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The genomes of DNA viruses are replicated by either host or virally encoded DNA polymerases. There is considerable diversity among DNA virus genomes and the relative stability of DNA allows for genomes much larger than possible for RNA viruses. Genomes of DNA viruses that infect animals range in size from less than 2 kb of single-stranded DNA to over 375 kb of double-stranded DNA. There are even larger DNA viruses that infect eukaryotic microorganisms. The genomes of DNA viruses vary in structure, size, and genome structure thus replication strategies are diverse. A feature common to many DNA viruses is their dependence on host DNA replication to provide enzymes (for example, DNA polymerases) and the deoxynucleotide triphosphate precursors required for DNA synthesis.

Replisome disassembly is the final step of eukaryotic DNA replication and is triggered by ubiquitylation of the CDC45–MCM–GINS (CMG) replicative helicase1–3. Despite being driven by evolutionarily diverse E3 ubiquitin ligases in different eukaryotes (SCFDia2 in budding yeast1, CUL2LRR1 in metazoa4–7), replisome disassembly is governed by a common regulatory principle, in which ubiquitylation of CMG is suppressed before replication termination, to prevent replication fork collapse. Recent evidence suggests that this suppression is mediated by replication fork DNA8–10. However, it is unknown how SCFDia2 and CUL2LRR1 discriminate terminated from elongating replisomes, to selectively ubiquitylate CMG only after termination. Here we used cryo-electron microscopy to solve high-resolution structures of budding yeast and human replisome–E3 ligase assemblies. Our structures show that the leucine-rich repeat domains of Dia2 and LRR1 are structurally distinct, but bind to a common site on CMG, including the MCM3 and MCM5 zinc-finger domains. The LRR–MCM interaction is essential for replisome disassembly and, crucially, is occluded by the excluded DNA strand at replication forks, establishing the structural basis for the suppression of CMG ubiquitylation before termination. Our results elucidate a conserved mechanism for the regulation of replisome disassembly in eukaryotes, and reveal a previously unanticipated role for DNA in preserving replisome integrity., A conserved mechanism for the regulation of replisome disassembly in eukaryotes is shown using cryo-electron microscopy, revealing a role for DNA in the preservation of replisome integrity.

Funder: Canadian Institutes of Health Research (Grant: FRN-142349) Natural Sciences and Engineering Research Council of Canada (Grant: RGPIN 05871-2014), Cells replicate and segregate their DNA with precision. Previous studies showed that these regulated cell-cycle processes were present in the last eukaryotic common ancestor and that their core molecular parts are conserved across eukaryotes. However, some metamonad parasites have secondarily lost components of the DNA processing and segregation apparatuses. To clarify the evolutionary history of these systems in these unusual eukaryotes, we generated a genome assembly for the free-living metamonad Carpediemonas membranifera and carried out a comparative genomics analysis. Here, we show that parasitic and free-living metamonads harbor an incomplete set of proteins for processing and segregating DNA. Unexpectedly, Carpediemonas species are further streamlined, lacking the origin recognition complex, Cdc6 and most structural kinetochore subunits. Carpediemonas species are thus the first known eukaryotes that appear to lack this suite of conserved complexes, suggesting that they likely rely on yet-to-be-discovered or alternative mechanisms to carry out these fundamental processes.

The minichromosome maintenance (MCM) protein family plays a key role in eukaryotic DNA replication and has been confirmed to be associated with the occurrence and progression of many tumors. However, the expression levels, functions, and prognostic values of MCMs in breast cancer (BC) have not been clearly and systematically explained. In this article, we studied the transcriptional levels of MCMs in BC based on the Oncomine database. Kaplan-Meier plotter was used to analyze prognostic value of MCMs in human BC patients. Furthermore, we constructed a MCM coexpression gene network and performed functional annotation analysis through DAVID to reveal the functions of MCMs and coexpressed genes. The data showed that the expression of MCM2–8 and MCM10 but not MCM1 and MCM9 was upregulated in BC. Kaplan-Meier plotter analysis revealed that high transcriptional levels of MCM2, MCM4–7, and MCM10 were significantly related to low relapse-free survival (RFS) in BC patients. In contrast, high levels of MCM1 and MCM9 predicted high RFS for BC patients. This study suggests that MCM2, MCM4–7, and MCM10 possess great potential to be valuable prognostic biomarkers for BC and that MCM1 and MCM9 may serve as potential treatment targets for BC patients.

DNA inversion is a type of site-specific recombination system that plays an important role in the generation of genetic diversity and phenotypic adaptation by programmed rearrangements in bacteria. However, no such inversion system exhibiting a strong directionality bias has been identified or developed in eukaryotes yet. Here, using directed evolution of Rci recombinase, a tyrosine recombinase from a bacterial DNA inversion system, we identified a mutant Rci8 with a ratio of inversion/deletion up to ∼4320 in yeast. Based on Rci8 recombinase and sfxa101 sites, we have established a DNA inversion system in yeast and mammalian cells, enabling specificity for DNA inversions between inverted sites over deletions between directly repeated sites. Our results validated that the reversible DNA inversion system can act as an on/off transcriptional switch. Moreover, we demonstrate that the inversion system can also work on linear chromosomes. The eukaryotic DNA inversion system would provide a new tool for fields of genetic circuits, cellular barcoding, and synthetic genomes.

