide crossover maps in two Arabidopsis hybrids, with and without MSH2 function. This revealed that total crossover number remained unchanged in the MMR-deficient hybrids, whilst crossovers redistributed into regions of reduced polymorphism density. This relationship was counter to my expectation that MMR would repress crossovers most strongly in divergent regions. However, this relationship was observed across varying physical scales, from 1–100 kilobases. This confirms a positive relationship between polymorphism and meiotic crossovers in Arabidopsis hybrids, and reveals a novel role for MSH2 in mediating this effect. In addition to the investigation of MSH2 in meiotic recombination, I present an analysis of the genome-wide distribution of MutS homolog 4 (MSH4). MSH4, and its binding partner MutS homolog 5, evolved from their ancestral role in MMR and now function exclusively to promote meiotic crossovers in the ZMM pathway. I present a genome-wide chromatin-immunoprecipitation-sequencing analysis of the binding profile of MSH4, and analyse its distribution at varying scales. This has revealed novel relationships between MSH4, the meiotic cohesin subunit REC8, and features of the chromatin and recombination landscapes. Together, these results advance our understanding of meiotic recombination in plants, and raise further questions about the regulation of crossovers in eukaryotes more broadly.

Meiosis is a specialised form of cell division, which halves the chromosome content of a cell. A single round of DNA replication is followed by two rounds of division. Meiosis utilises the key machinery of mitosis but with specific adaptations, particularly in the first division. In the first meiotic division, unlike mitosis or meiosis II, sister chromosomes segregate together. In budding yeast, the fusion of sister kinetochores allows them to attach to a single microtubule from one cell pole. The four-protein complex responsible for this fusion and therefore sister co-segregation in meiosis I is called monopolin. However, the mechanism by which monopolin acts is unclear. What is well understood about monopolin is its structure, it is a four-protein complex of two homodimers of Csm1 and two copies of the proteins Lrs4, Mam1 and the casein kinase, Hrr25. The complex forms a V-shaped structure, with a kinetochore binding region at each point of the V. This structure has led to the proposed mechanism of monopolin forming a mechanical bridge between the two kinetochores directly causing their fusion. There is, however, no direct evidence for this mechanism and many open questions remain, particularly as to how the bridging interaction would be regulated and the role of proteins, Mam1 and Hrr25, which do not form the structure of the V-shape. This project had several aims, firstly to dissect whether all components of the monopolin complex are required for monoorientation or if some only serve to target the complex to the kinetochore. By artificial tethering of monopolin to the kinetochore, I was able to demonstrate the requirement for a complete monopolin complex for the mono-orientation of sister chromatids. Secondly, I aimed to show the molecular basis for the interaction between monopolin and the kinetochore. This was done by following insight gained form the crystal structure of monopolin showing the interaction between Csm1 and its kinetochore receptor Dsn1, I generated mutants in Dsn1 that disrupt this interaction. Through predominantly assessment of cell viability, imaging of chromosome division in meiosis and chromatin immunoprecipitation of monopolin components I demonstrated that these sites within Dsn1 are essential for the localistion of monopolin to the kinetochore, sister chromatid mono-orientation and therefore meiosis. In addition, through the use of mass spectrometry I showed that two serines within this region of Dsn1 are phosphorylated in meiosis, and that they are good candidates for being a point of regulation for the interaction between monopolin and the kinetochore. Finally, although it is known that Hrr25 must be enzymatically active for mono-orientation of sister chromatids in meiosis, no in vivo targets of this phosphorylation have been described. I, therefore, utilized a mutant version of Hrr25 which is not recruited to the monopolin complex and mass spectrometry to identify possible targets of phosphorylation by Hrr25 at the meiotic kinetochore.

The organisation of microtubule networks into a bipolar spindle is essential for reliable chromosome segregation during cell division. A pair of centrioles surrounded by pericentriolar material (PCM), define the canonical centrosome that acts as the main microtubule organising centre (MTOC) during mitosis. In mammalian meiosis, centrioles are eliminated early on during oogenesis. Despite the absence of centrosomes, a large number of centrosomal proteins are highly expressed in mouse oocytes. Here, I characterise the localisation and function of centrosomal proteins at a previously undescribed meiotic spindle pole domain (MSPD). An initial protein screen identified a group of pericentriolar satellite proteins that localised to a previously undescribed spindle pole domain throughout meiotic maturation in mouse oocytes, including Pericentriolar material 1 protein (PCM1). This domain was distinct from spindle microtubules and the acentrosomal microtubule organising centres (aMTOCs). Initial characterisation focused on PCM1, the main centriolar satellite scaffold protein in somatic cells. Depletion of PCM1 revealed interdependence with the essential aMTOC component, Pericentrin. In the absence of PCM1, aMTOCs could no longer assemble or maintain their structural integrity. PCM1 degradation and disassembly of aMTOCs disrupted spindle assembly and reduced the total amount of nucleated microtubules throughout meiosis. In the absence of the main microtubule nucleating aMTOCs, oocytes relied on the Ran GTPase activity to form a small bipolar spindle. A similar mechanism was previously reported in human oocytes that lack prominent MTOCs. The extended centrosomal protein screen identified additional components of the MSPD. TACC3, under the regulation of Aurora-A at aMTOCs, drive assembly of the MSPD. This domain was absent in MTOC free human oocytes but a second population of TACC3 (identified in mouse oocytes) localised to the meiotic spindle and K-fibres was essential for maintaining spindle pole integrity. Establishing the Lightsheet Z.1 system for live cell imaging of human oocytes enabled us to observe the dynamic distribution of TACC3 in these oocytes. In the absence of prominent MTOCs and the MSPD, human oocytes likely rely on other spindle assembly factors and motor proteins to organise their spindle. Future work to address if the absence of the MSPD could account (in part) for the observed spindle instability in human oocytes is an exciting outlook.

Poor egg quality is the leading cause of pregnancy loss and Down's syndrome. While even eggs in young women frequently contain an incorrect number of chromosomes and are therefore unlikely to give rise to a viable pregnancy, the incidence of chromosomally abnormal eggs increases strikingly with advancing maternal age. Why egg quality declines dramatically as women approach their forties remains one of the outstanding questions in developmental biology. This PhD thesis demonstrates how unforeseen features of kinetochore organization that are unique to meiosis render this cell division process in mammals particularly prone to errors. Firstly, my results uncovered an unexpected multi-subunit organization of the meiotic kinetochore, which is widely conserved across mammals and biases eggs towards errors. Secondly, I identified two independent mechanisms that predispose eggs from older women to aneuploidy. The first mechanism affects the fidelity of meiosis I. My analysis revealed that human oocytes challenge the paradigm that sister kinetochores are fully fused. Instead, I demonstrated that sister kinetochores disjoin as women get older, which promoted erroneous kinetochore-microtubule attachments. This in turn allowed chromosomes to rotate on the spindle and provided a mechanistic explanation for reverse segregation - a recently discovered meiotic error that is unique to humans. Secondly, I pioneered the use of super-resolution microscopy to study chromosome architecture in human eggs and discovered that individual kinetochores during meiosis II in mammals are composed of previously unidentified subdomains. In young females, these subdomains are joined together by cohesin complexes. With age, kinetochores fragment into two pieces. Fragmented kinetochores frequently attach merotelically to spindle microtubules, which predisposes aged eggs to errors. What severely hinders our progress in identifying causes of human infertility is that numerous features of human meiosis are not represented in mice. To overcome this challenge, I developed an experimental platform to mimic the age-related changes that occur in humans in oocytes from young mice. I achieved this by extending the applications of Trim-Away, a novel method to degrade endogenous proteins even in primary cells, to partially deplete proteins. Furthermore, I established a new experimental model system to study human-like aspects of meiosis in live non-rodent cells in real time: pig oocytes. Together, these results set foundations for new therapeutic approaches to extend reproductive lifespan by counteracting the age-related loss in kinetochore integrity that this study identified. Furthermore, partial Trim-Away and studying meiosis in pigs opens new directions for meiotic research.

During meiosis, homologous chromosomes pair and undergo reciprocal genetic exchange, producing crossovers. This generates genetic diversity and is required for balanced homolog segregation. Despite the critical functions of crossovers, their frequency and distribution varies extensively within and between species. This crossover variation can be caused by trans-modifiers within populations, which encode diffusible molecules that influence crossover formation elsewhere in the genome. This project utilised natural accessions of Arabidopsis thaliana to identify trans-modifying loci underlying crossover variation within the species. I performed Quantitative Trait Loci (QTL) mapping using a fluorescence-based crossover reporter system to measure recombination frequency in a genomic interval on chromosome 3, termed 420. Mapping in a Col-420 × Bur-0 F2 population revealed four major recombination QTLs (rQTLs) that influence crossover frequency. A novel recessive rQTL on chromosome 1 that reduced crossovers within the interval was fine-mapped to a premature stop codon in TATA Binding Protein (TBP)-associated factor 4b (TAF4b) in Bur-0 (taf4b-1). TAF4b is a subunit of the TFIID complex, a multi-protein general transcription factor complex comprising TBP and numerous TAFs that forms a component of the pre-initiation complex that recruits RNA polymerase II to promoters. Transformation-based complementation experiments and the isolation of several independent taf4b alleles provided genetic proof that TAF4b is essential for wild-type levels of crossover within 420. Analysis of the prevalence of the taf4b-1 mutation in the global Arabidopsis accession collection demonstrated its specificity to three accessions in the British Isles. A combination of cytology, genetic analysis using additional fluorescent reporter lines, and sequencing in F2 recombinant populations demonstrated a genome-wide reduction in crossover frequency in taf4b-1. In addition, RNA sequencing identified numerous transcriptional changes in taf4b-1. Both up- and down-regulated gene sets displayed significant enrichment for genes that are predominantly expressed in meiocytes, and several gene ontology terms pertaining to protein modification and meiotic processes. These results further demonstrate the existence of genetic modifiers of crossover frequency in natural populations of A. thaliana, and the characterisation of a novel trans-modifier of recombination, TAF4b. This signifies a novel function for TAF4b in Arabidopsis, and further enhances our understanding of the molecular factors controlling the frequency and distribution of meiotic crossovers in plants.

