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BackgroundWhole blood is currently the most common DNA source for whole-genome sequencing (WGS), but for studies requiring non-invasive collection, self-collection, greater sample stability or additional tissue references, saliva or buccal samples may be preferred. However, the relative quality of sequencing data and accuracy of genetic variant detection from blood-derived, saliva-derived and buccal-derived DNA need to be thoroughly investigated.MethodsMatched blood, saliva and buccal samples from four unrelated individuals were used to compare sequencing metrics and variant-detection accuracy among these DNA sources.ResultsWe observed significant differences among DNA sources for sequencing quality metrics such as percentage of reads aligned and mean read depth (pConclusionFor WGS, we recommend using DNA extracted from blood rather than saliva or buccal swabs; if saliva or buccal samples are used, we recommend against using methylation-based eukaryotic DNA enrichment. All data used in this study are available for further open-science investigation.

This review covers the stages of studying left-helical Z-DNA form, from its discovery in 1979 until the present time. The repetitive nucleotide sequences that are capable of the B–Z transition and factors that initiate the transition and stabilization of Z-DNA, including non-histone proteins, which interact with the Z-sites, are described. The most important result of this study is the demonstration of the presence of this form in eukaryotic DNA and its role in the regulation of gene transcription. In addition, the facts of involvement of Z-DNA in the induction of a number of human pathologies are described.

Background:Guanine-rich DNA can fold into highly stable four-stranded DNA structures called G-quadruplexes (G4). In recent years, the G-quadruplex field has blossomed as new evidence strongly suggests that such alternately folded DNA structures are likely to exist in vivo. G4 DNA presents obstacles for the replication machinery, and both eukaryotic DNA helicases and polymerases have evolved to resolve and copy G4 DNA in vivo. In addition, G4-forming sequences are prevalent in gene promoters, suggesting that G4-resolving helicases act to modulate transcription.Methods:We have searched the PubMed database to compile an up-to-date and comprehensive assessment of the field’s current knowledge to provide an overview of the molecular interactions of Gquadruplexes with DNA helicases and polymerases implicated in their resolution.Results:Novel computational tools and alternative strategies have emerged to detect G4-forming sequences and assess their biological consequences. Specialized DNA helicases and polymerases catalytically act upon G4-forming sequences to maintain normal replication and genomic stability as well as appropriate gene regulation and cellular homeostasis. G4 helicases also resolve telomeric repeats to maintain chromosomal DNA ends. Bypass of many G4-forming sequences is achieved by the action of translesion DNS polymerases or the PrimPol DNA polymerase. While the collective work has supported a role of G4 in nuclear DNA metabolism, an emerging field centers on G4 abundance in the mitochondrial genome.Conclusion:Discovery of small molecules that specifically bind and modulate DNA helicases and polymerases or interact with the G4 DNA structure itself may be useful for the development of anticancer regimes.

Background Directed DNA methylation on N6-adenine (6mA), N4-cytosine (4mC), and C5-cytosine (5mC) can potentially increase DNA coding capacity and regulate a variety of biological functions. These modifications are relatively abundant in bacteria, occurring in about a percent of all bases of most bacteria. Until recently, 5mC and its oxidized derivatives were thought to be the only directed DNA methylation events in metazoa. New and more sensitive detection techniques (ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-ms/ms) and single molecule real-time sequencing (SMRTseq)) have suggested that 6mA and 4mC modifications could be present in a variety of metazoa. Results Here, we find that both of these techniques are prone to inaccuracies, which overestimate DNA methylation concentrations in metazoan genomic DNA. Artifacts can arise from methylated bacterial DNA contamination of enzyme preparations used to digest DNA and contaminating bacterial DNA in eukaryotic DNA preparations. Moreover, DNA sonication introduces a novel modified base from 5mC that has a retention time near 4mC that can be confused with 4mC. Our analyses also suggest that SMRTseq systematically overestimates 4mC in prokaryotic and eukaryotic DNA and 6mA in DNA samples in which it is rare. Using UHPLC-ms/ms designed to minimize and subtract artifacts, we find low to undetectable levels of 4mC and 6mA in genomes of representative worms, insects, amphibians, birds, rodents and primates under normal growth conditions. We also find that mammalian cells incorporate exogenous methylated nucleosides into their genome, suggesting that a portion of 6mA modifications could derive from incorporation of nucleosides from bacteria in food or microbiota. However, gDNA samples from gnotobiotic mouse tissues found rare (0.9–3.7 ppm) 6mA modifications above background. Conclusions Altogether these data demonstrate that 6mA and 4mC are rarer in metazoa than previously reported, and highlight the importance of careful sample preparation and measurement, and need for more accurate sequencing techniques. Electronic supplementary material The online version of this article (10.1186/s12864-019-5754-6) contains supplementary material, which is available to authorized users.