Translocases such as DNA/RNA polymerases, replicative helicases, and exonucleases are involved in eukaryotic DNA transcription, replication, and repair. Since eukaryotic genomic DNA wraps around histone octamers and forms nucleosomes, translocases inevitably encounter nucleosomes. A previous study has shown that a nucleosome repositions downstream when a translocase collides with the nucleosome. However, the molecular mechanism of the downstream repositioning remains unclear. In this study, we identified the lane-switch mechanism for downstream repositioning with molecular dynamics simulations and validated it with restriction enzyme digestion assays and deep sequencing assays. In this mechanism, after a translocase unwraps nucleosomal DNA up to the site proximal to the dyad, the remaining wrapped DNA switches its binding lane to that vacated by the unwrapping, and the downstream DNA rewraps, completing downstream repositioning. This mechanism may have broad implications for transcription through nucleosomes, histone recycling, and nucleosome remodeling.

The numerous enzymes and cofactors involved in eukaryotic DNA replication are conserved from yeast to human, and the budding yeast Saccharomyces cerevisiae (S.c.) has been a useful model organism for these studies. However, there is a gap in our knowledge of why replication origins in higher eukaryotes do not use a consensus DNA sequence as found in S.c. Using in vitro reconstitution and single-molecule visualization, we show here that S.c. origin recognition complex (ORC) stably binds nucleosomes and that ORC-nucleosome complexes have the intrinsic ability to load the replicative helicase MCM double hexamers onto adjacent nucleosome-free DNA regardless of sequence. Furthermore, we find that Xenopus laevis nucleosomes can substitute for yeast ones in engaging with ORC. Combined with re-analyses of genome-wide ORC binding data, our results lead us to propose that the yeast origin recognition machinery contains the cryptic capacity to bind nucleosomes near a nucleosome-free region and license origins, and that this nucleosome-directed origin licensing paradigm generalizes to all eukaryotes.

The pioneer event in eukaryotic DNA replication is binding of chromosomal DNA by the origin recognitioncomplex (ORC). The ORC-DNA complex directs the formation of origins, the specific chromosomal regions where DNA synthesis initiates. In all eukaryotes, incompletely understood features of chromatin promote ORC-DNA binding. Here, we uncover a role for the Fkh1 (Forkhead homolog) protein and its forkhead associated (FHA) domain in promoting ORC-origin binding and origin activity at a subset of origins in Saccharomyces cerevisiae. Several of the FHA-dependent origins examined required a distinct Fkh1 binding site located 5′ of and proximal to their ORC sites (5′-FKH-T site). Genetic and molecular experiments provided evidence that the Fkh1-FHA domain promoted origin activity directly through Fkh1 binding to this 5′ FKH-T site. Nucleotide substitutions within two relevant origins that enhanced their ORC-DNA affinity bypassed the requirement for their 5′ FKH-T sites and for the Fkh1-FHA domain. Significantly, assessment of ORC-origin binding by ChIPSeq provided evidence that this mechanism was relevant at ∼25% of yeast origins. Thus, the FHA domain of the conserved cell-cycle transcription factor Fkh1 enhanced origin selection in yeast at the level of ORC-origin binding.

Abstract The highly complex raw milk matrix challenges the sample preparation for amplicon-sequencing due to low bacterial counts and high amounts of eukaryotic DNA originating from the cow. In this study, we optimized the extraction of bacterial DNA from raw milk for microbiome analysis and evaluated the impact of cycle numbers in the library-PCR. The selective lysis of eukaryotic cells by proteinase K and digestion of released DNA before bacterial lysis resulted in a high reduction of mostly eukaryotic DNA and increased the proportion of bacterial DNA. Comparative microbiome analysis showed that a combined enzymatic and mechanical lysis procedure using the DNeasy® PowerFood® Microbial Kit with a modified protocol was best suitable to achieve high DNA quantities after library-PCR and broad coverage of detected bacterial biodiversity. Increasing cycle numbers during library-PCR systematically altered results for species and beta-diversity with a tendency to overrepresentation or underrepresentation of particular taxa. To limit PCR bias, high cycle numbers should thus be avoided. An optimized DNA extraction yielding sufficient bacterial DNA and enabling higher PCR efficiency is fundamental for successful library preparation. We suggest that a protocol using ethylenediaminetetraacetic acid (EDTA) to resolve casein micelles, selective lysis of somatic cells, extraction of bacterial DNA with a combination of mechanical and enzymatic lysis, and restriction of PCR cycles for analysis of raw milk microbiomes is optimal even for samples with low bacterial numbers. Key points • Sample preparation for high-throughput 16S rRNA gene sequencing of raw milk microbiota. • Reduction of eukaryotic DNA by enzymatic digestion. • Shift of detected microbiome caused by high cycle numbers in library-PCR.

The Origin Recognition Complex (ORC) binds to sites in chromosomes to specify the location of origins of DNA replication. The S. cerevisiae ORC binds to specific DNA sequences throughout the cell cycle but becomes active only when it binds to the replication initiator Cdc6. It has been unclear at the molecular level how Cdc6 activates ORC, converting it to an active recruiter of the Mcm2-7 hexamer, the core of the replicative helicase. Here we report the cryo-EM structure at 3.3 Å resolution of the yeast ORC–Cdc6 bound to an 85-bp ARS1 origin DNA. The structure reveals that Cdc6 contributes to origin DNA recognition via its winged helix domain (WHD) and its initiator-specific motif. Cdc6 binding rearranges a short α-helix in the Orc1 AAA+ domain and the Orc2 WHD, leading to the activation of the Cdc6 ATPase and the formation of the three sites for the recruitment of Mcm2-7, none of which are present in ORC alone. The results illuminate the molecular mechanism of a critical biochemical step in the licensing of eukaryotic replication origins., Eukaryotic DNA replication is mediated by many proteins which are tightly regulated for an efficient firing of replication at each cell cycle. Here the authors report a cryo-EM structure of the yeast ORC–Cdc6 bound to an 85-bp ARS1 origin DNA revealing additional insights into how Cdc6 contributes to origin DNA recognition.