Meiosis is a specialised form of cell division which results in the formation of haploid cells from a diploid progenitor, and thus meiosis halves the chromosome content of the parental cell. A tightly controlled sequence of chromosome segregation events is required to ensure that chromosome missegregation does not occur during meiosis. Chromosome missegregation causes aneuploidy, which in humans can result in the genetic disorder Down Syndrome, and is the leading cause of infertility and miscarriage. To avoid this, it is critical that in the first meiotic division homologous chromosomes are segregated, followed by sister chromatid segregation in meiosis II. Cohesin is a ring-shaped protein complex that holds that sister chromatids together during meiosis, and aids homologue pairing and recombination, as well as ensuring the correct timing of chromosome segregation. In meiosis I, cohesin must be cleaved by separase on the arms of the chromosomes, to allow homologue segregation, whilst at the centromere Sgo1-PP2A protects this pool of cohesin from cleavage and holds sister chromatids together. In meiosis II, the remaining centromeric cohesin is cleaved to allow segregation of sister chromatids. Therefore, correct regulation of Sgo1 and its interaction partners in meiosis is crucial to prevent aneuploidy. In this study I characterise the role of an Sgo1 binding partner, condensin, in meiotic chromosome segregation, and show that condensin is essential for faithful chromosome segregation in both meiosis I and II. Sgo1 is post-translationally modified in meiosis, and in this study Sgo1 phosphomutants were analysed, but were found to have no discernible effects on faithful chromosome segregation. However additional Sgo1 post-translational modifications were identified, leaving the regulation of Sgo1 by post-translational modification open to future study. Additional to the removal of cohesin by separase cleavage, there is a non-proteolytic pathway for cohesin removal. During mitotic prophase in higher eukaryotes, cohesin is destabilised from replicated sister chromatid arms through the action of Wapl. However, a subset of cohesin is protected from the destabilising effects of Wapl because acetylation of the Smc3 subunit of cohesin allows binding of sororin. Phosphorylation events prevent sororin association with chromosome arms, making cohesin susceptible to removal by Wapl. In contrast, at centromeres, shugoshin counteracts these phosphorylation events, thereby protecting centromeric cohesin from Wapl. During meiosis, Wapl is important in cohesin destabilisation in mouse, C. elegans and A. thaliana, and recent evidence suggests that this pathway is also active in budding yeast. However, it is unclear if the acetyltransferase, Eco1, and cohesin acetylation are important in the generation of cohesive cohesin in meiosis to allow faithful chromosome segregation. Additionally it is unclear if the destabilising activity of Wapl only contributes to cohesin removal on chromosome arms, or whether mechanisms are required to protect cohesin from destabilisation activity near centromeres. I aim to address these questions using budding yeast. Previous work showed that deletion of Wapl (Rad61) in meiosis prevents destabilisation of cohesin from the DNA prior to metaphase I. Consistently, I found Rad61 is regulated in meiosis, and has a role in promoting faithful chromosome segregation in tetranucleates. Additionally, Eco1 acetyltransferase is expressed during S phase of meiosis, and this expression coincides with Smc3 acetylation. In meiosis, disruption of Eco1 function and Smc3 acetylation has a detrimental impact on DNA segregation and cell viability. Further findings show that Sgo1 interacts with cohesin in budding yeast meiosis, and Sgo1 localisation to the chromatin is impacted in Eco1 mutants, but the precise interplay between Sgo1 and acetylated cohesin remains to be fully deduced.

During meiosis eukaryotes produce four haploid gametes from a single diploid parental cell. In meiotic S-phase homologous chromosomes, which were inherited from maternal and paternal parents, are replicated. Homologous chromosomes then pair and undergo reciprocal crossover, which generates new mosaics of maternal and paternal sequences. Meiosis also involves two rounds of chromosome segregation, meaning that only one copy of each chromosome is finally packaged into the resulting haploid gametes. In this work I sought to genetically engineer two elements of meiosis, in order to generate tools which may be useful for plant breeding. The first project sought to generate a second division restitution (SDR) population, where the second meiotic division is skipped. This is created by crossing an SDR mutant, omission of second division1, which produces diploid pollen due to a defective meiosis-II, to a haploid inducer line, whose chromosomes are lost from the zygote post-fertilisation. This was intended to give rise to diploid plants possessing chromosomes from just the SDR parent. Importantly, the SDR parent used was heterozygous, meaning that SDR progeny should show mostly homozygous chromosomes, but with regions of residual heterozygosity, determined by crossover locations. This project succeeded in creating a small number of plants with the predicted SDR genotype, although a range of aberrant genotypes were also observed. I present several hypotheses that could account for the observed progeny genotypes. In a second project I attempted to direct meiotic recombination using DNA double strand breaks targeted to specific sites. This project used a spo11-1 mutant, which is unable to produce the endogenous meiotic DNA DSBs that normally mature into crossovers. Instead, TALFokI nucleases (TALENs) were expressed from meiotic promoters in order to generate exogenous DSBs at sites determined by the DNA binding specificity of the TAL repeat domains. The project succeeded in transforming TALENs into spo11-1 mutants and confirming their expression. However, this was not sufficient to recover the spo11-1 mutant infertility or direct crossovers. Potential reasons for this non-complementation are discussed, as well as their implications for control of meiotic recombination in plant genomes.

Mitosis and meiosis are different forms of cell division. Mitosis is a non-reductive form of cell amplification whereby DNA chromosomes are replicated and segregated to form two progeny copies of the progenitor cell. Meiosis is a reductive form of cell division creating progeny containing half the chromosome copies of the progenitor cell. Improper chromosome segregation creates aneuploidy, which is poorly tolerated in cells. In cycling mitotic cells, aneuploidy leads to genome instability and cell death. Following meiosis, aneuploidy is associated with infertility, miscarriages, and birth defects. To segregate chromosome copies properly, pairs are physically organized and segregated to progeny cells by a mitotic spindle, whose functionality is tightly regulated. Kinesins are a family of highly conserved dimeric ATPase proteins which; organize spindle shape and size, facilitate chromosome capture and attachment to the spindle, and generate forces which are required for segregation. I investigated the molecular structure and function of human kinesin 13 family protein, Mitotic Centromere Associated Kinesin, MCAK. MCAK is a microtubule depolymerase whose full molecular structure and mechanism of depolymerization is not fully understood. Using in vitro biochemical assays and in vivo TIRF imaging, I found that altering MCAK molecular structure alters MCAK sub-spindle localization and by inference, alters global microtubule dynamics. This study suggests a potential mode for regulating of MCAK activity/function requiring further testing. Compared to over 30 kinesins in humans, showing a large amount of functional redundancy, yeast only has 6 identified kinesins whose function during meiotic cell division are still relatively unknown. I screened the importance and redundancy of yeast kinesins during meiosis. The results suggest similar roles and redundancies in meiosis to that during mitosis, despite different biochemical and biophysical spindle environments. Together, my investigations broaden the understanding of kinesin regulation and functional redundancy during different types of cell division.

Meiotic recombination, known as crossover, is a vital mechanism for generating genetic diversity in sexually reproducing populations. Recombination events are non-uniform across the genome, due to a variety of influences including chromatin structure, DNA-sequence, epigenetic marks and interference from other recombination events. These known factors do not fully explain the distribution of recombination events, and additionally do not account for all the variability in recombination frequency observed both between and within species. Furthermore, of the mechanisms that have been identified, many are not yet fully understood. In Arabidopsis thaliana, considerable variation is observed in recombination frequency and distribution between natural accessions. By investigating recombination events in A.thaliana, this project aimed to identify trans-acting modifiers of recombination frequency that varied between natural accessions. Identification of meiotic recombination modifiers was performed through Quantitative Trait Loci (QTL) mapping in A.thaliana natural-accession cross populations. Populations were generated from crosses between two accessions which differed significantly for recombination frequency as measured across a defined region of the genome flanked by a fluorescent-reporter system. F1 plants were then self-fertilised to produce segregating mosaic F2 populations for mapping. Recombination frequency for specific genomic intervals was determined for each individual in the population through measurement of the segregation of flanking fluorescence-genes expressed in the products of meiosis - seeds or pollen. Individuals were also genotyped using accession-specific markers across the genome, at a marker density of one marker per 2-5Mb, depending on the chromosome. Association of variation in recombination frequency with specific sections of the genome differing between the parental accessions through QTL mapping revealed significant modifiers of meiotic recombination segregating within the populations. This resulted in the identification of three significant large-effect modifiers that differed between Col-0 and Cvi-0 accessions, on chromosomes 1 ,2 and 5, affecting recombination in an interval in the sub-telomere region of chromosome 3. An additional modifier on chromosome 4 affecting the same sub-telomeric interval was identified that differed between the Col-0 and Can-0 accessions. Further fine-mapping of modifiers to improve location resolution was performed by repeated backcrosses into the Col-0 genetic background to remove the influence of other large-effect QTL and possible unknown small-effect modifiers. Improving the resolution provided a number of potential candidates for genes underlying the recombination phenotype for each QTL. Candidate testing was then performed, either through transformation of different accession alleles into the fluorescent-reporter system, or through analysis of T-DNA insertion lines that interrupted candidate genes. Preliminary results from T-DNA insertion mutants crossed to the fluorescent-reporter system suggest a potential role for the AT2G31510 gene in modification of meiotic recombination frequency, though the mode of action remains unknown. These results demonstrate the presence of large-effect modifiers of meiotic recombination frequency that vary between the natural A.thaliana accessions Col-0, Cvi-0 and Can-0. Confirmation of underlying genes or sequence elements and characterisation of their mechanism of action are opportunities for exploration in future experiments.