Eukaryotic DNA methylation has been receiving increasing attention for its crucial epigenetic regulatory function. The recently developed single-molecule real-time (SMRT) sequencing technology provides an efficient way to detect DNA N6-methyladenine (6mA) and N4-methylcytosine (4mC) modifications at a single-nucleotide resolution. The family Rosaceae contains horticultural plants with a wide range of economic importance. However, little is currently known regarding the genome-wide distribution patterns and functions of 6mA and 4mC modifications in the Rosaceae. In this study, we present an integrated DNA 6mA and 4mC modification database for the Rosaceae (MDR, http://mdr.xieslab.org). MDR, the first repository for displaying and storing DNA 6mA and 4mC methylomes from SMRT sequencing data sets for Rosaceae, includes meta and statistical information, methylation densities, Gene Ontology enrichment analyses, and genome search and browse for methylated sites in NCBI. MDR provides important information regarding DNA 6mA and 4mC methylation and may help users better understand epigenetic modifications in the family Rosaceae.

The 2019 Gairdner Prize will be given to John F.X. Diffley and Bruce Stillman for their groundbreaking work on the mechanisms and control of the initiation of eukaryotic DNA replication. No two people have contributed more extensively, or over a longer period of time, to enlighten us on how our genomes replicate themselves once and only once per cell cycle.

Nucleosomes represent the fundamental repeating unit of eukaryotic DNA, and comprise eight core histones around which DNA is wrapped in nearly two superhelical turns. Histones do not have the intrinsic ability to form nucleosomes; rather, they require an extensive repertoire of interacting proteins collectively known as ‘histone chaperones’. At a fundamental level, it is believed that histone chaperones guide the assembly of nucleosomes through preventing non-productive charge-based aggregates between the basic histones and acidic cellular components. At a broader level, histone chaperones influence almost all aspects of chromatin biology, regulating histone supply and demand, governing histone variant deposition, maintaining functional chromatin domains and being co-factors for histone post-translational modifications, to name a few. In this essay we review recent structural insights into histone-chaperone interactions, explore evidence for the existence of a histone chaperoning ‘pathway’ and reconcile how such histone-chaperone interactions may function thermodynamically to assemble nucleosomes and maintain chromatin homeostasis.

MCM8 and MCM9 are paralogues of the MCM2-7 eukaryotic DNA replication helicase proteins and play a crucial role in a homologous recombination-mediated repair process to resolve replication stress by fork stalling. Thus, deficiency of MCM8-9 sensitizes cells to replication stress caused, for example, by platinum compounds that induce interstrand cross-links. It is suggested that cancer cells undergo more replication stress than normal cells due to hyperstimulation of growth. Therefore, it is possible that inhibiting MCM8-9 selectively hypersensitizes cancer cells to platinum compounds and poly(ADP-ribose) polymerase inhibitors, both of which hamper replication fork progression. Here, we inhibited MCM8-9 in transformed and nontransformed cells and examined their sensitivity to cisplatin and olaparib. We found that knockout of MCM9 or knockdown of MCM8 selectively hypersensitized transformed cells to cisplatin and olaparib. In agreement with reported findings, RAS- and human papilloma virus type 16 E7-mediated transformation of human fibroblasts increased replication stress, as indicated by induction of multiple DNA damage responses (including formation of Rad51 foci). Such replication stress induced by oncogenes was further increased by knockdown of MCM8, providing a rationale for cancer-specific hypersensitization to cisplatin and olaparib. Finally, we showed that knocking out MCM9 increased the sensitivity of HCT116 xenograft tumors to cisplatin. Taken together, the data suggest that conceptual MCM8-9 inhibitors will be powerful cancer-specific chemosensitizers for platinum compounds and poly(ADP-ribose) polymerase inhibitors, thereby opening new avenues to the design of novel cancer chemotherapeutic strategies.