Telomeres are copied and reassembled each cell division cycle through a multistep process called telomere replication. Most telomeric DNA is duplicated semiconservatively during this process, but replication forks frequently pause or stall at telomeres in yeast, mouse and human cells, potentially causing chronic telomere shortening or loss in a single cell cycle. We have investigated the cause of this effect by examining the replication of telomeric templates in vitro. Using a reconstituted assay for eukaryotic DNA replication in which a complete eukaryotic replisome is assembled and activated with purified proteins, we show that budding yeast telomeric DNA is efficiently duplicated in vitro unless the telomere binding protein Rap1 is present. Rap1 acts as a roadblock that prevents replisome progression and leading strand synthesis, but also potently inhibits lagging strand telomere replication behind the fork. Both defects can be mitigated by the Pif1 helicase. Our results suggest that GC-rich sequences do not inhibit DNA replication per se, and that in the absence of accessory factors, telomere binding proteins can inhibit multiple, distinct steps in the replication process.

Summary Even though the interest in metabarcoding in environmental studies is growing, euglenids are still underrepresented in both sea and freshwater bodies researches. The reason for this situation could be the unsuitability of universal eukaryotic DNA barcodes and primers as well as the lack of a verified protocol, suitable to assess euglenid diversity. In this study, using specific primers for the V2 hypervariable region of 18S rDNA for metabarcoding resulted in obtaining a high fraction (85%) of euglenid reads and species‐level identification of almost 90% of them. Fifty species were detected by the metabarcoding method, including almost all species observed using a light microscope. We investigated three biomass harvesting methods (filtering, centrifugation and scraping the side of a collection vessel) and determined that centrifugation and filtration outperformed scrapes, but the choice between them is not crucial for the reliability of the analysis. In addition, eight DNA extraction methods were evaluated. We compared five commercially available DNA isolation kits, two CTAB‐based protocols and a chelating resin. For this purpose, the efficiency of extraction, quality of obtained DNA, preparation time and generated costs were taken into consideration. After examination of the aforementioned criteria, we chose the GeneMATRIX Soil DNA Purification Kit as the most suitable for DNA isolation.

As nucleotidyl transferases, formation of a covalent enzyme-adenylate intermediate is a common first step of all DNA ligases. While it has been shown that eukaryotic DNA ligases utilize ATP as the adenylation donor, it was recently reported that human DNA ligase IV can also utilize NAD+ and, to a lesser extent ADP-ribose, as the source of the adenylate group and that NAD+, unlike ATP, enhances ligation by supporting multiple catalytic cycles. Since this unexpected finding has significant implications for our understanding of the mechanisms and regulation of DNA double strand break repair, we attempted to confirm that NAD+ and ADP-ribose can be used as co-factors by human DNA ligase IV. Here, we provide evidence that NAD+ does not enhance ligation by pre-adenylated DNA ligase IV, indicating that this co-factor is not utilized for re-adenylation and subsequent cycles of ligation. Moreover, we find that ligation by de-adenylated DNA ligase IV is dependent upon ATP not NAD+ or ADP-ribose. Thus, we conclude that human DNA ligase IV cannot use either NAD+ or ADP-ribose as adenylation donor for ligation.

Genomic DNA is chemically reactive and therefore susceptible to damage by many exogenous and endogenous sources. Lesions produced from these damaging events can have various mutagenic and genotoxic consequences. This Perspective follows the journey of one particular lesion, 1,N6-ethenoadenine (eA), from its formation to replication and repair, and its role in cancerous tissues and inflammatory diseases. eA is generated by the reaction of adenine (A) with vinyl chloride or lipid peroxidation products. We present the miscoding properties of eA with an emphasis on how bacterial and mammalian cells can process lesions differently, leading to varied mutational spectra. But with information from these assays, we can better understand how the miscoding properties of eA lead to biological consequences and how genomic stability can be maintained via DNA repair mechanisms. We discuss how base excision repair (BER) and direct reversal repair (DRR) can minimize the biological consequences of eA lesions. Kinetic parameters of glycosylases and AlkB family enzymes are described, along with a discussion of the relative contributions of the BER and DRR pathways in the repair of eA. Because eukaryotic DNA is packaged in chromatin, we also discuss the impact of this packaging on BER and DRR, specifically in regards to repair of eA. Studying DNA lesions like eA in this context, from origin to biological implications, can provide crucial information to better understand prevention of mutagenesis and cancer.

In eukaryotes three DNA polymerases (Pols), α, δ, and ε, are tasked with bulk DNA synthesis of nascent strands during genome duplication. Most evidence supports a model where Pol α initiates DNA synthesis before Pol ε and Pol δ replicate the leading and lagging strands, respectively. However, a number of recent reports, enabled by advances in biochemical and genetic techniques, have highlighted emerging roles for Pol δ in all stages of leading-strand synthesis; initiation, elongation, and termination, as well as fork restart. By focusing on these studies, this review provides an updated perspective on the division of labor between the replicative polymerases during DNA replication.