Meiosis is a specialized form of cell division where homologous chromosomes are segregated in meiosis I before sister chromatids are segregated in meiosis II. To establish this pattern, a number of changes to the mitotic chromosome segregation machinery are put in place. Firstly, sister kinetochores orient towards the same pole in meiosis I (mono-orientation). Secondly, homologue recombination creates chiasmata, which link homologues together. And thirdly, cohesin, the molecule that holds sister chromatids together, is cleaved in a step-wise manner. This is achieved because the Shugoshin (Sgo1) protein recruits protein phosphatase 2A (PP2A) to centromeres to counteract cohesin phosphorylation, which is required for its cleavage. The work presented here has investigated two critical aspects of cohesin protection: firstly, how cohesin protection is deactivated in meiosis II and, secondly, how a meiosis-specific protein called Spo13 helps to set up cohesin protection in meiosis I. Previously, our lab had shown that Sgo1 is removed from chromosomes when sister chromatids come under tension during mitosis. I therefore sought to investigate whether sister kinetochore mono-orientation allows Sgo1 to stay on centromeres during meiosis I and carry out its protective function. To this end, I modified meiosis I chromosomes to lack both chiasmata and mono-oriented kinetochores. Under these conditions, where sister chromatids are forced to be under tension in metaphase I, Sgo1 is undetectable on chromosomes. As a consequence, centromeric cohesin is largely lost in anaphase I leading to the premature separation of sister chromatids in a fraction of cells. Since mono-orientation of sister kinetochores is exclusive to meiosis I, these findings suggest that Sgo1 localisation is influenced by sister kinetochore tension in both mitosis and meiosis. Therefore, our findings suggest a mechanism that could contribute to the deprotection of cohesin in meiosis II. However, loss of cohesin protection upon bi-orientation is not complete, suggesting that other factors are involved in the efficient protection and deprotection of cohesin. One such factor is the meiosis-specific protein Spo13, which had previously been shown to be required for cohesin protection as well as kinetochore monoorientation. Although it had been suggested that Spo13 regulates Sgo1 recruitment to centromeres, I could not find any evidence to support a loss of Sgo1, or PP2A, in spo13Δ cells. Additionally, even when Sgo1 is stabilised and clearly visible in anaphase I of spo13Δ mutants, pericentromeric cohesion is still defective. Therefore, I investigated the effect that polo kinase Cdc5, an interactor of Spo13, has on Sgo1. While cellular Sgo1 levels are increased in response to Cdc5 loss, this effect seems to be independent of Spo13. However, Spo13 is required for proper levels of Cdc5 at centromeres and the centromeric recruitment of Cdc5 by Spo13 is likely to be functionally important because tethering of Cdc5 to kinetochores rescued the mono-orientation phenotype of spo13Δ cells. In contrast, I found no evidence that the Spo13-Cdc5 interaction is required for cohesin protection. Meiotic overexpression of SPO13 enhances cohesin protection in meiosis I, apparently independent of its robust interaction with Cdc5, and causes increased Sgo1 enrichment at centromeres. This suggested that Spo13 might recruit Sgo1 to cohesin itself to facilitate its protection. Although I could not detect a loss of Sgo1-cohesin interaction in spo13Δ cells, tethering of Sgo1 to cohesin restores pericentromeric Rec8 to spo13Δ mutants in anaphase I. Surprisingly, sister chromatids still segregate in this case, suggesting that pericentromeric cohesion is defective, despite maintenance of Rec8. Furthermore, inhibition of either one of the cohesin kinases, DDK and Hrr25, restores sister chromatid cohesion to spo13Δ cells. Therefore, the findings in this study suggest that Spo13 is at the centre of a complex regulatory network that coordinates cohesin protection and sister chromatid cohesion in meiosis I.

Meiosis is the process by which sexually-reproducing organisms ensure that precisely half a chromosome set is passed from each parent to the following generation; this circumvents the doubling of the genome that would otherwise occur upon fertilisation. Meiosis occurs via a single round of DNA replication followed by two successive chromosome segregation events. In the first segregation, homologous chromosomes align and become physically linked through the process of meiotic recombination, which is crucial for the accurate segregation of homologous chromosomes. During the second round of segregation, sister chromatids are segregated to produce four haploid daughter cells. Failure to physically tether homologous chromosomes to each other through meiotic recombination can result in the aberrant segregation of homologous chromosomes, which can cause hereditary diseases (aneuploidies) and miscarriages in humans. Meiotic recombination also shuffles alleles of the parental chromosomes, which is crucial for evolution. The study of meiotic recombination, and its regulation, is thus paramount for our understanding of how genetic diversity is generated within populations. The work in this thesis has helped characterise factors, both genetic and environmental, that modulate meiotic recombination in the fission yeast, Schizosaccharomyces pombe. Here, I identify temperature as a major determinant of meiotic recombination outcome; when meiosis is performed at 16°C, significant reductions in meiotic recombination outcome are observed relative to meiosis performed at higher temperatures. Additionally, I present genetic and cytological evidence that the strand resection and strand invasion steps of meiotic recombination are impaired at 16°C relative to higher temperatures, but that double strand break levels appear not to be influenced by temperature. I have also characterised several novel genes predicted to be involved in meiotic recombination, and explored the genetic relationship between several genes already known to be crucial in modulating meiotic recombination. Finally, I have laid the foundations for a future project aiming to map the meiotic recombination landscape across the entire S. pombe genome.

Fetal germ cell development is a key stage of female reproductive life. The DAZ family proteins (DAZ, DAZL and BOLL) are RNA-binding proteins with critical roles in murine germ cell development but their expression and potential targets in the human are largely unknown. The studies in this Thesis investigated the expression and function of DAZL and BOLL in human fetal ovary. Both DAZL and BOLL mRNA are increased dramatically at the time of entry into meiosis. Immunohistochemical analysis with specific meiotic markers suggested that DAZL and BOLL have distinct spatial-temporal expression patterns, with minimal co-expression – BOLL expression was transient prior to follicle formation. This pattern was shown not to be present in the mouse fetal ovary, where Dazl and Boll are co-expressed, indicating a limitation of the mouse for exploring the function of Boll. Two human cell lines, embryonic kidney derived HEK293 cells and germ cell tumour derived TCam-2 cells were used as models to identify the mRNA targets of DAZL and BOLL after transfection of DAZL or BOLL vectors. In HEK293 cells, TEX19 and TEX14 were confirmed as potential targets of both DAZL and BOLL, and CDC25A as a potential DAZL target. Further experiments indicated that DAZL and BOLL did not increase target mRNA transcription but increased stabilisation. A DAZL/GFP co-transfection-FACS system for TCam-2 cells was established as this cell line has very low transfection efficiency. TEX14 and SYCP3 significantly increased in GFP+ve-DAZL+ve cells when compare to the GFP-ve-DAZL-ve cells, whilst SOX17 and DNMT3L significantly decreased in the GFP+ve-DAZL+ve cells. A 3'-UTR luciferase assay confirmed regulation of TEX14 and SOX17 by DAZL through their 3'-UTR. RNA immunoprecipitation further demonstrated direct binding between human TEX14, TEX19, SYCP3, SOX17 mRNA and DAZL protein, and that TEX14 binding is through its 3'-UTR. Dual fluorescence immunohistochemistry showed that SOX17 and DMNT3L are expressed in early germ cells with DAZL, and are later down-regulated co-incident with that of DAZL, consistent with the novel repressive effect of human DAZL on these two potential targets. These studies indicate that DAZL and BOLL are associated with different key meiotic stages of germ cell development in human fetal ovary. Several potential mRNA targets of DAZL and BOLL, and a novel repression function of human DAZL on its mRNA targets were identified giving further insight into the role of these factors in human ovarian development.

Meiosis is a specialized cell division that produces haploid gametes from a diploid progenitor cell. It consists of one round of DNA replication followed by two consecutive rounds of chromosome segregation. Homologous chromosomes segregate in meiosis I and sister chromatids segregate in meiosis II. Failure to correctly regulate meiosis can result in aneuploidy, where daughter cells inherit an incorrect number of chromosomes. Aneuploidy is usually poorly tolerated in eukaryotes, and is associated with infertility, miscarriages and birth defects. At the meiosis I to meiosis II transition, DNA replication does not occur between chromosome segregation steps despite the need for Spindle Pole Bodies (SPBs) to be re-licensed in order to build meiosis II spindles. The mechanisms that make this distinction are not yet known. In budding yeast, the protein phosphatase Cdc14 is essential for the progression of cells into meiosis II. Cdc14 is sequestered for the majority of the cell cycle in the nucleolus by the inhibitor Cfi1/Net, and is only released in anaphase. We have observed Cdc14 localizing to and interacting with SPB components when nucleolar sequestration is inhibited. Through fluorescence microscopy and EM analysis, we have determined that Cdc14 is required for the re-duplication of SPBs after meiosis I. Our data implies a role for Cdc14 in the phospho-regulation of SPB half-bridge component Sfi1. Cdc14 is therefore essential for the relicensing of SPB duplication, a crucial step necessary to ensure accurate chromosome segregation in meiosis.

The maintenance of genomic stability through suppression of retrotransposon activity is vital for the avoidance of potentially mutagenic genomic disruption caused by retrotransposition. Germline development is a particularly important phase for retrotransposon silencing as retrotransposition events here have the potential for transmission to the entire embryo, threatening the health of offspring. A collection of germline genome defence genes are required for the suppression of retrotransposons in the developing germline of male mice (e.g. Tex19.1, Dazl, Mili, Miwi2, Gasz, Mov10l1, Mael, Dnmt3l), all of which trigger meiotic prophase arrest when mutated. I have analysed the meiotic defects which arise in Tex19.1-/- male mice to contribute to the understanding of the fundamental mechanisms required for successful completion of meiosis and to investigate the involvement of retrotransposon silencing in this process. The absence of TEX19.1 in male mice causes infertility; with failed chromosome synapsis in ~50% of pachytene nuclei and associated apoptosis, as well as individual univalent chromosomes in 67% of remaining nuclei progressing to metaphase I. Where studied, failed chromosome synapsis is a common feature of germline genome defence mutant spermatocytes. One aim of my studies has been to better understand the mechanism responsible for this failed chromosome synapsis. I have demonstrated that unlike Mael-/- spermatocytes, additional SPO11-independent DNA damage potentially attributable to retrotransposition is not detectable in Tex19.1-/- spermatocytes. Rather, the formation of meiotic DNA double strand breaks (DSBs) is dramatically reduced in early prophase to around 50%, resulting in a reduction in nuclear γH2AX signal, production of SPO11- oligonucleotide complexes and foci formation by early recombination proteins RPA, DMC1 and RAD51. Despite this early reduction, DSB frequency recovers to more normal levels shortly after in zygotene. I have shown that defective pairing of homologous chromosomes by meiotic recombination is likely responsible for the asynapsis previously reported. The initial reduction in DSB frequency could be sufficient to cause failed chromosome synapsis in this mutant, assuming that late-forming DSBs cannot participate effectively in promoting homologous pairing. Alternative hypotheses include altered positioning of DSBs in response to altered chromatin organisation relating to retrotransposon upregulation, misguiding the pairing of homologous chromosomes. Such a model of disruption could also extend to other germline genome defence mutants. I have demonstrated that despite successful pairing of homologous chromosomes in a sub-population of Tex19.1-/- spermatocytes, subsequent progression of these cells through pachytene is delayed. Numerous diverse features of progression are all delayed, including recombination, ubiquitination on autosomes and sex chromosomes, expression of the mid-pachytene marker H1t, and chromosome organisation. The delay identified is related to recombination therefore this feature is likely to stem from the initial defect in DSB formation early in prophase. While some delayed features are probably directly related to recombination, others are not. The coordinated delay observed may suggest the presence of a recombination-sensitive cell-cycle checkpoint operating to regulate progression through pachytene. My research has also aimed to establish the cause of elevated univalent chromosomes not connected by chiasmata in metaphase I Tex19.1-/- spermatocytes. I have demonstrated that that absence of chiasmata is not due to failed crossover formation between synapsed chromosomes. Rather, the frequent observation of individual unsynapsed chromosomes during crossover formation suggests that some spermatocytes with low-level asynapsis are leaking through meiotic checkpoints and are unable to form a crossover before reaching metaphase. Therefore, again this later meiotic defect appears to stem from the initial defect in meiotic DSB formation, the consequences of which vary widely in severity. Remarkably the unsynapsed chromosomes present during crossover formation include both sex chromosomes, and autosomes. Tolerance of an unsynapsed autosome from pachytene into metaphase is an unusual observation in mice and this observation may aid the understanding of spermato cyte quality control mechanisms during this progression. Together these findings have greatly advanced the understanding of the infertility incurred during meiosis in Tex19.1-/- male mice. These findings may also extend to benefit the understanding of other germline genome defence mutants. Diverse observations made during my investigations also reveal a potential system of coordinated progression through pachytene relating to meiotic recombination. The variable severity of the synapsis defects incurred in this mutant appears to have variable effects on spermatocyte survival and could also inform the understanding of meiotic checkpoint sensitivity.