DNA damage response (DDR) constitutes a protein pathway to handle eukaryotic DNA lesions in the context of chromatin. DDR engages the recruitment of signaling, transducer, effector, chromatin modifiers and remodeling proteins, allowing cell cycle delay, DNA repair or induction of senescence or apoptosis. An early DDR-event includes the epigenetic phosphorylation of the histone variant H2AX on serine 139 of the C-termini, so-called gammaH2AX. GammaH2AX foci detected by immunolabeling on interphase nuclei have been largely studied; nonetheless gammaH2AX signals on mitotic chromosomes are less understood. The CHO9 cell line is a subclone of CHO (Chinese hamster ovary) cells with original and rearranged Z chromosomes originated during cell line transformation. As a result, homologous chromosome regions have been relocated in different Z-chromosomes. In a first quantitative analysis of gammaH2AX signals on immunolabeled mitotic chromosomes of cytocentrifuged metaphase spreads, we reported that gammaH2AX139 signals of both control and bleomycin-exposed cultures showed statistically equal distribution between CHO9 homologous chromosome regions, suggesting a possible dependence on the structure/function of chromatin. We have also demonstrated that bleomycin-induced gammaH2AX foci map preferentially to DNA replicating domains in CHO9 interphase nuclei. With the aim of understanding the role of gammaH2AX signals on metaphase chromosomes, the relation between 5-ethynyl-2'-deoxyuridine (EdU) labeled replicating chromosome regions and gammaH2AX signals in immunolabeled cytocentrifuged metaphase spreads from control and bleomycin-treated CHO9 cultures was analyzed in the present work. A quantitative analysis of colocalization between EdU and gammaH2AX signals based on the calculation of the Replication Related Damage Distribution Index (RDDI) on confocal metaphase images was performed. RDDI revealed a colocalization between EdU and gammaH2AX signals both in control and bleomycin-treated CHO9 metaphases, suggesting that replication may be involved in H2AX phosphorylation. The possible mechanisms implicated are discussed.

All the eukaryotic DNA ligases are known to use adenosine triphosphate (ATP) for DNA ligation. Here, we report that human DNA ligase IV, a key enzyme in DNA double-strand break (DSB) repair, is able to use NAD+ as a substrate for double-stranded DNA ligation. In the in vitro ligation assays, we show that the recombinant Ligase IV can use both ATP and NAD+ for DNA ligation. For NAD+-mediated ligation, the BRCA1 C-terminal (BRCT) domain of Ligase IV recognizes NAD+ and facilitates the adenylation of Ligase IV, the first step of ligation. Although XRCC4, the functional partner of Ligase IV, is not required for the NAD+-mediated adenylation, it regulates the transfer of AMP moiety from Ligase IV to the DNA end. Moreover, cancer-associated mutation in the BRCT domain of Ligase IV disrupts the interaction with NAD+, thus abolishes the NAD+-mediated adenylation of Ligase IV and DSB ligation. Disrupting the NAD+ recognition site in the BRCT domain impairs non-homologous end joining (NHEJ) in cell. Taken together, our study reveals that in addition to ATP, Ligase IV may use NAD+ as an alternative adenylation donor for NHEJ repair and maintaining genomic stability.

Cells must replicate and segregate their DNA with precision. In eukaryotes, these processes are part of a regulated cell-cycle that begins at S-phase with the replication of DNA and ends after M-phase. Previous studies showed that these processes were present in the last eukaryotic common ancestor and the core parts of their molecular systems are conserved across eukaryotic diversity. However, some unicellular parasites, such as the metamonad Giardia intestinalis, 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 high-quality draft genome assembly for the free-living metamonad Carpediemonas membranifera and carried out a comparative genomics analysis. We found that parasitic and free-living metamonads harbor a conspicuously incomplete set of canonical proteins for processing and segregating DNA. Unexpectedly, Carpediemonas species are further streamlined, lacking the origin recognition complex, Cdc6 and other replisome components, most structural kinetochore subunits including the Ndc80 complex, as well as several canonical cell-cycle checkpoint proteins. Carpediemonas is the first eukaryote known to have lost this large suite of conserved complexes, suggesting that it has a highly unusual cell cycle and that unlike any other known eukaryote, it must rely on novel or alternative set of mechanisms to carry out these fundamental processes.