Eukaryotic DNA polymerase β (Pol β) plays an important role in cellular DNA repair, as it fills short gaps in dsDNA that result from removal of damaged bases. Since defects in DNA repair may lead to cancer and genetic instabilities, Pol β has been extensively studied, especially its mechanisms for substrate binding and a fidelity-related conformational change referred to as "fingers closing." Here, we applied single-molecule FRET to measure distance changes associated with DNA binding and prechemistry fingers movement of human Pol β. First, using a doubly labeled DNA construct, we show that Pol β bends the gapped DNA substrate less than indicated by previously reported crystal structures. Second, using acceptor-labeled Pol β and donor-labeled DNA, we visualized dynamic fingers closing in single Pol β-DNA complexes upon addition of complementary nucleotides and derived rates of conformational changes. We further found that, while incorrect nucleotides are quickly rejected, they nonetheless stabilize the polymerase-DNA complex, suggesting that Pol β, when bound to a lesion, has a strong commitment to nucleotide incorporation and thus repair. In summary, the observation and quantification of fingers movement in human Pol β reported here provide new insights into the delicate mechanisms of prechemistry nucleotide selection.

Transcriptional control is exerted primarily through the binding of transcription factor proteins to regulatory elements in DNA. By virtue of eukaryotic DNA being complexed with histones, transcription factor binding to DNA alters or eliminates histone-DNA contacts, leading to increased accessibility of the DNA region to nuclease enzymes. This hypersensitivity to nuclease digestion has been used to define DNA binding events and regulatory elements across genomes, and to compare these attributes between cell types or conditions. These approaches make it possible to define the regulatory elements in a genome as well as to predict the regulatory networks of transcription factors and their target genes in a given cell state. As these chromatin accessibility assays are increasingly used, it is important to consider how to analyze the resulting data to avoid artifactual results or misinterpretation. In this review, we focus on some of the key technical and computational caveats associated with plant chromatin accessibility data, including strategies for sample preparation, sequencing, read mapping, and downstream analyses.

DNA replication is a central process in all living organisms. Polyomavirus DNA replication serves as a model system for eukaryotic DNA replication and has considerably contributed to our understanding of basic replication mechanisms. However, the details of the involved processes are still unclear, in particular regarding lagging strand synthesis. To delineate the complex mechanism of coordination of various cellular proteins binding simultaneously or consecutively to DNA to initiate replication, we investigated single-stranded DNA (ssDNA) interactions by the SV40 large T antigen (Tag). Using single molecule imaging by atomic force microscopy (AFM) combined with biochemical and spectroscopic analyses we reveal independent activity of monomeric and oligomeric Tag in high affinity binding to ssDNA. Depending on ssDNA length, we obtain dissociation constants for Tag-ssDNA interactions (K-D values of 10-30 nM) that are in the same order of magnitude as ssDNA binding by human replication protein A (RPA). Furthermore, we observe the formation of RPA-Tag-ssDNA complexes containing hexameric as well as monomeric Tag forms. Importantly, our data clearly show stimulation of primase function in lagging strand Okazaki fragment synthesis by monomeric Tag whereas hexameric Tag inhibits the reaction, redefining DNA replication initiation on the lagging strand. Else Kröner-Fresenius-Stiftung (EKFS) [2013_A215]; PML Consortium (Washington, USA) [RIB1099] to H.P.N. Funding for open access charge: Funding by NUI Galway and University of Würzburg. peer-reviewed

Replication protein A (RPA) plays a critical role in all eukaryotic DNA processing involving single-stranded DNA (ssDNA). Contrary to the notion that RPA provides solely inert protection to transiently formed ssDNA, the RPA–ssDNA complex acts as a dynamic DNA processing unit. Here, we studied the diffusion of RPA along 60 nt ssDNA using a coarse-grained model in which the ssDNA–RPA interface was modeled by both aromatic and electrostatic interactions. Our study provides direct evidence of bulge formation during the diffusion of ssDNA along RPA. Bulges can form at a few sites along the interface and store 1–7 nt of ssDNA whose release, upon bulge dissolution, leads to propagation of ssDNA diffusion. These findings thus support the reptation mechanism, which involves bulge formation linked to the aromatic interactions, whose short range nature reduces cooperativity in ssDNA diffusion. Greater cooperativity and a larger diffusion coefficient for ssDNA diffusion along RPA are observed for RPA variants with weaker aromatic interactions and for interfaces homogenously stabilized by electrostatic interactions. ssDNA propagation in the latter instance is characterized by lower probabilities of bulge formation; thus, it may fit the sliding-without-bulge model better than the reptation model. Thus, the reptation mechanism allows ssDNA mobility despite the extensive and high affinity interface of RPA with ssDNA. The short-range aromatic interactions support bulge formation while the long-range electrostatic interactions support the release of the stored excess ssDNA in the bulge and thus the overall diffusion.

