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EFTUD2 is mutated in patients with mandibulofacial dysostosis with microcephaly (MFDM). We generated a mutant mouse line with conditional mutation in Eftud2 and used Wnt1-Cre2 to delete it in neural crest cells. Homozygous deletion of Eftud2 causes brain and craniofacial malformations, affecting the same precursors as in MFDM patients. RNAseq analysis of embryonic heads revealed a significant increase in exon skipping and increased levels of an alternatively spliced Mdm2 transcript lacking exon 3. Exon skipping in Mdm2 was also increased in O9-1 mouse neural crest cells after siRNA knock-down of Eftud2 and in MFDM patient cells. Moreover, we found increased nuclear P53, higher expression of P53-target genes and increased cell death. Finally, overactivation of the P53 pathway in Eftud2 knockdown cells was attenuated by overexpression of non-spliced Mdm2, and craniofacial development was improved when Eftud2-mutant embryos were treated with Pifithrin-α, an inhibitor of P53. Thus, our work indicates that the P53-pathway can be targeted to prevent craniofacial abnormalities and shows a previously unknown role for alternative splicing of Mdm2 in the etiology of MFDM.

In the past decade, there has been a growing appreciation for R-loop structures as important regulators of the epigenome, telomere maintenance, DNA repair, and replication. Given these numerous functions, dozens, or potentially hundreds, of proteins could serve as direct or indirect regulators of R-loop writing, reading, and erasing. In order to understand common properties shared amongst potential R-loop binding proteins, we mined published proteomic studies and distilled 10 features that were enriched in R-loop binding proteins compared with the rest of the proteome. Applying an easy-ensemble machine learning approach, we used these R-loop binding protein-specific features along with their amino acid composition to create random forest classifiers that predict the likelihood of a protein to bind to R-loops. Known R-loop regulating pathways such as splicing, DNA damage repair and chromatin remodeling are highly enriched in our datasets, and we validate 2 new R-loop binding proteins LIG1 and FXR1 in human cells. Together these datasets provide a reference to pursue analyses of novel R-loop regulatory proteins.

About 10–15% of all human cancer cells employ a telomerase-independent recombination-based telomere maintenance method, known as alternative lengthening of telomere (ALT), of which the full mechanism remains incompletely understood. While implicated in previous studies as the initiating signals for ALT telomere repair, the prevalence of non-canonical nucleic acid structures in ALT cancers remains unclear. Extending earlier reports, we observe higher levels of DNA/RNA hybrids (R-loops) in ALT-positive (ALT+) compared to telomerase-positive (TERT+) cells. Strikingly, we observe even more pronounced differences for an associated four-stranded nucleic acid structure, G-quadruplex (G4). G4 signals are found at the telomere and are broadly associated with telomere length and accompanied by DNA damage markers. We establish an interdependent relationship between ALT-associated G4s and R-loops and confirm that these two structures can be spatially linked into unique structures, G-loops, at the telomere. Additionally, stabilization of G4s and R-loops cooperatively enhances ALT-activity. However, co-stabilization at higher doses resulted in cytotoxicity in a synergistic manner. Nuclear G4 signals are significantly and reproducibly different between ALT+ and TERT+ low-grade glioma tumours. Together, we present G4 as a novel hallmark of ALT cancers with potential future applications as a convenient biomarker for identifying ALT+ tumours and as therapeutic targets., Graphical Abstract Graphical abstractALT+ cells promote a telomeric environment that leads to increased formation of G-quadruplexes and R-loops. Co-formation of G-quadruplexes and R-loops results in special structures, G-loops, which are postulated to facilitate the ALT recombination process.

R loops are three-stranded nucleic acid structures consisting of an RNA molecule that has invaded duplex DNA. R-loop structures have normal functions in regulating gene expression, class-switch recombination, telomere stability, and mitochondrial DNA replication. However, unscheduled R-loop accumulation is a driver of DNA replication stress and genome instability. Meanwhile, R loops and associated transcription-replication conflicts have been observed in cells that have lost tumor-suppressor genes or have activated oncogenes. While ectopic R loops can both disrupt epigenetic states, and promote genome instability, in most cases the hinted-at direct links between R loops and cancer development are lacking. Here, we review the possible influences of altered R-loop stability and metabolism on cancer development and discuss how R-loop accumulation might be exploited to benefit cancer patients.

The spliceosome has been implicated in genome maintenance for decades. Recently, a surge in discoveries in cancer has suggested that the oncogenic mechanism of spliceosomal defects may involve defective genome stability. The action of the core spliceosome prevents R-loop accumulation, and regulates the expression of genome stability factors. At the same time, specific spliceosomal components have non-canonical functions in genome maintenance. Here we review these different models, highlighting their discovery in different model systems, and describing their potential impact on human disease states.