Minichromosome maintenance protein 10 (MCM10) is essential for eukaryotic DNA replication. Here, we describe compound heterozygous MCM10 variants in patients with distinctive, but overlapping, clinical phenotypes: natural killer (NK) cell deficiency (NKD) and restrictive cardiomyopathy (RCM) with hypoplasia of the spleen and thymus. To understand the mechanism of MCM10-associated disease, we modeled these variants in human cell lines. MCM10 deficiency causes chronic replication stress that reduces cell viability due to increased genomic instability and telomere erosion. Our data suggest that loss of MCM10 function constrains telomerase activity by accumulating abnormal replication fork structures enriched with single-stranded DNA. Terminally-arrested replication forks in MCM10-deficient cells require endonucleolytic processing by MUS81, as MCM10:MUS81 double mutants display decreased viability and accelerated telomere shortening. We propose that these bi-allelic variants in MCM10 predispose specific cardiac and immune cell lineages to prematurely arrest during differentiation, causing the clinical phenotypes observed in both NKD and RCM patients., Minichromosome maintenance protein 10 (MCM10) is critical for eukaryotic DNA replication. Here, by modelling MCM10 variants in human cell lines, the authors reveal a mechanism of MCM10-associated disease, finding that loss of MCM10 function constrains telomerase activity.

The pioneer event in eukaryotic DNA replication is binding of chromosomal DNA by the origin recognition complex (ORC), which directs the formation of origins, the specific chromosomal regions where DNA will be unwound for the initiation of DNA synthesis. In all eukaryotes, incompletely understood features of chromatin promote ORC-DNA binding. Here, we uncover a role for the Fkh1 (forkhead homolog) protein, and, in particular, its forkhead associated (FHA) domain in promoting ORC-origin binding and origin activity at a subset of origins inSaccharomyces cerevisiae.The majority of the FHA-dependent origins within the experimental subset examined contain a distinct Fkh1 binding site located 5’ of and proximal to their ORC sites (5’-FKH-T site). Epistasis experiments using selected FHA-dependent origins provided evidence that the FHA domain promoted origin activity through Fkh1 binding directly to this 5’ FKH-T site. Nucleotide substitutions within two of these origins that enhanced the affinity of their ORC sites for ORC bypassed these origins’ requirement for their 5’ FKH-T sites and for the FHA domain. Significantly, direct assessment of ORC-origin binding by ChIPSeq provided evidence that this mechanism affected ~25% of yeast origins. Thus, this study reveals a new mechanism to enhance ORC-origin binding in budding yeast that requires the FHA domain of the conserved cell-cycle transcription factor Fkh1.

Like all DNA viruses, human cytomegalovirus (HCMV) infection is known to result in profound effects on host cell cycle. Infection of fibroblasts with HCMV is known to induce an advance in cell cycle through the G(0)-G(1) phase and then a subsequent arrest of cell cycle in early S-phase, presumably resulting in a cellular environment optimum for high levels of viral DNA replication whilst precluding replication of cellular DNA. Although the exact mechanisms used to arrest cell cycle by HCMV are unclear, they likely involve a number of viral gene products and evidence points to the ability of the virus to prevent licensing of cellular DNA synthesis. One viral protein known to profoundly alter cell cycle is the viral immediate early 86 (IE86) protein--an established function of which is to initially drive cells into early S phase but then inhibit cellular DNA synthesis. Here we show that, although IE86 interacts with the cellular licensing factor Cdt1, it does not inhibit licensing of cellular origins. Instead, IE86-mediated inhibition of cellular DNA synthesis requires mini-chromosome-maintenance 3 (MCM3) associated protein (MCM3AP), which can cause subsequent inhibition of initiation of cellular DNA synthesis in a licensing-independent manner.

Although HCMV encodes many genes required for the replication of its DNA genome, no HCMV-encoded orthologue of the origin binding protein, which has been identified in other herpesviruses, has been identified. This has led to speculation that HCMV may use other viral proteins or possibly cellular factors for the initiation of DNA synthesis. It is also unclear whether cellular replication factors are required for efficient replication of viral DNA during or after viral replication origin recognition. Consequently, we have asked whether cellular pre-replication (pre-RC) factors that are either initially associated with cellular origin of replication (e.g. ORC2), those which recruit other replication factors (e.g. Cdt1 or Cdc6) or those which are subsequently recruited (e.g. MCMs) play any role in the HCMV DNA replication. We show that whilst RNAi-mediated knock-down of these factors in the cell affects cellular DNA replication, as predicted, it results in concomitant increases in viral DNA replication. These data show that cellular factors which initiate cellular DNA synthesis are not required for the initiation of replication of viral DNA and suggest that inhibition of cellular DNA synthesis, in itself, fosters conditions which are conducive to viral DNA replication.