Cell division relies on many steps, precisely synchronised, to ensure the fidelity of chromosome segregation. To achieve such complex and multiple functions, cells have evolved mechanisms by which one protein can participate in numerous events on the cell life. Over the past few years, an increasing number of functions have been assigned to the proteins of the nuclear pore complex (NPC) also called nucleoporins. NPCs are large complexes studded in the nuclear envelope, which control the nucleocytoplasmic transport. It is now known that nucleoporins participate in spindle assembly, kinetochore organisation, spindle assembly checkpoint, and all processes important for genome integrity maintenance. This work demonstrates that the nucleoporin ALADIN participates in mitosis, meiosis and in cilia. In both mitosis and meiosis, ALADIN is important for proper spindle assembly. In mitosis, it was also discovered that ALADIN is a novel factor in the spatial regulation of the mitotic regulator Aurora A kinase. Without ALADIN, active Aurora A spreads from centrosomes onto spindle microtubules, which affects the distribution of a subset of microtubule regulators and slows spindle assembly and chromosome alignment. Interestingly, mutations in ALADIN causes triple A syndrome and some of the mitotic phenotypes observed after ALADIN depletion also occur in cells from triple A syndrome patients. In meiosis, ALADIN contributes to trigger the resumption of meiosis in female mouse. Impairment of ALADIN from mouse oocyte slows spindle assembly, migration and reduces oocytes ability to extrude polar bodies during meiosis I, which concomitantly affects the robustness of oocyte maturation and impairs mouse embryo development. Nucleoporins were also found at the base of the cilia, a centriole-derived organelle that participates in differentiation, migration, cell growth from development to adulthood. Here it is shown that ALADIN is also localised at the base of the cilia. With this work, new ALADIN’s functions have been identified across cell division, as well as uncovered an unexpected relation between triple A syndrome and cell division.

Meiotic recombination is a fundamental biological process in sexually reproducing organisms, enabling offspring to inherit novel combinations of mutations, and ensuring even segregation of chromosomes into gametes. Recombination is initiated by programmed Double Strand Breaks (DSBs), the genomic locations of which are determined in most mammals by PRDM9, a rapidly evolving DNA-binding protein. In crosses between different mouse subspecies, certain Prdm9 alleles cause infertility in hybrid males, implying a critical role in fertility and speciation. Upon binding to DNA, PRDM9 deposits a histone modification (H3K4me3) typically found in the promoters of expressed genes, suggesting that binding might alter the expression of nearby genes. Many other questions have remained about how PRDM9 initiates recombination, how it causes speciation, and why it evolves so rapidly. This body of work investigates these questions using complementary experimental and analytical methodologies. By generating a map of human PRDM9 binding sites and applying novel sequence analysis methods, I uncovered new DNA-binding modalities of PRDM9 and identified sequence-independent factors that predict binding and recombination outcomes. I also confirmed that PRDM9 can affect gene expression by binding to promoters, identifying candidate regulatory targets in meiosis. Furthermore, I showed that PRDM9’s DNA-binding domain also mediates strong protein-protein interactions that produce PRDM9 multimers, which may play an important functional role. Finally, by generating high-resolution maps of PRDM9 binding in hybrid mice, I provide evidence for a mechanism to explain PRDM9-mediated speciation as a consequence of the joint evolution of PRDM9 and its binding targets. This work reveals that PRDM9 binding on one chromosome strongly impacts DSB formation and/or repair on the homologue, suggesting a novel role for PRDM9 in promoting efficient homology search and DSB repair, both critical for meiotic progression and fertility. One consequence is that PRDM9 may play a wider role in mammalian speciation.

Errors in chromosome segregation during the first division of female meiosis are very common in humans and result in aneuploidy leading to reproduction problems. Chromosome segregation depends on the formation and function of the meiotic spindle as well as the structure of chromosomes, which need to condense to be able to orient and segregate properly. It is important to understand the mechanisms underlying the female meiotic spindle function and chromosome condensation to gain insight into female fertility problems. The female meiotic spindle assembles without centrosomes, so the mechanisms ensuring microtubule nucleation, spindle assembly and establishment of bipolarity act differently from those of mitosis or male meiosis. I identified a set of genes that are required for microtubule nucleation, spindle maintenance and centromere orientation in Drosophila female meiosis. This was accomplished by mapping previously uncharacterized Drosophila mutants and depleting already known genes by RNAi. I discovered that several proteins have a different role in female meiosis as compared to mitosis, which provides insight into the major differences between these systems. Little is known about the molecular mechanisms of chromosome condensation. The roles of only a few factors, such as condensin complexes, have been studied previously, and the evidence suggests that there are more molecular players required for chromosome condensation. To discover molecular mechanisms critical to this process, I depleted various chromosomal proteins by RNAi and screened for abnormalities of metaphase chromosome morphology in Drosophila oocytes by immunostaining and live imaging. I found that the conserved kinase NHK-1 plays a role in chromosome condensation in female meiosis. BAF is a critical NHK-1 substrate in this process and its phosphorylation is required for detachment of the chromosomes from the nuclear envelope to allow proper condensation. Also, I discovered that the nucleosome remodelling complex NuRD is crucial for chromosome condensation, especially for the chromosome arms. As a result of my PhD project I identified multiple factors required for meiotic spindle function. I also discovered two novel pathways of chromosome condensation that require the NuRD complex and NHK-1 activity.

The spliceosomal U5 snRNP contains several proteins with well characterised functions in splicing, including: Brr2, an ATPase/RNA helicase that disrupts U4/U6 and U2/U6 snRNA base pairing during activation of the spliceosome; Snu114, a GTPase that controls the action of Brr2; and Prp8, the largest and most conserved protein considered to have a central role in the spliceosome, which interacts directly with Snu114 and Brr2. Yeast Cwc21 is one of twelve Bact complex proteins that associate with spliceosomes shortly before the first step of splicing catalysis. Cwc21 interacts directly with Prp8 and Snu114, as does its human orthologue, the SR protein SRm300/SRRM2. Although, Cwc21 is not essential for yeast cell viability, it is required for sporulation. This work aims to identify the function of Cwc21 during meiosis. PP1 is a protein phosphatase required for both steps of splicing. Multiple sequence alignments of Snu114 and Prp8 revealed the presence of putative PP1 binding motifs that are well conserved among different species. This led me to hypothesize that PP1 may interact with Snu114 and/or Prp8 to regulate these or other interacting proteins. By screening intron-containing genes that are expressed in meiosis, I found that Cwc21 is required for splicing HRB1 transcripts. In addition, I show that HRB1 is also required during meiosis. The HRB1 intron contains an unusual branchsite sequence, TACTAATG, which when changed to the consensus branchsite sequence restores sporulation in the absence of Cwc21. Therefore, it is likely that Cwc21 promotes the expression of HRB1 during an early stage of meiosis by stabilising its pre-mRNA in the catalytic centre of the spliceosome. This study demonstrates a novel function for Cwc21 during meiosis. Using yeast two hybrid assay I have identified the interacting regions of Cwc21, PP1 and Brr2 in Snu114. Through biochemical studies I provide evidence for mutually exclusive interaction of Cwc21 and PP1 in the putative PP1 binding motif situated in Snu114 domain ‘IVa’. In the case of yeast Snu114, the PP1 binding motif has a novel sequence ‘YGVQYK’. I also show that the affinity of Cwc21 and PP1 for Snu114 is influenced by the different nucleotide-bound states of Snu114. Furthermore, I show that mutations in Snu114 domain ‘IVa’ restrict Snu114 function during meiosis and affect the MER1 splicing regulatory network. Therefore, Snu114 may play a role in modulating the conformational state of the catalytic spliceosome through its interactions with Cwc21/PP1 in regulating subsets of genes during meiosis. Finally, I show that PP1 is a putative regulator of Prp8.