In this study, novel p-benzimidazole-derived calix[4]arene compounds with different structures, and a benzothiazole-derived calix[4]arene compound, were synthesized by a microwave-assisted method and their structures were determined by FTIR, 1H NMR, 13C NMR, MALDI-TOF mass spectroscopy, and elemental analysis. The effects of functional calixarenes against bacterial (pBR322 plasmid DNA) and eukaryotic DNA (calf thymus DNA = CT-DNA) were investigated. The studies with plasmid DNA have shown that compounds 6 and 10 containing methyl and benzyl groups, respectively, have DNA cleavage activity at the highest concentrations (10 000 μM). Interactions with plasmid DNA using some restriction enzymes (BamHI and HindIII) were also investigated. The binding ability of p-substituted calix[4]arene compounds towards CT-DNA was examined using UV-vis and fluorescence spectroscopy and it was determined that some compounds showed efficiency. In particular, it was observed that the functional compounds (10 and 5) containing benzyl and chloro-groups had higher activity (Kb binding constants were found to be 7.1 × 103 M−1 and 9.3 × 102 M−1 respectively) on DNA than other compounds. Competitive binding experiments using ethidium bromide also gave an idea about the binding properties. Docking studies of the synthesized compounds with DNA were performed to predict the binding modes, affinities and noncovalent interactions stabilizing the DNA–compound complexes at the molecular level. Docking results were in good agreement with the experimental findings on the DNA binding activities of compounds. Based on these results, this preliminary study could shed light on future experimental antibacterial and/or anticancer research.

Fungi are found in diverse ecological niches as primary decomposers, mutualists, or parasites of plants and animals. Although animals and fungi share a common ancestor, fungi dramatically diversified their life cycle, cell biology, and metabolism as they evolved and colonized new niches. This review focuses on a family of fungal transcription factors (Swi4/Mbp1, APSES, Xbp1, Bqt4) derived from the lateral gene transfer of a KilA-N domain commonly found in prokaryotic and eukaryotic DNA viruses. These virus-derived fungal regulators play central roles in cell cycle, morphogenesis, sexual differentiation, and quiescence. We consider the possible origins of KilA-N and how this viral DNA binding domain came to be intimately associated with fungal processes.

The initiation of Okazaki fragment synthesis during cellular DNA replication is a crucial step for lagging strand synthesis, which is carried out by the primase function of DNA polymerase ¿-primase (Pol-prim). Since cellular replication protein A (RPA) prevents primase from starting RNA synthesis on single-stranded DNA (ssDNA), primase requires auxiliary factors, such as the simian virus 40 (SV40) T antigen (Tag), for the initiation reaction on RPA-bound ssDNA. Here, we investigated the ability of Tag variants and Tag protein complexes to bind to ssDNA and their resulting effects on the stimulation of Pol-prim on free and RPA-bound ssDNA. Atomic force microscopy imaging showed that while Tag(131-627) (V350E/P417D) and Tag(131-627) (L286D/R567E) (abbreviated as M1 and M2, respectively) could bind to ssDNA as monomers, these monomeric Tags could come together and bind to ssDNA as dimers as well. In a model assay for the initiation of Okazaki fragment synthesis, full-length Tag SV40 Tag(1-708) and monomeric M2 stimulated DNA synthesis of Pol-prim on ssDNA and on RPA-bound ssDNA. In contrast, neither monomeric M1 nor M1-M2 dimers could stimulate Pol-prim, on ssDNA or on RPA-bound ssDNA. Overall, we show that a lack of stimulatory activity of monomeric M1 and M1-M2 dimers suggests that residues V350 and P417 are not only important for interactions between Tag molecules but also for protein-protein interactions within Okazaki fragment initiation complexes. Thus, we highlight that mutations in M1 are dominant negative with regard to Okazaki fragment initiation. This work was supported by funding from the Deutsche Forschungsgemeinschaft (DFG, grant TE-671/4-2 to IT) and from the Else Kröner-Fresenius-Stiftung (EKFS 2013_A215) and the PML Consortium (Washington, USA; RIB1099) to HPN. The authors thank Patricia Nyland for expert technical assistance and Dr Lars Schönemann (Recombinant Protein Expression Facility, University of Würzburg) for his expert purification of wild type and mutant Tag131-627. We acknowledge Angela Borst for her contributions to AFM data collection and analyses, and Maciej Doczyk (NUI Galway) for his graphical work. We would also like to acknowledge the FP7 WeNMR (project# 261572), H2020 West-Life (project# 675858), the EOSC-hub (project #777536) and the EGI-ACE (project# 101017567) European e-Infrastructure projects for the use of their web portals, which make use of the EGI infrastructure with the dedicated support of CESNET-MCC, INFN-PADOVA-STACK, INFN-LNL-2, NCG-INGRID-PT, TW-NCHC, CESGA, IFCA-LCG2, UA-BITP, SURFsara and NIKHEF, and the additional support of the national GRID Initiatives of Belgium, France, Italy, Germany, the Netherlands, Poland, Portugal, Spain, UK, Taiwan and the US Open Science Grid. peer-reviewed

R-loops are a major source of genome instability associated with transcription-induced replication stress. However, how R-loops inherently impact replication fork progression is not understood. Here, we characterize R-loop-replisome collisions using a fully reconstituted eukaryotic DNA replication system. We find that RNA:DNA hybrids and G-quadruplexes at both co-directional and head-on R-loops can impact fork progression by inducing fork stalling, uncoupling of leading strand synthesis from replisome progression, and nascent strand gaps. RNase H1 and Pif1 suppress replication defects by resolving RNA:DNA hybrids and G-quadruplexes, respectively. We also identify an intrinsic capacity of replisomes to maintain fork progression at certain R-loops by unwinding RNA:DNA hybrids, repriming leading strand synthesis downstream of G-quadruplexes, or utilizing R-loop transcripts to prime leading strand restart during co-directional R-loop-replisome collisions. Collectively, the data demonstrates that the outcome of R-loop-replisome collisions is modulated by R-loop structure, providing a mechanistic basis for the distinction of deleterious from non-deleterious R-loops.