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 core complexes and forms nucleosomes, translocases inevitably encounter nucleosomes. Previous studies have shown that a histone core complex repositions upstream (downstream) when SP6RNA or T7 RNA polymerase (bacterial exonuclease, RecBCD) partially unwraps nucleosomal DNA. However, the molecular mechanism of the downstream repositioning remains unclear. In this study, we identify the lane-shift mechanism for downstream nucleosome repositioning via coarse-grained molecular dynamics simulations, which we validated by 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 region (lane) to that vacated by the unwrapping, and the downstream DNA rewraps, completing downstream repositioning. This mechanism may have crucial implications for transcription through nucleosomes, histone recycling, and nucleosome remodeling.SIGNIFICANCEEukaryotic chromosomes are composed of repeating subunits termed nucleosomes. Thus, proteins that translocate along the chromosome, DNA translocases, inevitably collide with nucleosomes. Previous studies revealed that a translocase repositions a nucleosome upstream or downstream upon their collision. Though the molecular mechanisms of the upstream repositioning have been extensively studied, that of downstream repositioning remains elusive. In this study, we performed coarse-grained molecular dynamics simulations, proposed the lane-shift mechanism for downstream repositioning, and validated this mechanism by restriction enzyme digestion assays and deep sequencing assays. This mechanism has broad implications for how translocases deal with nucleosomes for their functions.

4-methylcytosine (4mC) is an important DNA modification in prokaryotes, but its relevance, and even presence in eukaryotes have been mysterious. Here we show that spermatogenesis in the liverwortMarchantia polymorphainvolves two waves of extensive DNA methylation reprogramming. First, 5-methylcytosine (5mC), a well-known eukaryotic DNA modification, expands from transposons to the entire genome. Notably, the second wave installs 4mC throughout genic regions, covering over 50% of CG sites in sperm. 4mC is catalyzed by two novel methyltransferases (MpDN4MT1a and MpDN4MT1b) specifically expressed during late spermiogenesis. Deletion of MpDN4MT1seliminates 4mC, alters the sperm transcriptome, and produces sperm with swimming defects. Our results reveal extensive 4mC in a eukaryote and define a new family of eukaryotic methyltransferases, thereby expanding the repertoire of functional eukaryotic DNA modifications.

Minichromosome maintenance complex component 7 (MCM7) belongs to the minichromosome maintenance family that is important for the initiation of eukaryotic DNA replication. Overexpression of the MCM7 protein is relative to cellular proliferation and responsible for aggressive malignancy in various cancers. Mechanistically, inhibition of MCM7 significantly reduces the cellular proliferation associated with cancer. To date, no effective small molecular candidate has been identified that can block the progression of cancer induced by the MCM7 protein. Therefore, the study has been designed to identify small molecular-like natural drug candidates against aggressive malignancy associated with various cancers by targeting MCM7 protein. To identify potential compounds against the targeted protein a comprehensive in silico drug design including molecular docking, ADME (Absorption, Distribution, Metabolism and Excretion), toxicity, and molecular dynamics (MD) simulation approaches has been applied. Seventy phytochemicals isolated from the neem tree (Azadiractha indica) were retrieved and screened against MCM7 protein by using the molecular docking simulation method, where the top four compounds have been chosen for further evaluation based on their binding affinities. Analysis of ADME and toxicity properties reveals the efficacy and safety of the selected four compounds. To validate the stability of the protein–ligand complex structure MD simulations approach has also been performed to the protein–ligand complex structure, which confirmed the stability of the selected three compounds including CAS ID:105377-74-0, CID:12308716 and CID:10505484 to the binding site of the protein. In the study, a comprehensive data screening process has performed based on the docking, ADMET properties, and MD simulation approaches, which found a good value of the selected four compounds against the targeted MCM7 protein and indicates as a promising and effective human anticancer agent.