RNA processing mutants have been broadly implicated in genome stability, but mechanistic links are often unclear. Two predominant models have emerged: one involving changes in gene expression that perturb other genome maintenance factors and another in which genotoxic DNA:RNA hybrids, called R-loops, impair DNA replication. Here we characterize genome instability phenotypes in yeast splicing factor mutants and find that mitotic defects, and in some cases R-loop accumulation, are causes of genome instability. In both cases, alterations in gene expression, rather than direct cis effects, are likely to contribute to instability. Genome instability in splicing mutants is exacerbated by loss of the spindle-assembly checkpoint protein Mad1. Moreover, removal of the intron from the α-tubulin gene TUB1 restores genome integrity. Thus, differing penetrance and selective effects on the transcriptome can lead to a range of phenotypes in conditional mutants of the spliceosome, including multiple routes to genome instability.

The exosome functions to regulate the cellular transcriptome through RNA biogenesis, surveillance, and decay. Mutations in Dis3, a catalytic subunit of the RNA exosome with separable endonuclease and exonuclease activities, are linked to multiple myeloma. Here we report that a cancer-associated DIS3 allele, dis3E729K, provides evidence for DIS3 functioning in mitotic fidelity in yeast. This dis3E729K allele does not induce defects in 7S→5.8S rRNA processing, although it elicits a requirement for P-body function. While it does not significantly influence cell cycle progression alone, the allele reduces the efficiency of cell cycle arrest in strains with defects in kinetochore assembly. Finally, point mutations in the exonuclease domains of yeast Dis3 elicit genome instability phenotypes; however, these DIS3 mutations do not increase DNA damage or RNA processing defects that lead to the accumulation of polyadenylated RNA in the nucleus. These data suggest that specific DIS3 activities support mitotic fidelity in yeast.

The nucleus is a highly organized organelle with an intricate substructure of chromatin, RNAs, and proteins. This environment represents a challenge for maintaining protein quality control, since non-native proteins may interact inappropriately with other macromolecules and thus interfere with their function. Maintaining a healthy nuclear proteome becomes imperative during times of stress, such as upon DNA damage, heat shock, or starvation, when the proteome must be remodeled to effect cell survival. This is accomplished with the help of nuclear-specific chaperones, degradation pathways, and specialized structures known as protein quality control (PQC) sites that sequester proteins to help rapidly remodel the nuclear proteome. In this review, we focus on the current knowledge of PQC sites in Saccharomyces cerevisiae, particularly on a specialized nuclear PQC site called the intranuclear quality control site, a poorly understood nuclear inclusion that coordinates dynamic proteome triage decisions in yeast.

The GPN proteins are a conserved family of GTP-binding proteins that are involved in the assembly and subsequent import of RNA polymerase II and III. In this study, we sought to ascertain the specificity of yeast GPN2 for RNA polymerases by screening the localization of a collection of 1350 GFP-tagged nuclear proteins in WT or GPN2 mutant cells. We found that the strongest mislocalization occurred for RNA polymerase II and III subunits and only a handful of other RNAPII associated proteins were altered in GPN2 mutant cells. Our screen identified Ess1, an Rpb1 C-terminal domain (CTD) prolyl isomerase, as mislocalized in GPN2 mutants. Building on this observation we tested for effects of mutations in other factors which regulate Rpb1-CTD phosphorylation status. This uncovered significant changes in nuclear-cytoplasmic distribution of Rpb1-GFP in strains with disrupted RNA polymerase CTD kinases or phosphatases. Overall, this screen shows the exquisite specificity of GPN2 for RNA polymerase transport, and reveals a previously unappreciated role for CTD modification in RNAPII nuclear localization.

Genome instability is a hallmark of cancer that arises through a panoply of mechanisms driven by oncogene and tumour-suppressor gene mutations. Oncogenic mutations in the core splicing factor SF3B1 have been linked to genome instability. Since SF3B1 mutations alter the selection of thousands of 3' splice sites affecting genes across biological pathways, it is not entirely clear how they might drive genome instability. Here we confirm that while R-loop formation and associated replication stress may account for some of the SF3B1-mutant genome instability, a mechanism involving changes in gene expression also contributes. An SF3B1-H662Q mutant cell line mis-splices the 5'UTR of the DNA repair regulator DYNLL1, leading to higher DYNLL1 protein levels, mis-regulation of DNA repair pathway choice and PARP inhibitor sensitivity. Reduction of DYNLL1 protein in these cells restores genome stability. Together these data highlight how SF3B1 mutations can alter cancer hallmarks through subtle changes to the transcriptome.

ARID1A is a core DNA-binding subunit of the BAF chromatin remodeling complex, and is lost in up to 7% of all cancers. The frequency of ARID1A loss increases in certain cancer types, such as clear cell ovarian carcinoma where ARID1A protein is lost in about 50% of cases. While the impact of ARID1A loss on the function of the BAF chromatin remodeling complexes is likely to drive oncogenic gene expression programs in specific contexts, ARID1A also binds genome stability regulators such as ATR and TOP2. Here we show that ARID1A loss leads to DNA replication stress associated with R-loops and transcription-replication conflicts in human cells. These effects correlate with altered transcription and replication dynamics in ARID1A knockout cells and to reduced TOP2A binding at R-loop sites. Together this work extends mechanisms of replication stress in ARID1A deficient cells with implications for targeting ARID1A deficient cancers., Author summary DNA is an incredibly busy molecule. It is bound by an ever-changing array of proteins, which control how our cells read the instructions encoded within DNA, through a process called transcription. DNA also must be replicated, condensed, and segregated every time a cell divides. These processes of DNA replicating and transcribing must not interfere with one another or the cell risks damage to DNA and potentially changes to the DNA code called mutations. In cancer many DNA transactions are perturbed, and this has been associated with damaging collisions between replication and transcription. Here we find that a gene called ARID1A, which is frequently lost in cancer cells, prevents such collisions between replication and transcription machinery. Loss of ARID1A has many effects on the cell, but in this context it seems to change the location and activity of an important regulator of DNA twisting and untangling called Topoisomerase 2A. Understanding how loss of ARID1A creates stresses on dividing cancer cells provides new opportunities to develop or apply therapies that could exploit this stress.