Eukaryotic DNA Mismatch Repair (MMR) involves redundant exonuclease 1 (Exo1)-dependent and Exo1-independent pathways, of which the Exo1-independent pathway(s) is not well understood. The exo1Δ440-702 mutation, which deletes the MutS Homolog 2 (Msh2) and MutL Homolog 1 (Mlh1) interacting peptides (SHIP and MIP boxes, respectively), eliminates the Exo1 MMR functions but is not lethal in combination with rad27Δ mutations. Analyzing the effect of different combinations of the exo1Δ440-702 mutation, a rad27Δ mutation and the pms1-A99V mutation, which inactivates an Exo1-independent MMR pathway, demonstrated that each of these mutations inactivates a different MMR pathway. Furthermore, it was possible to reconstitute a Rad27- and Msh2-Msh6-dependent MMR reaction in vitro using a mispaired DNA substrate and other MMR proteins. Our results demonstrate Rad27 defines an Exo1-independent eukaryotic MMR pathway that is redundant with at least two other MMR pathways., Defects in DNA mismatch repair (MMR) have been linked to inherited and sporadic cancers. Here the authors demonstrate that the DNA repair protein Rad27 (human FEN1) functions in one of three redundant mispair excision pathways, where its flap endonuclease activity catalyzes mispair excision.

In eukaryotes, 5-methylcytosine is the predominant DNA base modification, followed by N6-methyladenine. However, N4-methylcytosine (4mC) is confined to bacteria. Here we report that 4mC can serve as an epigenetic mark in eukaryotes. Bdelloid rotifers, freshwater invertebrates with transposon-poor genomes that are rich in foreign genes, lack C5-methyltransferases but encode an amino-methyltransferase, N4CMT, captured from bacteria >60 Mya. N4CMT introduces 4mC into DNA, and its chromodomain shapes the "histone-read-DNA-write" architecture together with a "DNA-read-histone-write" SETDB1/eggless H3K9me3 histone methyltransferase variant preferentially binding 4mC-DNA, to maintain 4mC and silent chromatin at transposons and tandem repeats. Our results bring the third base modification into the eukaryotic repertoire, demonstrate how non-native DNA methyl groups can reshape complex epigenetic systems to suppress transposon proliferation, and establish horizontal gene transfer as the source of regulatory innovation in eukaryotes.

Formation of gametes in the malaria parasite occurs in the midgut of the mosquito and is critical to onward parasite transmission. Transformation of the male gametocyte into microgametes, called microgametogenesis, is an explosive cellular event and one of the fastest eukaryotic DNA replication events known. The transformation of one microgametocyte into eight flagellated microgametes requires reorganisation of the parasite cytoskeleton, replication of the 22.9 Mb genome, axoneme formation and host erythrocyte egress, all of which occur simultaneously in Plasmodium falciparum, our live-cell approach combines three-dimensional imaging through time (4D imaging) and covers early microgametocyte development through to microgamete release. Combining live-cell stains for DNA, tubulin and the host erythrocyte membrane, 4D imaging enables definition of the positioning of newly replicated and segregated DNA. It also shows the microtubular cytoskeleton, location of newly formed basal bodies and elongation of axonemes, as well as behaviour of the erythrocyte membrane, including its specific perforation prior to microgamete egress. 4D imaging was additionally undertaken in the presence of known transmission-blocking inhibitors and the untested proteasomal inhibitor bortezomib. Here, for the first time we find that bortezomib inhibition results in a clear block of DNA replication, full axoneme nucleation and elongation. These data not only define a framework for understanding microgametogenesis in general but also suggest that the process is critically dependent on proteasomal activity, helping to identify potentially novel targets for transmission-blocking antimalarial drug development.

A fundamental concept in eukaryotic DNA replication is the temporal separation of G1 origin licensing from S phase origin firing. Re-replication and genome instability ensue if licensing occurs after DNA synthesis has started. In humans and other vertebrates, the E3 ubiquitin ligase CRL4Cdt2 starts to degrade the licensing factor Cdt1 after origins fire, raising the question of how cells prevent re-replication in early S phase. Here, using quantitative microscopy, we show that Cdt1 inhibits DNA synthesis during an overlap period when cells fire origins while Cdt1 is still present. Cdt1 inhibits DNA synthesis by suppressing CMG helicase progression at replication forks through the MCM-binding domain of Cdt1, and DNA synthesis commences once Cdt1 is degraded. Thus, instead of separating licensing from firing to prevent re-replication in early S phase, cells separate licensing from DNA synthesis through Cdt1-mediated inhibition of CMG helicase after firing.Highlights– Cdt1 is present together with fired origins of replication at the start of S phase– Cdt1 delays DNA synthesis by inhibiting CMG helicase progression after origins fire– Cdt1 inhibits CMG helicase progression through the MCM-binding domain of Cdt1