In eukaryotes, recombination plays a critical role in both the production of viable gametes and as a population genetic process. Here, we are interested in studying recombination as it provides insight into a process that has shaped variation. To this end, we study the evolution of cross-over rates in chimpanzees and humans through two experiments. Components of the recombination machinery are well described in yeast and C. elegans, but less so in other species. In humans, cross-over rates vary across physical scales and occur predominantly in narrow ∼2 kb regions called hotspots, where hotspot usage differs considerably between individuals. Differential hotspot usage is associated with specific DNA motifs, and DNA-contacting zinc finger array variants in the transacting PRDM9 H3K4 trimethyltransferase. The precise relationship between DNA motifs, PRDM9 and hotspot activity is not completely understood. Experiment 1. To investigate the importance of PRDM9 motif recognition, which is predicted be different between humans and chimpanzees, and the effect of PRDM9 on the evolution of fine-scale cross-over rates, we sequenced 10 unrelated Pan troglodytes verus (Western chimpanzee) genomes to moderate coverage (∼10×). I validate the approach by demonstrating that fine-scale maps estimated from 10 human genomes of each African and European ancestry recapitulate independently estimated maps. Then I characterise the error modes in sequencing data arising from errors in chemistry, alignment, variant calling, and genotyping. I identify several cryptic error modes missed by state-of-the-art filters and develop methods to counteract them. To guard against genotype error arising from stochastic variation in low to moderate coverage sequencing, I develop methods to incorporate the underlying statistical uncertainty into recombination analyses, evaluate the approaches through simulation (estimated 11% improvement) and empirical assessment (estimated 4% improvement), and discover that the reported genotype uncertainty is poorly calibrated, which limits the approaches. Consequently, a filtering approach was applied to the hard-called chimpanzee genotypes. I estimate recombination rates in chimpanzees through an existing LD-based method. In contrast to humans, there is no increased cross-over localisation around chimpanzee PRDM9 binding predictions, nor motifs consistently associated with activity. Hotspots do not overlap between the two species, indicating that rates evolved rapidly and consistent with PRDM9 localising all hotspots. In contrast, gene pro- moters and CpG islands are common attractors of recombination (2.7-fold increase in rate in chimpanzee, 1.5-fold increase in human), suggesting chromatin state influences hotspot placement but to varying degree in the species. I discuss the potential implications for PRDM9 mechanism. Experiment 2. To enable a more representative characterisation of the spectrum of genome changes occurring in chimpanzee genomes, I analyse data from an extended three generation Western chimpanzee pedigree sequenced at high coverage (∼30×). I use Mendel transmission to filter variants, infer haplotypes, and identify recombination events through a Hidden Markov Model approach. We detect 375 recombination events, of which 3 are double cross-over events. Sex-specific recombination rate estimates in chimpanzees mirror sex differences in humans (N♂/N♀ = 0.58) and have similar levels of total recombination. We resolve recombination events typically at ∼ 856 base-pair resolution. Additionally, analyses of Mendel inconsistencies suggest that extended pedigree sequencing opens the door on studying complex genome changes. These experiments demonstrate the power of comparative analyses, the utility of high throughput sequencing in enabling the study of recombination in almost any species of interest, the challenges in sifting signal from noise in these data, and the need for experimental and algorithmic methods to guard against error.

Chromosome segregation errors that occur in the developing germline generate aneuploidies which are among the leading causes of embryonic lethality, spontaneous abortions and chromosomal disorders, such as Down’s syndrome. Compared to other species, human oocytes appear to be particularly prone to suffer chromosome missegregation and the risk of aneuploid pregnancies in humans increases drastically with maternal age. Despite its particular importance for human health, relatively little is known about the basis for the high incidence of aneuploidies in human oocytes and the maternal-age effect. The identification and analysis of molecular pathways that promote genetic and chromosomal stability is important for our understanding of mechanisms that lead to aneuploidy and how it can be prevented. Here, I examine the role of the pluripotency associated Tex19.1 gene, in preventing aneuploidy during mouse female germ cell development. I demonstrate that Tex19.1-/- females are subfertile when mated with wild type males due to defects in chromosome segregation during meiosis. In contrast to Tex19.1-/- male gem cells, synaptonemal complex formation appears to be completed normally in Tex19.1-/- females but high levels of aneuploidy are evident during the second meiotic stages of oogenesis. The Tex19.1-/- females transmit these aneuploidies to their offspring likely resulting in the observed embryonic death and subfertility. In addition to its role in the female germline, I investigated the function of Tex19.1 during embryonic development. I found that Tex19.1-/- knockout mice are born at a sub- Mendelian frequency and this reduction is exacerbated in diapaused embryos, suggesting that Tex19.1 plays a role during a stage where a pluripotent state is maintained for a prolonged period of time. Furthermore, I identified high levels of aneuploidy accumulating in pluripotent stem cells in the absence of Tex19.1.

The interval between meiotic nuclear divisions can be regarded as a modified mitotic cell cycle where DNA replication is blocked. Mechanisms regulating this critical aspect of meiosis that allows haploid cells to be generated from a diploid progenitor were investigated in this project. Licensing is restricted after meiosis I due to down-regulation of Cdc18 and Cdt1. Late meiotic expression of Cdc18 and Cdt1, which load the MCM helicase onto replication origins, can lead to partial DNA replication after meiosis I. This implies that block to initiation via licensing forms an important component of this regulation. As detecting any minor DNA re-replication after meiosis I requires a technique more sensitive than flow cytometry for detection of total cell DNA contents, I also investigated a procedure to allow incorporation and detection of 5-ethynyl-2'-deoxyuridine (EdU) in fission yeast. Additional inactivation of Spd1 or stabilization of Dfp1 after MI when Cdc18 and Cdt1 are also expressed does not enhance re-replication, but cyclin-dependent kinase Cdc2 plays a role in preventing re-replication during the MI-MII interval. Unexpectedly, when the licensing block is subverted, replication forks only move a short distance in the interval between meiosis I and II, implying that the elongation step of DNA replication is also inefficient. In addition, I show that the regulation of entry into meiosis II is not delayed by a partial round of DNA replication or DNA damage, indicating that replication and DNA damage checkpoints do not operate in late meiosis.

Specification of a germ cell lineage and appropriate maturation are essential for the transfer of genetic information from one generation to the next. Germ cells form from pluripotent precursor cells that migrate into the gonadal ridge and undergo commitment to either the female or male lineage. In the fetal ovary, germ cells enter meiotic prophase I, then arrest at the diplotene stage; in the testis germ cells do not begin meiosis until puberty. Abnormal differentiation of germ cells can result in malignant transformation. Somatic cells play a key role in modulating the developmental fate of the germ cells. Research into germ cell development during fetal life has almost exclusively focused on studies in rodents, but we, and others, have reported several fundamental differences in the expression of germ cell specific markers in the human compared with the mouse. The studies described in this thesis have investigated germ cell-specific gene expression and the possible impact of the somatic cells during development. This was achieved by studying human fetal gonads obtained during the 1st and 2nd trimesters of pregnancy and through the use of both wild-type and mutant mouse ES cell lines. Studies on germ cells in the human fetal testis have extended the findings of others, and confirmed that germ cell populations at different stages of maturation co-exist in the human fetal testis, a situation that is in contrast to that in rodents. For example expression of M2A and AP2γ was restricted to the OCT4-positive gonocyte population, while VASA and NANOS1 were localised exclusively to the to the OCT4-negative prespermatogonia. DAZL was expressed in both populations. Analysis also revealed that both the gonocyte and prespermatogonial populations proliferate throughout the 2nd trimester. Recent studies have implicated retinoic acid (RA) in the control of meiotic entry in germ cells of the fetal mouse ovary. In this study we demonstrated for the first time that two genes implicated in the action of RA in mouse gonad, STRA8 and NANOS2, are also expressed in a similar sexspecific- manner in the human fetal gonads, and that the RA receptors are present in both somatic and germ cells suggesting that RA may regulate germ cell function in the human as well as the mouse. However, whilst the mesonephros appears to be the primary site of RA synthesis in the mouse our initial studies indicate that in the human the gonad itself may be a more likely site of RA biosynthesis. In the fetal mouse testis, RA is degraded by the enzyme Cyp26b1 present in the somatic cells and germ cells do not enter meiosis, our novel findings suggest that CYP26B1 is more abundant in the human fetal ovary than the testis, suggesting that meiotic entry may be controlled by an alternative signalling pathway in the human. One of the methods that can aid our understanding of somatic cell gene expression in the gonad is in vitro culture. To date, there have been no published reports of the successful in vitro culture of somatic cells from the human fetal testis. In the current study, populations of human somatic cells were dissociated and maintained in vitro and characterised. Analysis demonstrated that cells expressing mRNAs characteristic of Sertoli cells, Leydig cells and peritubular myoid (PTM) cells were present initially, but long-term culture resulted in downregulation in expression of mRNAs specific for Sertoli cells and Leydig cells, suggesting that these cells either failed to survive or underwent alterations to their phenotype. In contrast PTM/fibroblast cells proliferated in vitro and initially maintained androgen receptor expression. These cultures therefore hold promise for studies into the signalling or cell-cell interactions in testicular somatic cells especially those relevant to the PTM population. Several studies have claimed differentiation of putative germ cells from ES cells. In the current study, analysis of mouse ES cell lines has expanded on results showing that ES cells and early germ cells express a number of genes in common. Kit signalling was shown to be important for ES cell survival as they differentiate although expression of Kit was heterogeneous. We also demonstrated that ES cells that did not express Kit displayed a decreased expression of the early germ cell genes Blimp1, Fragilis and Stella, implicating Kit signalling in the control of germ cell-associated gene expression in ES cells. This may be important to future studies optimising germ cell derivation from ES cells. In conclusion, this study has demonstrated important differences in protein expression patterns in germ cells of the human fetal testis compared to the mouse, and has raised questions about whether the proposed mechanism controlling meiotic entry of germ cells in the mouse can be applied to the human. The establishment of a system for culturing human fetal gonadal somatic cells may lead to further understanding of gene expression and development in the human fetal testis, and data suggest that the Kit/Kitl signalling system may influence germ cell gene expression in mouse ES cells.

Microtubule-based spindle formation is essential to faithful chromosome segregation during cell division. In many animal species, the oocyte meiotic spindle forms without centrosomes (the major microtubule organizing centers), unlike most mitotic cells. Even in mitotic cells, centrosomes are sometimes dispensable for bipolar spindle formation, indicating a redundant pathway initiating spindle assembly. In this study, we examined meiotic spindle assembly in C. elegans oocytes. We have demonstrated: First, metaphase I spindle formation is Ran-GEF and Ran-GAP independent in meiotic embryos. Second, free tubulin, also called soluble tubulin, concentrates in the nuclear volume during Germinal Vesicle Breakdown (GVBD) as well as in the spindle region during metaphase I and metaphase II. We then showed that the concentration of free tubulin at metaphase II spindle region is enclosed by dense ER sheets which exclude cytoplasmic organelles including mitochondria and yolk granules from the meiotic spindle. Similar observations are also shown in early mitotic cells. Together, this suggests free tubulin concentrating in the nuclear region might be a common mechanism promoting spindle formation through volume exclusion in both meiotic and early mitotic embryos in C. elegans. Moreover, the movement of molecules during GVBD depends on the size and the charge of the molecules.