In the past decade there has been a growing appreciation for R-loop structures as important regulators of the epigenome, telomere maintenance, DNA repair and replication. Given these numerous functions, dozens, or potentially hundreds, of proteins could serve as direct or indirect regulators of R-loop writing, reading, and erasing. In order to understand common properties shared amongst potential R-loop binding proteins (RLBPs) we mined published proteomic studies and distilled 10 features that were enriched in RLBPs compared to the rest of the proteome. We used these RLBP-specific features along with their amino acid composition to create a random forest classifier which predicts the likelihood of a protein to bind to R-loops. In parallel, we employed a whole-genome CRISPR screen coupled with flow-cytometry using the S9.6 monoclonal antibody to sort guide RNAs associated with induction of high S9.6 staining. Known R-loop regulating pathways such as splicing and DNA damage repair are highly enriched in our datasets, and we validate two new R-loop modulating proteins. Together these resources provide a reference to pursue analyses of novel R-loop regulatory proteins.

SummaryEFTUD2, a GTPase and core component of the splicesome, is mutated in patients with mandibulofacial dysostosis with microcephaly (MFDM). We generated a mutant mouse line with conditional mutation inEftud2and usedWnt1-Cre2to delete it in neural crest cells. Homozygous deletion ofEftud2leads to neural crest cell death and malformations in the brain and craniofacial region of embryos. RNAseq analysis of embryonic mutant heads revealed a significant increase in exon skipping, in retained introns and enriched levels ofMdm2transcripts lacking exon 3. Mutants also had increased nuclear P53, higher expression of P53-target genes, and increased cell death. Their craniofacial development was significantly improved when treated with Pifithrin-α, an inihibitor of P53. We propose that craniofacial defects caused by mutations ofEFTUD2are a result of mis-splicing ofMdm2and P53-associated cell death. Hence, drugs that reduce P53 activity may help prevent craniofacial defects associated with spliceosomopathies.

Cdc48 (known as VCP in mammals) is a highly conserved ATPase chaperone that plays an essential role in the assembly and disassembly of protein-DNA complexes and in degradation of misfolded proteins. We find that in

DNA replication stress describes a state of impaired replication fork progress that triggers a cellular stress response to maintain genome stability and complete DNA synthesis. Replication stress is a common state that must be tolerated in many cancers. One promising therapeutic approach is targeting replication stress response factors such as the ataxia telangiectasia and rad 3-related kinase (ATR) or checkpoint kinase 1 (CHK1) kinases that some cancers depend upon to survive endogenous replication stress. However, research revealing the complexity of the replication stress response suggests new genetic interactions and candidate therapeutic targets. Many of these candidates regulate DNA transactions around reversed replication forks, including helicases, nucleases and alternative polymerases that promote fork stability and restart. Here we review emerging strategies to exploit replication stress for cancer therapy.

Cdc48/VCP is a highly conserved ATPase chaperone that plays an essential role in the assembly or disassembly of protein-DNA complexes and in degradation of misfolded proteins. We find that Cdc48 accumulates during cellular stress at intranuclear protein quality control (INQ) sites. Cdc48 function is required to suppress INQ formation under non-stress conditions and to promote recovery following genotoxic stress. Cdc48 physically associates with the INQ substrate and splicing factor Hsh155 and regulates its assembly with partner proteins. Accordingly, cdc48 mutants have defects in splicing and show spontaneous distribution of Hsh155 to INQ aggregates where it is stabilized. Overall, this study shows that Cdc48 regulates deposition of proteins at INQ and suggests a previously unknown role for Cdc48 in the regulation or stability of splicing subcomplexes.

R-loops are formed when replicative forks collide with the transcriptional machinery and can cause genomic instability. However, it is unclear how R-loops are regulated at transcription-replication conflict (TRC) sites and how replisome proteins are regulated to prevent R-loop formation or mediate R-loop tolerance. Here, we report that ATAD5, a PCNA unloader, plays dual functions to reduce R-loops both under normal and replication stress conditions. ATAD5 interacts with RNA helicases such as DDX1, DDX5, DDX21 and DHX9 and increases the abundance of these helicases at replication forks to facilitate R-loop resolution. Depletion of ATAD5 or ATAD5-interacting RNA helicases consistently increases R-loops during the S phase and reduces the replication rate, both of which are enhanced by replication stress. In addition to R-loop resolution, ATAD5 prevents the generation of new R-loops behind the replication forks by unloading PCNA which, otherwise, accumulates and persists on DNA, causing a collision with the transcription machinery. Depletion of ATAD5 reduces transcription rates due to PCNA accumulation. Consistent with the role of ATAD5 and RNA helicases in maintaining genomic integrity by regulating R-loops, the corresponding genes were mutated or downregulated in several human tumors.