Eukaryotic DNA replication begins at genomic loci termed origins, which are bound by the origin recognition complex (ORC). Although ORC is conserved across species, the sequence composition of origins is more varied. In the budding yeast Saccharomyces cerevisiae, the ORC-binding motif consists of an A/T-rich 17 bp “extended ACS” sequence adjacent to a B1 element composed of two 3-bp motifs. Similar sequences occur at origins in closely related species, but it is not clear when this type of replication origin arose and whether it predated a whole-genome duplication that occurred around 100 Ma in the budding yeast lineage. To address these questions, we identified the ORC-binding sequences in the nonduplicated species Torulaspora delbrueckii. We used chromatin immunoprecipitation followed by sequencing and identified 190 ORC-binding sites distributed across the eight T. delbrueckii chromosomes. Using these sites, we identified an ORC-binding motif that is nearly identical to the known motif in S. cerevisiae. We also found that the T. delbrueckii ORC-binding sites function as origins in T. delbrueckii when cloned onto a plasmid and that the motif is required for plasmid replication. Finally, we compared an S. cerevisiae origin with two T. delbrueckii ORC-binding sites and found that they conferred similar stabilities to a plasmid. These results reveal that the ORC-binding motif arose prior to the whole-genome duplication and has been maintained for over 100 Myr.

Summary MNase-seq (micrococcal nuclease sequencing) is used to map nucleosome positions in eukaryotic genomes to study the relationship between chromatin structure and DNA-dependent processes. Current protocols require at least two days to isolate nucleosome-protected DNA fragments. We have developed a streamlined protocol for S. cerevisiae and other fungi which takes only three hours. Modified protocols were developed for wild fungi and mammalian cells. This method for rapidly producing sequencing-ready nucleosome footprints from several organisms makes MNase-seq faster and easier, with less chemical waste., Graphical abstract, Highlights • A fast way to prepare micrococcal nuclease nucleosome footprints for MNase-seq • Eliminates use of phenol and chloroform and reduces the amount of cells required • Adaptable for a variety of organisms, MNase-seq is used to map nucleosome positions in eukaryotic genomes to study the relationship between chromatin structure and DNA-dependent processes. Current protocols require at least two days to isolate nucleosome-protected DNA fragments. We have developed a streamlined protocol for S. cerevisiae and other fungi which takes only three hours. Modified protocols were developed for wild fungi and mammalian cells. This method for rapidly producing sequencing-ready nucleosome footprints from several organisms makes MNase-seq faster and easier, with less chemical waste.

As we explore beyond Earth, astronauts may be at risk for harmful DNA damage caused by ionizing radiation. Double-strand breaks are a type of DNA damage that can be repaired by two major cellular pathways: non-homologous end joining, during which insertions or deletions may be added at the break site, and homologous recombination, in which the DNA sequence often remains unchanged. Previous work suggests that space conditions may impact the choice of DNA repair pathway, potentially compounding the risks of increased radiation exposure during space travel. However, our understanding of this problem has been limited by technical and safety concerns, which have prevented integral study of the DNA repair process in space. The CRISPR/Cas9 gene editing system offers a model for the safe and targeted generation of double-strand breaks in eukaryotes. Here we describe a CRISPR-based assay for DNA break induction and assessment of double-strand break repair pathway choice entirely in space. As necessary steps in this process, we describe the first successful genetic transformation and CRISPR/Cas9 genome editing in space. These milestones represent a significant expansion of the molecular biology toolkit onboard the International Space Station.

DNA methylation is one of the most important epigenetic modifications and is closely related with several biological processes such as regulation of gene transcription and the development of non-malignant diseases. The prevailing dogma states that DNA methylation in eukaryotes occurs essentially through 5-methylcytosine (5mC) but recently adenine methylation was also found to be present in eukaryotes. In mouse embryonic stem cells, 6-methyladenine (6mA) was associated with the repression and silencing of genes, particularly in the X-chromosome, known to play an important role in cell fate determination. Here, we have demonstrated that 6mA is a ubiquitous eukaryotic epigenetic modification that is put in place during epigenetically sensitive periods such as embryogenesis and fetal development. In somatic cells there are clear tissue specificity in 6mA levels, with the highest 6mA levels being observed in the brain. In zebrafish, during the first 120 h of embryo development, from a single pluripotent cell to an almost fully formed individual, 6mA levels steadily increase. An identical pattern was observed over embryonic days 7-21 in the mouse. Furthermore, exposure to a neurotoxic environmental pollutant during the same early life period may led to a decrease in the levels of this modification in female rats. The identification of the periods during which 6mA epigenetic marks are put in place increases our understanding of this mammalian epigenetic modification, and raises the possibility that it may be associated with developmental processes. ispartof: FRONTIERS IN GENETICS vol:12 ispartof: location:Switzerland status: published

Proliferating cell nuclear antigen (PCNA) plays a critical role as a processivity clamp for eukaryotic DNA polymerases and a binding platform for many DNA replication and repair proteins. The enzymatic activities of PCNA loading and unloading have been studied extensively in vitro. However, the subcellular locations of PCNA loaders, replication complex C (RFC) and CTF18-RFC-like-complex (RLC), and PCNA unloader ATAD5-RLC remain elusive, and the role of their subunits RFC2-5 is unknown. Here we used protein fractionation to determine the subcellular localization of RFC and RLCs and affinity purification to find molecular requirements for the newly defined location. All RFC/RLC proteins were detected in the nuclease-resistant pellet fraction. RFC1 and ATAD5 were not detected in the non-ionic detergent-soluble and nuclease-susceptible chromatin fractions, independent of cell cycle or exogenous DNA damage. We found that small RFC proteins contribute to maintaining protein levels of the RFC/RLCs. RFC1, ATAD5, and RFC4 co-immunoprecipitated with lamina-associated polypeptide 2 (LAP2) α which regulates intranuclear lamin A/C. LAP2α knockout consistently reduced detection of RFC/RLCs in the pellet fraction, while marginally affecting total protein levels. Our findings strongly suggest that PCNA-mediated DNA transaction occurs through regulatory machinery associated with nuclear structures, such as the nuclear matrix.