The process of gametogenesis is orchestrated by a dynamic gene expression program, where a vital subset constitutes the early meiotic genes (EMGs). In budding yeast, the transcription factor Ume6 represses EMG expression during mitotic growth. However, during the transition from mitotic to meiotic cell fate, EMGs are activated in response to the transcriptional regulator Ime1 through its interaction with Ume6. While it is known that binding of Ime1 to Ume6 promotes EMG expression, the mechanism of EMG activation remains elusive. Two competing models have been proposed whereby Ime1 either forms an activator complex with Ume6 or promotes Ume6 degradation. Here, we resolve this controversy using a combination of depletion and tethering strategies to functionally characterize both Ume6 and Ime1 (Chapter 2 and Chapter 3). Much of the research surrounding Ume6 function, and the genes it regulates, has come from using a null allele (ume6∆). Accordingly, constitutive loss of UME6 function leads to derepression of several meiosis specific genes during mitosis. Expression of these meiotic genes during the mitotic cell cycle causes conflicts in cellular machinery and leads to pleiotropic consequences. Instead of ume6∆ to assess UME6 function, here we leverage the auxin inducible degron (AID) system to construct a depletable allele of UME6 (Ume6-AID). This allele of UME6 maintains repression of its targets during the mitotic cell cycle. Using this approach, we identify the set of genes that are directly regulated by Ume6, including UME6 itself (Chapter 2).With the development of Ume6-AID, we next investigated the functional relationship between Ume6 and Ime1 by combining our Ume6-AID with a method of synchronizing expression for IME1, which encodes the meiotic transcription factor, and IME4, which encodes an mRNA N6-adenosine methyltransferase (Chapter 3). This increased control allowed careful monitoring of how Ume6 responds to IME1 and IME4 expression and allowed functional dissection of Ume6’s role in the mechanism of early meiotic gene (EMG) expression. We find that, while Ume6 protein levels increase in response to Ime1, Ume6 degradation occurs much later in meiosis, in a manner dependent on another meiotic transcription factor called Ndt80. Importantly, we found that depletion of Ume6 shortly before meiotic entry is detrimental to EMG activation and gamete formation. Finally, to explore the functional role of Ime1 in EMG expression, we employed a tethering strategy. We find that tethering of Ume6 to a heterologous activation domain is sufficient to trigger EMG expression and produce viable gametes in the absence of Ime1. These data indicate that Ime1 and Ume6 form an activator complex in meiosis. While Ume6 is indispensable for EMG expression, Ime1 primarily serves as a transactivator for Ume6.This dissertation unifies observations from two disparate models involving Ime1 and Ume6 and their involvement in meiotic initiation. Our work unveils the impact Ime1 has on Ume6 through its binding to Ume6 and how this influences EMG expression. In doing so we elevate Ume6 to a primary determinant of cell state through its exchange of transcriptional cofactors and significantly advance our understanding of meiotic gene regulation.

Aneuploidy, or abnormal number of chromosomes in a haploid set, in XY/XX organisms has consequences that can impede physical and cognitive development. To prevent aneuploidy, cellular division relies on the correct position of paired chromosomes and subsequent segregation in the meiotic program. If connections between paired chromosomes sever or errors in chromosome contraction towards spindle poles arise, aneuploidy occurs. Many arthropod species have chromosomes that naturally do not pair, or that pair differently than typical autosomes. Through the evaluation of such systems, novel insights into chromosomal coordination and positioning may be revealed. The objective of this study is twofold: (1) investigate univalent X chromosome behavior in the two-striped planthopper Acanalonia bivittata (X0/XX) and (2) explore a sex trivalent model (XXY/XX) in the giant shield mantis Rhombodera megaera. Here, we will investigate both systems using live-cell and immunofluorescence imaging to reveal mechanisms regulating chromosome coordination and segregation to further understand translational implications across all species.

Meiosis is a tightly regulated series of events leading to gamete formation. A key player in this process is the protease Separase (SEP-1). Known for its role in chromosome segregation, we have defined a role for SEP-1 in vesicular trafficking during cell division. Securin (IFY-1) is an inhibitory chaperone of SEP-1 that is degraded at anaphase onset after ubiquitination by the Anaphase Promoting Complex/Cyclosome (APC/C). After IFY-1 degradation, activated SEP-1 cleaves a subunit of cohesion to allow chromosome segregation. SEP-1 also displays dynamic localization during the cell cycle. In prometaphase I, IFY-1 localizes to kinetochores and mysterious cortical filaments. During anaphase I, SEP-1 transfers to the midbivalent, the region between homologous chromosomes where Cohesin localizes, and concurrently appears on vesicles, called cortical granules, in the cortex. How the dynamic localization of SEP-1 is regulated is currently unknown. Here, I investigated how the APC/C and IFY-1 regulate Separase during meiosis I. First, I observed that IFY-1 is degraded by anaphase I and is not present on vesicles. Second, APC/C activity is required for SEP-1 to localize to vesicles. apc/c RNAi results in arrested embryos with SEP-1 and IFY-1 remaining colocalized on cortical filaments. Third, partial ify-1 RNAi causes precocious SEP-1 vesicle localization. These observations suggest that IFY-1 degradation is required for SEP-1 to localize to vesicles during anaphase I. To further test this, Non-Degradable Securin fused to GFP (GFP::IFY-1DM) was generated. GFP::IFY-1DM is stably expressed during anaphase I and causes embryonic lethality. GFP::IFY-1DM causes chromosome segregation defects, polar body extrusion failure and inhibits cortical granule exocytosis. Importantly, expression of GFP::IFY-1DM causes a reduced and delayed localization of SEP-1 to vesicles during anaphase I. I conclude that degradation of IFY-1 regulates the localization of SEP-1 to vesicles to coordinate exocytosis with chromosome segregation. My findings suggest a novel role for the APC/C and IFY-1 in controlling the localization of SEP-1, in addition to regulating its protease activity for chromosome segregation and cortical granule exocytosis during anaphase I.

Plant breeders rely on heritable genetic variation for trait improvement, and the two primary novel sources of this variation are recombination and mutation of genetic material during meiosis. These independent processes can have overlapping significance for breeders, as the genetic resolution generated by recombination influences our ability to locate mutations. In addition, population improvement often results in inbreeding, which reduces the effective recombination and increases the likelihood of deleterious mutation hitchhiking via repulsion linkages. This is especially true in regions of chromosomes with low recombination rates, like the pericentromere. Polypoid crops pose an additional challenge to pinpointing the genetic control of desirable traits, as the phenotypic consequence of a single-variable locus (e.g., genomic structural variation) can be masked by the redundant copies of other homoeologous genomes. Here I address how geneticists can improve the accuracy of identifying targets for positional cloning, and whether breeders should seek to manipulate recombination to further harness the power of selection, using hexaploid wheat (Triticum aestivum L.) as a model. First, I present a study with a traditional approach to positionally clone a quantitative trait locus for yield components on wheat chromosome arm 5A. Leveraging a fine-mapping population, genomic data, phenotypic associations, early grain development transcriptome profiles, and predicted gene function, it was determined the quantitative trait locus was a result of strong linkage disequilibrium with a large deletion on wheat chromosome arm 5AS. This study highlights the phenotypic resiliency of polyploids harboring structural variants and actionable recommendations to increase the likelihood of identifying causal variants in wheat. Second, I used simulation models with empirical data to assess the potential of controlled recombination for genomic selection breeding programs. While controlled recombination remains under research and development, initial reports have prompted interest in evaluating increased recombination in the pericentromere to disrupt repulsion linkages. Comparing high and low values for a range of simulation parameters identified that few combinations under increased recombination retained greater genetic variation and fewer still achieved higher genetic gain. More recombination was associated with loss of genomic prediction accuracy, which often outweighed the benefits of disrupting repulsion linkages. Irrespective of recombination frequency and distribution and QTL location, enhanced response to selection under increased recombination largely depended on quantitative trait architecture, high heritability, more repulsion than coupling linkages, and greater than six cycles of genomic selection. Altogether, the results discourage a controlled recombination approach to genomic selection in wheat as a more efficient path to retaining genetic variation and increasing genetic gains compared to existing breeding methods.

Meiosis is the conserved cell division process used by sexually reproducing organisms to produce gametes, which are the cells that give rise to the next generation. In humans, meiosis generates eggs and sperm, and in the model budding yeast Saccharomyces cerevisiae, meiosis generates spores. Despite considerable evolutionary divergence, the general mechanics of meiotic chromosome division are largely conserved from yeast to humans. This conservation has made yeast an invaluable tool for understanding meiosis. In addition to important discoveries related to chromosome division, yeast have shaped our knowledge of other meiotic processes, including gene regulatory changes, organelle dynamics and inheritance, protein quality control, and cellular rejuvenation.Changes in expression impact virtually every gene in the yeast genome during meiosis and spore development. In addition to increases and decreases in the transcription of classically defined genes and mRNAs, meiotic gene expression also involves the transcription of extended transcripts called Long Undecoded Transcript Isoforms (LUTIs), which have 5' extended sequences relative to their canonical counterparts. LUTIs are repressive, acting to transcriptionally interfere with canonical mRNA expression and translationally block protein synthesis from their main ORF via competitive translation of upstream open reading frames (uORFs). LUTIs are also pervasive and used to drive protein levels for approximately 8% of all yeast genes during meiosis. The majority of LUTI mRNAs have yet to be characterized in detail, and many of the proteins impacted by their expression are reported to exhibit stable expression in mitosis.Superoxide dismutase 1 (Sod1) is a highly abundant and primarily cytosolic antioxidant that detoxifies superoxide radicals (O2•–), a type of reactive oxygen species (ROS), from cells. We became interested in studying Sod1 after analysis of mRNA-sequencing and ribosome profiling data that suggested a LUTI mRNA is expressed from the SOD1 locus during meiosis. During mitotic growth, yeast express a canonical SOD1 mRNA (SOD1canon.) of approximately 600 nucleotides. During the meiotic divisions, cells initiate transcription from an extremely distal start site, resulting in the production of a putative LUTI mRNA that is nearly four times the length of SOD1canon.. We confirmed that this extended isoform (SOD1LUTI) is in fact produced as a continuous, ~2.2 kb transcript, and that LUTI expression functions to repress canonical mRNA levels. Although naturally produced during meiosis, SOD1LUTI is also made by mitotic cells experiencing ER stress, raising the possibility that the meiotic unfolded protein response (UPRER) is responsible for turning on the LUTI.In addition to highly dynamic changes in the expression of SOD1canon. and SOD1LUTI, we found that Sod1 protein levels and localization change during meiosis. During the transition from nutrient-rich media to starvation conditions, Sod1 levels decrease considerably and discrete foci of Sod1 appear. As cells go through meiosis, the brightness and number of these foci decreases, suggesting their degradation. The formation of these foci is independent of entry into meiosis, but their disappearance from cells relies on meiotic progression. During the meiotic divisions, due to the combination of LUTI expression and the degradation of preexisting protein, Sod1 levels reach their lowest levels. Once the chromosome divisions have completed, cells begin to express SOD1canon. once more, and new Sod1 protein is produced. Altogether, these data support a model in which old Sod1 protein is systematically cleared from cells to facilitate the generation of new protein. We propose that the regulation of this important antioxidant outlined in this dissertation may play a role the lifespan resetting that takes place during gametogenesis. Many important questions remain unanswered regarding the meiotic regulation of Sod1. What is the significance of this transient dip in expression during meiosis? What is the nature of the relationship between SOD1LUTI and the UPRER? Are the foci of Sod1 we observe true protein aggregates? Answering these and other questions will improve our understanding of the biology of this important antioxidant.