Cdc48/VCP is a highly conserved ATPase chaperone that plays an essential role in the assembly or disassembly of protein-DNA complexes and in degradation of misfolded proteins. We find that Cdc48 accumulates during cellular stress at intranuclear protein quality control (INQ) sites. Cdc48 function is required to suppress INQ formation under non-stress conditions and to promote recovery following genotoxic stress. Cdc48 physically associates with the INQ substrate and splicing factor Hsh155 and regulates its assembly with partner proteins. Accordingly, cdc48 mutants have defects in splicing and show spontaneous distribution of Hsh155 to INQ aggregates where it is stabilized. Overall, this study shows that Cdc48 regulates deposition of proteins at INQ and suggests a previously unknown role for Cdc48 in the regulation or stability of splicing subcomplexes.

Chromosomal rearrangements involving the mixed-lineage leukemia (MLL1) gene are common in a unique group of acute leukemias, with more than 100 fusion partners in this malignancy alone. However, do these fusions occur or have a role in solid tumors? We performed extensive network analyses of MLL1-fusion partners in patient datasets, revealing that multiple MLL1-fusion partners exhibited significant interactions with the androgen-receptor signaling pathway. Further exploration of tumor sequence data from TCGA predicts the presence of MLL1 fusions with truncated SET domain in prostate tumors. To investigate the physiological relevance of MLL1 fusions in solid tumors, we engineered a truncated version of MLL1 by fusing it with one of its known fusion partners, ZC3H13, to use as a model system. Functional characterization with cell-based assays revealed that MLL1-ZC3H13 fusion induced chromosomal instability, affected mitotic progression, and enhanced tumorsphere formation. The MLL1-ZC3H13 chimera consistently increased the expression of a cancer stem cell marker (CD44); in addition, we detected potential collateral lethality between DOT1L and MLL1 fusions. Our work reveals that MLL1 fusions are likely prevalent in solid tumors and exhibit a potential pro-tumorigenic role.

Chang et al. link the RECQ-like helicase BLM and its yeast orthologue Sgs1 to preventing DNA damage caused by the accumulation of DNA:RNA hybrid structures called R-loops. This adds to a growing family of helicases implicated in R-loop resolution., Sgs1, the orthologue of human Bloom’s syndrome helicase BLM, is a yeast DNA helicase functioning in DNA replication and repair. We show that SGS1 loss increases R-loop accumulation and sensitizes cells to transcription–replication collisions. Yeast lacking SGS1 accumulate R-loops and γ-H2A at sites of Sgs1 binding, replication pausing regions, and long genes. The mutation signature of sgs1Δ reveals copy number changes flanked by repetitive regions with high R-loop–forming potential. Analysis of BLM in Bloom’s syndrome fibroblasts or by depletion of BLM from human cancer cells confirms a role for Sgs1/BLM in suppressing R-loop–associated genome instability across species. In support of a potential direct effect, BLM is found physically proximal to DNA:RNA hybrids in human cells, and can efficiently unwind R-loops in vitro. Together, our data describe a conserved role for Sgs1/BLM in R-loop suppression and support an increasingly broad view of DNA repair and replication fork stabilizing proteins as modulators of R-loop–mediated genome instability.

DNA repair defects create cancer predisposition in humans by fostering a higher rate of mutations. While DNA repair is quite well characterized, recent studies have identified previously unrecognized relationships between DNA repair and R-loop-mediated genome instability. R-loops are three-stranded nucleic acid structures in which RNA binds to genomic DNA to displace a loop of single-stranded DNA. Mutations in homologous recombination, nucleotide excision repair, crosslink repair, and DNA damage checkpoints have all now been linked to formation and function of transcription-coupled R-loops. This perspective will summarize recent literature linking DNA repair to R-loop-mediated genomic instability and discuss how R-loops may contribute to mutagenesis in DNA-repair-deficient cancers.

Ectopic R-loop accumulation causes DNA replication stress and genome instability. To avoid these outcomes, cells possess a range of anti-R-loop mechanisms, including RNaseH that degrades the RNA moiety in R-loops. To comprehensively identify anti-R-loop mechanisms, we performed a genome-wide trigenic interaction screen in yeast lacking RNH1 and RNH201. We identified >100 genes critical for fitness in the absence of RNaseH, which were enriched for DNA replication fork maintenance factors including the MRE11-RAD50-NBS1 (MRN) complex. While MRN has been shown to promote R-loops at DNA double-strand breaks, we show that it suppresses R-loops and associated DNA damage at transcription–replication conflicts. This occurs through a non-nucleolytic function of MRE11 that is important for R-loop suppression by the Fanconi Anemia pathway. This work establishes a novel role for MRE11-RAD50-NBS1 in directing tolerance mechanisms at transcription–replication conflicts., Accumulations of R-loops can lead to genome instability. Here the authors reveal a role for the MRN complex in suppressing R-loops and associated DNA damage at transcription–replication conflicts.