Histones are classically known to organize the eukaryotic DNA into chromatin. They are one of the key players in regulating transcriptionally permissive and non-permissive states of the chromatin. Nevertheless, their context-dependent appearance within the cytoplasm and systemic circulation has also been observed. The past decade has also witnessed few scientific communications on the existence of vesicle-associated histones. Diverse groups have attempted to determine the significance of these extra-nuclear histones so far, with many of those studies still underway. Of note amongst these are interactions of extra-nuclear or free histones with cellular membranes, mediated by mutual cationic and anionic natures, respectively. It is here aimed to consolidate the mechanism of formation of extra-nuclear histones; implications of histone-induced membrane destabilization and explore the mechanisms of their association/release with extracellular vesicles, along with the functional aspects of these extra-nuclear histones in cell and systemic physiology.

G-quadruplexes (G4s) are tetrahelical DNA structures stabilized by four guanines paired via Hoogsteen hydrogen bonds into quartets. While their presence within eukaryotic DNA is known to play a key role in regulatory processes, their functional mechanisms are still under investigation. In the present work, we analysed the nanomechanical properties of three G4s present within the promoter of the KIT proto-oncogene from a single-molecule point of view through the use of magnetic tweezers (MTs). The study of DNA extension fluctuations under negative supercoiling allowed us to identify a characteristic fingerprint of G4 folding. We further analysed the energetic contribution of G4 to the double-strand denaturation process in the presence of negative supercoiling, and we observed a reduction in the energy required for strands separation.

Priming of single stranded templates is essential for DNA replication. In recent years, significant progress was made in understanding how DNA primase fulfils this fundamental function, particularly with regard to the initiation. Equally intriguing is the unique property of archeao-eukaryotic primases to terminate primer formation at a well-defined unit length. The apparent ability to “count” the number of bases incorporated prior to primer release is not well understood, different mechanisms having been proposed for different species. We report a mechanistic investigation of primer termination by the pRN1 primase from Sulfolobus islandicus. Using an HPLC-based assay we determined structural features of the primer 5′-end that are required for consistent termination. Mutations within the unstructured linker connecting the catalytic domain to the template binding domain allowed us to assess the effect of altered linker length and flexibility on primer termination., Frontiers in Microbiology, 12, ISSN:1664-302X

Cell cycle checkpoints and DNA repair pathways contribute to maintaining genome integrity and are thought to be evolutionarily ancient and broadly conserved. For example, in the yeast Saccharomyces cerevisiae and humans, DNA damage induces activation of a checkpoint effector kinase, Rad53p (human homolog Chk2), to promote cell cycle arrest and transcription of DNA repair genes., Cell cycle checkpoints and DNA repair pathways contribute to maintaining genome integrity and are thought to be evolutionarily ancient and broadly conserved. For example, in the yeast Saccharomyces cerevisiae and humans, DNA damage induces activation of a checkpoint effector kinase, Rad53p (human homolog Chk2), to promote cell cycle arrest and transcription of DNA repair genes. However, recent studies have revealed variation in the DNA damage response networks of some fungi. For example, Shor et al. (mBio 11:e03044-20, 2020, https://doi.org/10.1128/mBio.03044-20) demonstrate that in comparison to S. cerevisiae, the fungal pathogen Candida glabrata has reduced activation of Rad53p in response to DNA damage. Consequently, some downstream targets that contribute to S. cerevisiae genome maintenance, such as DNA polymerases, are transcriptionally downregulated in C. glabrata. Downregulation of genome maintenance genes likely contributes to higher rates of mitotic failure and cell death in C. glabrata. This and other recent findings highlight evolutionary diversity in eukaryotic DNA damage responses.

Present in all organisms, DNA ligases catalyse the formation of a phosphodiester bond between a 3′ hydroxyl and a 5′ phosphate, a reaction that is essential for maintaining genome integrity during replication and repair. Eubacterial DNA ligases use NAD+ as a cofactor and possess low sequence and structural homology relative to eukaryotic DNA ligases which use ATP as a cofactor. These key differences enable specific targeting of bacterial DNA ligases as an antibacterial strategy. In this study, four small molecule accessible sites within functionally important regions of Escherichia coli ligase (EC-LigA) were identified using in silico methods. Molecular docking was then used to screen for small molecules predicted to bind to these sites. Eight candidate inhibitors were then screened for inhibitory activity in an in vitro ligase assay. Five of these (geneticin, chlorhexidine, glutathione (reduced), imidazolidinyl urea and 2-(aminomethyl)imidazole) showed dose-dependent inhibition of EC-LigA with half maximal inhibitory concentrations (IC50) in the micromolar to millimolar range (11–2600 µM). Two (geneticin and chlorhexidine) were predicted to bind to a region of EC-LigA that has not been directly investigated previously, raising the possibility that there may be amino acids within this region that are important for EC-LigA activity or that the function of essential residues proximal to this region are impacted by inhibitor interactions with this region. We anticipate that the identified small molecule binding sites and inhibitors could be pursued as part of an antibacterial strategy targeting bacterial DNA ligases.