The nucleus, the defining organelle of the eukaryotic cell, must be remodeled during every cell division in order to accommodate the division of genetic material. While this remodeling has been extensively studied during mitosis, it remains poorly understood how the nucleus is remodeled during meiosis. In this study, we provide a comprehensive analysis of nuclear behavior during meiosis in the model organism budding yeast. First, we find that the nuclear envelope undergoes a five-way division, forming a nuclear-envelope bound compartment that is excluded from gametes. This compartment, termed the Gametogenesis Uninherited Nuclear Compartment or GUNC, contains nuclear pore complexes (NPCs) in all cells and age-induced damage in old cells. The material sequestered to the GUNC is subsequently eliminated by release of vacuolar proteases, establishing this remodeling event as a novel nuclear quality control mechanism that contributes to meiotic cellular rejuvenation. Second, we find that the NPC undergoes two mechanistically-distinct meiotic remodeling events: partial nuclear basket detachment during meiosis I and full nuclear basket detachment during meiosis II. Meiosis I detachment, which involves Nup60 and its binding partner Nup2, is driven by Polo kinase-dependent phosphorylation of Nup60 at its binding interface with the NPC core. Notably, this remodeling event also occurs in the distantly related Schizosaccharomyces pombe, suggesting basket detachment involves conserved nuclear basket organizational principles and fulfills an important function. Finally, we find that the nuclear permeability barrier is transiently disrupted during meiosis II, driving intermixing of the cytoplasm and nucleoplasm. Since the nuclear envelope stays intact throughout meiosis, regulation of nuclear transport machinery is likely involved in this event. Several meiotic regulators disrupt barrier loss or return, although their precise mechanistic contributions remain unclear. In all, this work establishes the nuclear periphery as a highly dynamic and regulated entity during budding yeast meiosis. Studying the budding yeast meiotic nucleus will continue to improve our understanding of nuclear organization and its contributions to cellular health for years to come.

All eukaryotic cells depend on faithful cell division to give rise to daughter cells that contain the correct number of chromosomes. Chromosome segregation errors during meiosis in germ cells can lead to aneuploid gametes, which can cause infertility, genetic disorders such as Down Syndrome and miscarriages. Additionally, chromosome segregation errors during mitosis can lead to aneuploid somatic cells, which are a hallmark of cancer. Despite the importance of tightly regulating these two types of cell division, the mechanisms that ensure fidelity during these processes remain active areas of investigation. Proteins that have been shown to be important regulators of fidelity during meiosis in Caenorhabditis elegans (C. elegans) include ZHP-3 and PCH-2. ZHP-3 is a putative SUMO or ubiquitin ligase required for crossover recombination, but whether or not ZHP-3 is regulated to promote recombination remains poorly understood. PCH-2 is a conserved AAA+ ATPase required for timely coordination of the meiotic prophase events pairing, synapsis and recombination, but the mechanism for how it accomplishes this coordination is currently unknown. Additionally, PCH-2 has been shown to be required for both silencing and activation of the spindle assembly checkpoint during mitosis, but how it regulates the checkpoint is still an active area of investigation. Here, I use C. elegans as a model system to investigate the molecular mechanisms of these two proteins during meiotic and mitotic chromosome segregation. In Chapter 1, I investigate whether ZHP-3 is phosphorylated by a conserved DNA damage kinase, CHK-1, to determine if this phosphorylation event is required for ZHP-3’s localization or function during meiosis. I find that while ZHP-3 is phosphorylated by CHK-1 kinase in vitro, mutation of these phosphorylation sites in vivo does not affect ZHP-3’s localization to meiotic chromosomes, or its function in promoting crossover recombination. In Chapter 2, I investigate a different regulator of meiosis, PCH-2, to determine how it coordinates meiotic prophase events and whether it acts on meiotic HORMADS to accomplish this. I find that mutations in PCH-2 genetically interact with mutations in the meiotic HORMADS HTP-3 and HIM-3, suggesting that these proteins work together to coordinate meiotic prophase events. Additionally, I find that PCH-2 acts on HTP-3 to coordinate pairing and synapsis, but acts on HIM-3 during recombination, indicating that the meiotic HORMADS are cleanly delegated to regulate specific meiotic prophase events. Finally, in Chapter 3, I explore PCH-2’s role during somatic cell division and investigate how PCH-2 regulates the strength of the spindle assembly checkpoint. I find that PCH-2 is more enriched in P1 cells compared to AB cells in the early C. elegans embryo, potentially explaining why cells that give rise to the germline have stronger checkpoints than somatic cells. Additionally, we find that this enrichment depends on cell fate factors and PCH-2’s adaptor protein CMT-1. Taken together, these results contribute mechanistic insight for how these proteins promote fidelity during meiosis and mitosis by determining post-translational modifications of meiotic recombination factors (Chapter 1), identifying potential substrates of meiotic prophase regulators (Chapter 2), and demonstrating how spindle checkpoint regulators are differentially inherited in specific cell types to give rise to differences in spindle checkpoint strength (Chapter 3).

Homologous chromosome pairing is an important first step in Meiosis. It physically aligns the homologs at a close distance along their length so that they can exchange genetic information. This exchange of information increases genetic diversity and forms the basis for Mendelian inheritance. Homologous chromosome pairing also disentangles the chromosomeswhich prepares them for chromosome segregation later in the cell cycle. Errors in chromosome segregation lead to infertility, meiotic arrest, and chromosome number disorders. It is thus important to understand how this crucial first step in Meiosis is achieved. While a lot is known about the genetics of homologous chromosome pairing, much less is known about the physics of chromosome pairing. Even in an organism with relatively short chromosomes such as S. cerivisea, pairing is still a limiting step in Meiosis, taking hours to complete. At these timescales, photobleaching and phototoxicity are problematic, even when using very low intensity light. For these reasons, it has proved difficult to study this process live. The difficulty of studying this process experimentally motivated us to build a physical model. Our physical model simulates not only meiotic chromosomes, but the physical environment, constraints, and active motion that the chromosomes experience during pairing. We used this model to investigate how different features of Meiosis affect the rate of pairing and the resolution of interlocks. By modeling three strains of S. cerivisea in silico, and comparing our results to experimental data, we were also able to make a prediction about the distribution of binding sites along the length of the chromosomes. Finally, our model gives us detailed movies of interlock resolution events which are then used to build a step-by-step recipe for interlock resolution. Together, these results bring new insights to the physical process of homologous chromosome pairing.

Maintenance of genome integrity is critical for cell proliferation, organism survival and fertility. The DNA damage response (DDR) is an important aspect of genome maintenance that coordinates a range of cellular processes including DNA replication, cell cycle regulation and DNA repair. The phosphatidylinositol 3’ kinase (PI3K)-related kinase ATR, has emerged as a central regulator of the DDR by phosphorylating a diverse range of targets in response to DNA damage to coordinate DNA repair pathway choice with the cell cycle and other DNA metabolic activities. Recently, ATR has emerged as an attractive target for cancer treatment and ATR inhibitors are currently in clinical trials. Importantly, the ATR-activating scaffold protein TOPBP1 has also been implicated in regulating DNA repair pathway choice as well as coordinating distinct branches of ATR signaling. In this thesis, I will present a range of evidence that extends our understanding of the mechanism of the ATR-TOPBP1 signaling axis in somatic DNA repair.In addition to the roles of TOPBP1 and ATR in the somatic DNA damage response, TOPBP1 and ATR are essential for male meiotic progression by promoting DNA repair by homologous recombination, chromosome synapsis, and transcriptional silencing of unsynapsed chromosomes including the X and Y, which can only partially synapse by a small region known as the PAR (pseudoautosomal region). First, I present a characterization of ATR signaling events in mouse meiosis by using a dual approach to inhibit ATR followed by mass spectrometry. Specifically, we used a RAD1 conditional knockout model, which prevents TOPBP1-mediated ATR activation in meiocytes, and mice treated with ATR inhibitors. In this way, we can take advantage of both tissue-specific and acute ATR inhibition to generate a set of high-confidence ATR-dependent phosphorylation events. Second, in chapter 3, I utilize this database of ATR and RAD1-dependent events to understand the status of ATR activation in a new separation-of-function TOPBP1 model mouse with eight charge reversal mutations in BRCT5 of TOPBP1. These Topbp1KE/KE mice are grossly indistinguishable from wild type littermates and have no checkpoint or cell sensitivity phenotypes, yet males are completely sterile. By comparing the set of ATR and RAD1-dependent phosphosites to a testes phosphoproteome from the Topbp1KE/KE mice, we find that between 80-90% of ATR signaling remains unchanged, indicating that a small, but critical set of ATR targets are lost in the Topbp1KE/KE testes. Furthermore, in chapter 3, I present the characterization of the Topbp1KE/KE phenotype and describe several hypothesis and future directions to identify the critical ATR targets or changes in TOPBP1 protein interactions that contribute to the total loss of male fertility in these mice. Next, in chapter 4, I present my efforts towards understanding the role of the ATR signaling axis in the DNA damage response in cancer cell lines. Specifically, how ATR promotes the repair of DNA lesions by the error-free pathway homologous recombination (HR) by maintaining the expression of a set of HR factors. I established a protocol of low-dose, long-term treatment of ATR inhibitors that results in a depletion of HR factors which subsequently results in sensitivity to DNA damage induced by PARP inhibitors. I establish that this sensitivity is due to increased DNA-PK dependent mutagenic repair through the non-homologous end joining pathway (NHEJ) and both sensitivity and chromosomal aberrations can be rescued by DNA-PKcs inhibition. Finally, in Chapter 5, I present evidence that TOPBP1 regulates DNA repair pathway choice. Specifically, TOPBP1 coordinates the actions of the scaffold proteins 53BP1 and BRCA1 in regulating the decision between non-homologous end joining (NHEJ) or homologous recombination (HR). I show that by modulating the stability of the TOPBP1-53BP1 or TOPBP1-BRCA1 complexes by expression of protein fusion constructs that artificially strengthen these interactions or by mutating TOPBP1 BRCT domains to disrupt the interaction of TOPBP1 to phosphorylated 53BP1 or BRCA1, in mouse models. Furthermore, I present evidence that it is not possible to complement a loss of ATR activity after knockdown of endogenous TOPBP1 with expression of ectopic TOPBP1 in several cell culture systems. This result implies the existence of an unknown mechanism of TOPBP1 regulation that only occurs after expression from the endogenous locus. Overall, this thesis represents a body of work that extends our understanding of the role of the ATR-TOPBP1 signaling axis in spermatogenesis and in somatic DNA repair pathway choice.