Alterations of the transcriptome and proteome enable stress recovery, but coordination of these events under stress is only partly understood. Mathew et al. report that under stress, an RNA splicing complex disassembles and the splicing factor Hsh155 moves to protein aggregates, coinciding with a drop in splicing and concomitant repression of ribosome production., Upon genotoxic stress, dynamic relocalization events control DNA repair as well as alterations of the transcriptome and proteome, enabling stress recovery. How these events may influence one another is only partly known. Beginning with a cytological screen of genome stability proteins, we find that the splicing factor Hsh155 disassembles from its partners and localizes to both intranuclear and cytoplasmic protein quality control (PQC) aggregates under alkylation stress. Aggregate sequestration of Hsh155 occurs at nuclear and then cytoplasmic sites in a manner that is regulated by molecular chaperones and requires TORC1 activity signaling through the Sfp1 transcription factor. This dynamic behavior is associated with intron retention in ribosomal protein gene transcripts, a decrease in splicing efficiency, and more rapid recovery from stress. Collectively, our analyses suggest a model in which some proteins evicted from chromatin and undergoing transcriptional remodeling during stress are targeted to PQC sites to influence gene expression changes and facilitate stress recovery.

Mutations that cause genome instability are considered important predisposing events that contribute to initiation and progression of cancer. Genome instability arises either due to defects in genes that cause an increased mutation rate (mutator phenotype), or defects in genes that cause chromosome instability (CIN). To extend the catalog of genome instability genes, we systematically explored the effects of gene overexpression on mutation rate, using a forward-mutation screen in budding yeast. We screened ∼5100 plasmids, each overexpressing a unique single gene, and characterized the five strongest mutators, MPH1 (mutator phenotype 1), RRM3, UBP12, PIF1, and DNA2. We show that, for MPH1, the yeast homolog of Fanconi Anemia complementation group M (FANCM), the overexpression mutator phenotype is distinct from that of mph1Δ. Moreover, while four of our top hits encode DNA helicases, the overexpression of 48 other DNA helicases did not cause a mutator phenotype, suggesting this is not a general property of helicases. For Mph1 overexpression, helicase activity was not required for the mutator phenotype; in contrast Mph1 DEAH-box function was required for hypermutation. Mutagenesis by MPH1 overexpression was independent of translesion synthesis (TLS), but was suppressed by overexpression of RAD27, a conserved flap endonuclease. We propose that binding of DNA flap structures by excess Mph1 may block Rad27 action, creating a mutator phenotype that phenocopies rad27Δ. We believe this represents a novel mutator mode-of-action and opens up new prospects to understand how upregulation of DNA repair proteins may contribute to mutagenesis.

SUMMARYEctopic R-loop accumulation causes DNA replication stress and genome instability. To avoid these outcomes, cells possess a range of anti-R-loop mechanisms, including RNaseH that degrades the RNA moiety in R-loops. To comprehensively identify anti-R-loop mechanisms, we performed a genome-wide trigenic interaction screen in yeast lacking RNH1 and RNH201. We identified >100 genes critical for fitness in the absence of RNaseH, which were enriched for DNA replication fork maintenance factors such as RAD50. We show in yeast and human cells that R-loops accumulate during RAD50 depletion. In human cancer cell models, we find that RAD50 and its partners in the MRE11-RAD50-NBS1 complex regulate R-loop-associated DNA damage and replication stress. We show that a non-nucleolytic function of MRE11 is important for R-loop suppression via activation of PCNA-ubiquitination by RAD18 and recruiting anti-R-loop helicases in the Fanconi Anemia pathway. This work establishes a novel role for MRE11-RAD50-NBS1 in directing tolerance mechanisms of transcription-replication conflicts.

Eukaryotic DNA replication occurs in the context of chromatin. Recent years have seen major advances in our understanding of histone supply, histone recycling and nascent histone incorporation during replication. Furthermore, much is now known about the roles of histone remodellers and post-translational modifications in replication. It has also become clear that nucleosome dynamics during replication play critical roles in genome maintenance and that chromatin modifiers are important for preventing DNA replication stress. An understanding of how cells deploy specific nucleosome modifiers, chaperones and remodellers directly at sites of replication fork stalling has been building more slowly. Here we will specifically discuss recent advances in understanding how chromatin composition contribute to replication fork stability and restart.

To achieve genome stability cells must coordinate the action of various DNA transactions including DNA replication, repair, transcription and chromosome segregation. How transcription and RNA processing enable genome stability is only partly understood. Two predominant models have emerged: one involving changes in gene expression that perturb other genome maintenance factors, and another in which genotoxic DNA:RNA hybrids, called R-loops, impair DNA replication. Here we characterize genome instability phenotypes in a panel yeast splicing factor mutants and find that mitotic defects, and in some cases R-loop accumulation, are causes of genome instability. Genome instability in splicing mutants is exacerbated by loss of the spindle-assembly checkpoint protein Mad1. Moreover, removal of the intron from the α-tubulin gene TUB1 restores genome integrity. Thus, while R-loops contribute in some settings, defects in yeast splicing predominantly lead to genome instability through effects on gene expression.

Deep sequencing has impacted on cancer research by enabling routine sequencing of genomes and exomes to identify genetic changes associated with carcinogenesis. Researchers can now use the frequency, type, and context of all mutations in tumor genomes to extract mutation signatures that reflect the driving mutational processes. Identifying mutation signatures, however, may not immediately suggest a mechanism. Consequently, several recent studies have employed deep sequencing of model organisms exposed to discrete genetic or environmental perturbations. These studies exploit the simpler genomes and availability of powerful genetic tools in model organisms to analyze mutation signatures under controlled conditions, forging mechanistic links between mutational processes and signatures. We discuss the power of this approach and suggest that many such studies may be on the horizon.

The ability to rapidly assess chromosome instability (CIN) has enabled profiling of most yeast genes for potential effects on genome stability. The A-like faker (ALF) assay is one of several qualitative and quantitative marker loss assays that indirectly measure loss or conversion of genetic material using a counterselection step. The ALF assay relies on the ability to count spurious mating events that occur upon loss of the MATα locus of haploid Saccharomyces cerevisiae strains. Here, we describe the deployment of the ALF assay for both rapid and simple qualitative, and more in-depth quantitative analysis allowing determination of absolute ALF frequencies.

The ability to rapidly assess chromosome instability (CIN) has enabled profiling of most yeast genes for potential effects on genome stability. The A-like faker (ALF) assay is one of several qualitative and quantitative marker loss assays that indirectly measure loss or conversion of genetic material using a counterselection step. The ALF assay relies on the ability to count spurious mating events that occur upon loss of the MATα locus of haploid Saccharomyces cerevisiae strains. Here, we describe the deployment of the ALF assay for both rapid and simple qualitative, and more in-depth quantitative analysis allowing determination of absolute ALF frequencies.

Sgs1 is a yeast DNA helicase functioning in DNA replication and repair, and is the orthologue of the human Bloom’s syndrome helicase BLM. Here we analyze the mutation signature associated with SGS1 deletion in yeast, and find frequent copy number changes flanked by regions of repetitive sequence and high R-loop forming potential. We show that loss of SGS1 increases R-loop accumulation and sensitizes cells to replication-transcription collisions. Accordingly, in sgs1Δ cells the genome-wide distribution of R-loops shifts to known sites of Sgs1 action, replication pausing regions, and to long genes. Depletion of the orthologous BLM helicase from human cancer cells also increases R-loop levels, and R-loop-associated genome instability. In support of a direct effect, BLM is found physically proximal to DNA:RNA hybrids in human cells, and can efficiently unwind R-loops in vitro. Together our data describe a conserved role for Sgs1/BLM in R-loop suppression and support an increasingly broad view of DNA repair and replication fork stabilizing proteins as modulators of R-loop mediated genome instability.

Upon genotoxic stress, dynamic relocalization events control DNA repair, and alterations of the transcriptome and proteome enabling stress recovery. How these events may influence one another is only partly known. Beginning with a cytological screen for genome maintenance proteins that move under stress, we find that, upon alkylation stress, the splicing factor Hsh155 localizes to both intranuclear and cytoplasmic protein quality control aggregates. Under stress, an ordered sequestration of Hsh155 occurs at nuclear and then cytoplasmic aggregates in a manner that is regulated by molecular chaperones. This dynamic behavior is preceded by a decrease in splicing efficiency. While DNA replication stress signaling is not required for Hsh155 sequestration, Hsh155 aggregation is cell cycle and TOR pathway dependent. Indeed, loss of a TORC1 regulated ribosomal protein gene transcription factor Sfp1 allows general aggregate formation but prevents Hsh155 recruitment. Together, our analyses suggest a model in which some proteins evicted from chromatin undergoing transcriptional remodeling during stress are targeted to protein quality control sites.

Student creation of educational materials has the capacity both to enhance learning and to decrease costs. Three successive honors-style classes of undergraduate students in a cancer genetics class worked with a new software system, CuboCube, to create an e-textbook. CuboCube is an open-source learning materials creation system designed to facilitate e-textbook development, with an ultimate goal of improving the social learning experience for students. Equipped with crowdsourcing capabilities, CuboCube provides intuitive tools for nontechnical and technical authors alike to create content together in a structured manner. The process of e-textbook development revealed both strengths and challenges of the approach, which can inform future efforts. Both the CuboCube platform and the Cancer Genetics E-textbook are freely available to the community.