Meiosis is a central mechanism in sexual reproduction, through which the diploid precursor cells in the germline produce haploid gametes. After fertilization, a new set of diploid genome forms in the offspring with the characteristics of mother and father. The faithful transmission of genetic material to next generation relies on the fidelity of chromosome segregation during meiosis. A variety of mechanisms regulate the chromosome segregations in meiosis, including homolog interaction, chromosome cohesion, and sister-chromatid orientation. In most eukaryotic organisms, homolog interactions are built by the formation of chiasmata resulted from crossovers, but it is absent in male Drosophila in which an alternative conjunction complex is obligate to stabilize homologous chromosome. In addition, Drosophila also deploys a three-member complex, called SOS consisting of SOLO, ORD and SUNN, to establish and maintain the sister-chromatid cohesion in meiosis, while this responsibility is executed by a well-known cohesin complex composed of four subunits (SMC1, SMC3, REC8 and SA) in other eukaryotes. A conserved meiosis-specific sister-chromatid mono-orientation is evident in Drosophila, as in the meiosis I of other eukaryotes, but the regulatory pathway has been elusive until now in both sexes of Drosophila. In this investigation, we used conventional genetic and advanced fluorescence microscopy to investigate the role of checkpoint Rough Deal (ROD) in Drosophila male meiosis by using trans-heterozygous rod mutants. Based on our results, ROD protein is required for the maintenance, but not the establishment, of centromere cohesion by prohibiting the redistribution of Shugoshin protein MEI-S332. More importantly, ROD modulates the orientation of sister chromatid in meiosis I, might through the regulation and coordination of microtubule attachments on kinetochores. Therefore, dysfunction of ROD causes a high frequency of chromosome mis-segregation, which gives rise to premature equational segregation and 4/O chromosome nondisjunction in the first meiotic division. To get more insights into the ROD function in meiosis, we further generate a conditional ROD-depleted line in which we expect to induce the degradation of ROD protein in Drosophila spermatocytes, which could help us to fully understand the role of ROD in a null-like background.

RAD51 and BRCA2 are conserved proteins involved in homology-directed DNA double-strand break (DSB) repair. This form of DSB repair occurs in somatic cells and is also the essence of meiotic recombination, which allows gametes to have different combinations of alleles and facilitates proper division of chromosomes into haploid gametes. Organisms with rad51 or brca2 mutations often have fertility defects Additionally, there is emerging evidence of a link between DSB repair and plant defense. This dissertation focuses on the RAD51A1, RAD51A2, and BRCA2 proteins in maize. Little is known about the interactions between RAD51A1 and other maize proteins. A series of in vitro affinity experiments were performed: phage display (a discovery-driven molecular biology technique typically used for biomedical applications which finds short amino acid sequences with in vitro affinity for a bait protein) followed by dot-blotting and ELISA to verify affinity. To validate whether these alignments with maize proteins signified possible affinity for RAD51A1, short peptides were synthesized based on the aligning sequences found in maize. Of these 32 synthesized peptides, 14 bound RAD51A1 in vitro, including four peptides that match transcription factors. This is a promising avenue of investigation—in Arabidopsis, RAD51 appears to function as a transcription factor during defense. Mutants were used to investigate the function of RAD51A1, RAD51A2, and BRCA2 in maize reproduction. Previous evidence of sterility in these mutants relied on the macroscopically visible phenotypes of defective male tassels and low female seed sets. In contrast, this dissertation involved direct examination of reproductive cells. Male sterility was examined by pollen staining. In addition to the expected finding of almost complete sterility in rad51a1/rad51a2 and brca2 mutants, there was an unexpected finding of reduced fertility in rad51a1 single mutants. This reduced male fertility, despite the fact that these plants had one or two wild-type RAD51A2 alleles, suggests that RAD51A1 may have some key reproductive function partially lost in RAD51A2. The basis of the male sterility phenotype was also investigated in male zygotene and pachytene meiocytes. ZYP1, a protein involved in pairing homologous chromosomes, was localized in these cells. As predicted, rad51a1/rad51a2 and brca2 mutants had impaired ZYP1 axis formation compared to wild type; ZYP1 co-localized with DNA but with a more punctate and fragmented appearance compared to wild type. This suggests faulty pairing of chromosomes in these mutants, which could explain their failure to form fertile pollen. Finally, this thesis discusses transcriptomic and reverse genetics investigations of the potential link between DNA repair and plant defense. Wild-type maize plants were treated with UV-B or salicylic acid (SA), inoculated with Xanthomonas vasicola, or used as a control. RNA-seq data from this experiment showed that the defense genes PR1(PATHOGENESIS-RELATED PROTEIN 1, or Zm00001d018738) and PRP3 (PATHOGENESIS-RELATED PROTEIN 3, or Zm00001d048947) had increased transcription in inoculated samples. There was also increased transcription of the Bowman-Birk type trypsin inhibitor Zm00001d024960. This experiment also uncovered several genes of interest involved in maize response to UV, including genes involved in diterpenoid metabolism. Only one gene met the adjusted p-value (padj)

DNA damage response (DDR) pathways give cells the ability to sense and repair DNA lesions or initiate apoptosis when the damage is unrepairable. The RAD9A-HUS1-RAD1 (9A-1-1) complex is a heterotrimeric DNA clamp that plays an important role in checkpoint signaling and DNA repair. The 9A-1-1 complex interacts with TOPBP1 at the site of damaged DNA, which promotes CHK1 phosphorylation by stimulating the upstream kinase ATR. The 9A-1-1 complex also directly interacts with multiple components of several DNA repair pathways, such as base excision repair and homologous recombination. Loss of any subunit of the 9A-1-1 complex causes severe genomic instability, heightened sensitivity to genotoxic stress, and embryonic lethality. In order to study this complex in vivo I am using two genetic approaches to determine 1) how the 9A-1-1 complex along with alternative 9-1-1 complexes act during male mammalian meiosis and 2) what the physiological requirements are for 9A-1-1 complex-mediated checkpoint signaling in mitotic and meiotic cells. Our first approach utilized a genetic model to study the functions of 9A-1-1 and alternative 9-1-1 complexes throughout prophase I of meiosis. Previous studies revealed that conditional knockout of Hus1 in the testes causes germ cell loss and infertility due to unresolved meiotic DSBs. Surprisingly, RAD1 was able to localize to meiotic chromosome cores even in the absence of Hus1. This led us to hypothesize that RAD1 interacts with paralogs of HUS1 and RAD9A, termed HUS1B and RAD9B respectively to form alternative 9-1-1 complexes during meiosis. We created a testis-specific conditional knockout of Rad1 and found that Rad1 loss led to significantly increased asynapsis of homologous chromosomes, compromised DSB repair and impaired phosphorylation of known ATR targets such as CHK1. Phosphoproteomic analysis of testes from Rad1 CKO mice or ATR inhibitor-treated wild-type mice further implicated RAD1 and ATR signaling in the regulation of cohesin subunits, synaptonemal complex proteins and various DNA repair factors. To differentiate between the DNA repair and checkpoint signaling roles of the 9A-1-1 complex, a RAD9A separation-of-function mutant was created. Serine 385 of the C-terminal tail of RAD9A is the main phosphorylation site responsible for interacting with TOPBP1, ultimately leading to activation of ATR and its downstream substrate CHK1. By creating a phospho-dead mutant (RAD9A-S385A) we selectively disrupted checkpoint signaling without compromising other DNA repair functions of the 9A-1-1 complex. Although Rad9aSA/SA mice were viable, they were born at less than expected frequency and were smaller in size as compared to control littermates. Rad9aSA/SA mice had a 10-fold increase in micronucleated red blood cells, a marker of genomic instability, and were hypersensitive to the DNA interstrand crosslinking agent mitomycin C (MMC). Cultured Rad9aSA/SA cells demonstrated reduced genotoxin-induced CHK1 phosphorylation as compared to control cell lines, confirming that ATR signaling and CHK1 activation were disrupted as anticipated. Exposure to DNA damaging agents also triggered increased chromosomal aberrations, including radials, fusions and breaks, in Rad9aSA/SA cells relative to controls. Rad9aSA/SA cells also showed defects in replication fork protection, reflected by significantly increased MRE11-dependent nascent strand degradation. Together, these studies provide a deeper understanding of the multifaceted functions that the canonical 9A-1-1 complex and alternative clamps play in maintaining genomic stability both within the germline and in somatic cells.