Gene-gene or gene-drug interactions are typically quantified using fitness as a readout because the data are continuous and easily measured in high throughput. However, to what extent fitness captures the range of other phenotypes that show synergistic effects is usually unknown. Using Saccharomyces cerevisiae and focusing on a matrix of DNA repair mutants and genotoxic drugs, we quantify 76 gene-drug interactions based on both mutation rate and fitness and find that these parameters are not connected. Independent of fitness defects, we identified six cases of synthetic hypermutation, where the combined effect of the drug and mutant on mutation rate was greater than predicted. One example occurred when yeast lacking RAD1 were exposed to cisplatin, and we characterized this interaction using whole-genome sequencing. Our sequencing results indicate mutagenesis by cisplatin in rad1Δ cells appeared to depend almost entirely on interstrand cross-links at GpCpN motifs. Interestingly, our data suggest that the following base on the template strand dictates the addition of the mutated base. This result differs from cisplatin mutation signatures in XPF-deficient Caenorhabditis elegans and supports a model in which translesion synthesis polymerases perform a slippage and realignment extension across from the damaged base. Accordingly, DNA polymerase ζ activity was essential for mutagenesis in cisplatin-treated rad1Δ cells. Together these data reveal the potential to gain new mechanistic insights from nonfitness measures of gene-drug interactions and extend the use of mutation accumulation and whole-genome sequencing analysis to define DNA repair mechanisms.

Bisulfite, in the form of sodium bisulfite or metabisulfite, is used commercially as a food preservative. Bisulfite is used in the laboratory as a single-stranded DNA mutagen in epigenomic analyses of DNA methylation. Recently it has also been used on whole yeast cells to induce mutations in exposed single-stranded regions in vivo. To understand the effects of bisulfite on live cells we conducted a genome-wide screen for bisulfite sensitive mutants in yeast. Screening the deletion mutant array, and collections of essential gene mutants we define a genetic network of bisulfite sensitive mutants. Validation of screen hits revealed hyper-sensitivity of transcription and RNA processing mutants, rather than DNA repair pathways and follow-up analyses support a role in perturbation of RNA transactions. We propose a model in which bisulfite-modified nucleotides may interfere with transcription or RNA metabolism when used in vivo.

In addition to environmental factors and intrinsic variations in base substitution rates, specific genome-destabilizing mutations can shape the mutational trajectory of genomes. How specific alleles influence the nature and position of accumulated mutations in a genomic context is largely unknown. Understanding the impact of genome-destabilizing alleles is particularly relevant to cancer genomes where biased mutational signatures are identifiable. We first created a more complete picture of cellular pathways that impact mutation rate using a primary screen to identify essential Saccharomyces cerevisiae gene mutations that cause mutator phenotypes. Drawing primarily on new alleles identified in this resource, we measure the impact of diverse mutator alleles on mutation patterns directly by whole-genome sequencing of 68 mutation-accumulation strains derived from wild-type and 11 parental mutator genotypes. The accumulated mutations differ across mutator strains, displaying base-substitution biases, allele-specific mutation hotspots, and break-associated mutation clustering. For example, in mutants of POLα and the Cdc13–Stn1–Ten1 complex, we find a distinct subtelomeric bias for mutations that we show is independent of the target sequence. Together our data suggest that specific genome-instability mutations are sufficient to drive discrete mutational signatures, some of which share properties with mutation patterns seen in tumors. Thus, in a population of cells, genome-instability mutations could influence clonal evolution by establishing discrete mutational trajectories for genomes.

The GPN proteins are a poorly characterized and deeply evolutionarily conserved family of three paralogous small GTPases, Gpn1, 2, and 3. The founding member, GPN1/NPA3/XAB1, is proposed to function in nuclear import of RNA polymerase II along with a recently described protein called Iwr1. Here we show that the previously uncharacterized protein Gpn2 binds both Gpn3 and Npa3/Gpn1 and that temperature-sensitive alleles of Saccharomyces cerevisiae GPN2 and GPN3 exhibit genetic interactions with RNA polymerase II mutants, hypersensitivity to transcription inhibition, and defects in RNA polymerase II nuclear localization. Importantly, we identify previously unrecognized RNA polymerase III localization defects in GPN2, GPN3, and IWR1 mutant backgrounds but find no localization defects of unrelated nuclear proteins or of RNA polymerase I. Previously, it was unclear whether the GPN proteins and Iwr1 had overlapping function in RNA polymerase II assembly or import. In this study, we show that the nuclear import defect of iwr1Δ, but not the GPN2 or GPN3 mutant defects, is partially suppressed by fusion of a nuclear localization signal to the RNA polymerase II subunit Rpb3. These data, combined with strong genetic interactions between GPN2 and IWR1, suggest that the GPN proteins function upstream of Iwr1 in RNA polymerase II and III biogenesis. We propose that the three GPN proteins execute a common, and likely essential, function in RNA polymerase assembly and transport.

The concept of synthetic lethality has gained popularity as a rational guide for predicting chemotherapeutic targets based on negative genetic interactions between tumor-specific somatic mutations and a second-site target gene. One hallmark of most cancers that can be exploited by chemotherapies is chromosome instability (CIN). Because chromosome replication, maintenance, and segregation represent conserved and cell-essential processes, they can be modeled effectively in simpler eukaryotes such as Saccharomyces cerevisiae. Here we analyze and extend genetic networks of CIN cancer gene orthologs in yeast, focusing on essential genes. This identifies hub genes and processes that are candidate targets for synthetic lethal killing of cancer cells with defined somatic mutations. One hub process in these networks is DNA replication. A nonessential, fork-associated scaffold, CTF4, is among the most highly connected genes. As Ctf4 lacks enzymatic activity, potentially limiting its development as a therapeutic target, we exploited its function as a physical interaction hub to rationally predict synthetic lethal interactions between essential Ctf4-binding proteins and CIN cancer gene orthologs. We then validated a subset of predicted genetic interactions in a human colorectal cancer cell line, showing that siRNA-mediated knockdown of MRE11A sensitizes cells to depletion of various replication fork-associated proteins. Overall, this work describes methods to identify, predict, and validate in cancer cells candidate therapeutic targets for tumors with known somatic mutations in CIN genes using data from yeast. We affirm not only replication stress but also the targeting of DNA replication fork proteins themselves as potential targets for anticancer therapeutic development.

G-quadruplex DNAs form four-stranded helical structures and are proposed to play key roles in different cellular processes. Targeting G-quadruplex DNAs for cancer treatment is a very promising prospect. Here, we show that CX-5461 is a G-quadruplex stabilizer, with specific toxicity against BRCA deficiencies in cancer cells and polyclonal patient-derived xenograft models, including tumours resistant to PARP inhibition. Exposure to CX-5461, and its related drug CX-3543, blocks replication forks and induces ssDNA gaps or breaks. The BRCA and NHEJ pathways are required for the repair of CX-5461 and CX-3543-induced DNA damage and failure to do so leads to lethality. These data strengthen the concept of G4 targeting as a therapeutic approach, specifically for targeting HR and NHEJ deficient cancers and other tumours deficient for DNA damage repair. CX-5461 is now in advanced phase I clinical trial for patients with BRCA1/2 deficient tumours (Canadian trial, NCT02719977, opened May 2016)., Stabilization of DNA quadruplex structures (G4) is lethal for cells with a compromised DNA repair pathway. Here, the authors show that CX-5461, a small molecule in clinical trials as RNA polymerase inhibitor, has G4-stablization properties and can be repurposed to target DNA repair-defective cancers cells.

Upon genotoxic stress, dynamic relocalization events control DNA repair as well as alterations of the transcriptome and proteome, enabling stress recovery. How these events may influence one another is only partly known. Beginning with a cytological screen of genome stability proteins, we find that the splicing factor Hsh155 disassembles from its partners and localizes to both intranuclear and cytoplasmic protein quality control (PQC) aggregates under alkylation stress. Aggregate sequestration of Hsh155 occurs at nuclear and then cytoplasmic sites in a manner that is regulated by molecular chaperones and requires TORC1 activity signaling through the Sfp1 transcription factor. This dynamic behavior is associated with intron retention in ribosomal protein gene transcripts, a decrease in splicing efficiency, and more rapid recovery from stress. Collectively, our analyses suggest a model in which some proteins evicted from chromatin and undergoing transcriptional remodeling during stress are targeted to PQC sites to influence gene expression changes and facilitate stress recovery.

Gene-gene or gene-drug interactions are typically quantified using fitness as readout because the data is continuous and easily measured in high-throughput. However, to what extent fitness captures the range of other phenotypes that show synergistic effects is usually unknown. Using Saccharomyces cerevisiae, and focusing on a matrix of DNA repair mutants and genotoxic drugs, we quantify 76 gene-drug interactions based on both mutation rate and fitness and find that these parameters are not necessarily overlapping. Independent of fitness defects we identified six cases of synthetic hypermutation, where the combined effect of the drug and mutant on mutation rate was greater than predicted. One example occurred when yeast lacking RAD1 were exposed to cisplatin and we characterized this interaction using whole-genome sequencing. Our sequencing results indicate mutagenesis by cisplatin in rad1Δ cells appeared to depend almost entirely on interstrand crosslinks at GpCpN motifs. Interestingly, our data suggests that the 3’ base in these motifs templates the addition of the mutated base. This result differs from cisplatin mutation signatures in XPF-deficient C. elegans and supports a model in which translesion synthesis polymerases perform a slippage and realignment extension across from the damaged base. Accordingly, DNA polymerase ζ activity was essential for mutagenesis in cisplatin treated rad1Δ cells. Together these data reveal the potential to gain new mechanistic insights from non-fitness measures of gene-drug interactions and extend the use of mutation accumulation and whole-genome sequencing analysis to define DNA repair mechanisms.

The eukaryotic cytosolic chaperonin containing TCP-1 (CCT) has an important function in maintaining cellular homoeostasis by assisting the folding of many proteins, including the cytoskeletal components actin and tubulin. Yet the nature of the proteins and cellular pathways dependent on CCT function has not been established globally. Here, we use proteomic and genomic approaches to define CCT interaction networks involving 136 proteins/genes that include links to the nuclear pore complex, chromatin remodelling, and protein degradation. Our study also identifies a third eukaryotic cytoskeletal system connected with CCT: the septin ring complex, which is essential for cytokinesis. CCT interactions with septins are ATP dependent, and disrupting the function of the chaperonin in yeast leads to loss of CCT–septin interaction and aberrant septin ring assembly. Our results therefore provide a rich framework for understanding the function of CCT in several essential cellular processes, including epigenetics and cell division.