The DEK oncoprotein regulates cellular chromatin function via a number of protein-protein interactions. However, the biological relevance of its unique pseudo-SAP/SAP-box domain, which transmits DNA modulating activities in vitro, remains largely speculative. As hypothesis-driven mutations failed to yield DNA-binding null (DBN) mutants, we combined random mutagenesis with the Bacterial Growth Inhibition Screen (BGIS) to overcome this bottleneck. Re-expression of a DEK-DBN mutant in newly established human DEK knockout cells failed to reduce the increase in nuclear size as compared to wild type, indicating roles for DEK-DNA interactions in cellular chromatin organization. Our results extend the functional roles of DEK in metazoan chromatin and highlight the predictive ability of recombinant protein toxicity in E. coli for unbiased studies of eukaryotic DNA modulating protein domains. published

International audience; Eukaryotic DNA replication initiates during S phase from origins that have been licensed in the preceding G1 phase. Here, we compare ChIP-seq profiles of the licensing factors Orc2, Orc3, Mcm3, and Mcm7 with gene expression, replication timing, and fork directionality profiles obtained by RNA-seq, Repli-seq, and OK-seq. Both, the origin recognition complex (ORC) and the minichromosome maintenance complex (MCM) are significantly and homogeneously depleted from transcribed genes, enriched at gene promoters, and more abundant in early- than in late-replicating domains. Surprisingly, after controlling these variables, no difference in ORC/MCM density is detected between initiation zones, termination zones, unidirectionally replicating regions, and randomly replicating regions. Therefore, ORC/MCM density correlates with replication timing but does not solely regulate the probability of replication initiation. Interestingly, H4K20me3, a histone modification proposed to facilitate late origin licensing, was enriched in late-replicating initiation zones and gene deserts of stochastic replication fork direction. We discuss potential mechanisms specifying when and where replication initiates in human cells.

DNA polymerase delta (Pol δ) plays several essential roles in eukaryotic DNA replication and repair. At the replication fork, Pol δ is responsible for the synthesis and processing of the lagging-strand. At replication origins, Pol δ has been proposed to initiate leading-strand synthesis by extending the first Okazaki fragment. Destabilizing mutations in human Pol δ subunits cause replication stress and syndromic immunodeficiency. Analogously, reduced levels of Pol δ in Saccharomyces cerevisiae lead to pervasive genome instability. Here, we analyze how the depletion of Pol δ impacts replication origin firing and lagging-strand synthesis during replication elongation in vivo in S. cerevisiae. By analyzing nascent lagging-strand products, we observe a genome-wide change in both the establishment and progression of replication. S-phase progression is slowed in Pol δ depletion, with both globally reduced origin firing and slower replication progression. We find that no polymerase other than Pol δ is capable of synthesizing a substantial amount of lagging-strand DNA, even when Pol δ is severely limiting. We also characterize the impact of impaired lagging-strand synthesis on genome integrity and find increased ssDNA and DNA damage when Pol δ is limiting; these defects lead to a strict dependence on checkpoint signaling and resection-mediated repair pathways for cellular viability., Author summary DNA replication in eukaryotes is carried out by the replisome–a multi-subunit complex comprising the enzymatic activities required to generate two intact daughter DNA strands. DNA polymerase delta (Pol δ) is a multi-functional replisome enzyme responsible for synthesis and processing of the lagging-strand. Mutations in Pol δ cause a variety of human diseases: for example, destabilizing mutations lead to immunodeficiency. We titrate the concentration of Pol δ in budding yeast–a simple model eukaryote with conserved DNA replication machinery. We characterize several replication defects associated with Pol δ scarcity. The defects we observe provide insight into how destabilizing Pol δ mutations lead to genome instability.

The in vitro reconstitution of origin firing was a key step toward the biochemical reconstitution of eukaryotic DNA replication in budding yeast. Today the basic replication assay involves proteins purified from 24 separate protocols that have evolved since their first publication, and as a result, the efficiency and reliability of the in vitro replication system has improved. Here we will present protocols for all 24 purifications together with a general protocol for the in vitro replication assay and some tips for troubleshooting problems with the assay.

Eukaryotic DNA replication initiates during S phase from origins that have been licensed in the preceding G1 phase. Here, we compare ChIP-seq profiles of the licensing factors Orc2, Orc3, Mcm3, and Mcm7 with gene expression, replication timing and fork directionality profiles obtained by RNA-seq, Repli-seq and OK-seq. ORC and MCM are strongly and homogeneously depleted from transcribed genes, enriched at gene promoters, and more abundant in early-than in late-replicating domains. Surprisingly, after controlling these variables, no difference in ORC/MCM density is detected between initiation zones, termination zones, unidirectionally replicating and randomly replicating regions. Therefore, ORC/MCM density correlates with replication timing but does not solely regulate the probability of replication initiation. Interestingly, H4K20me3, a histone modification proposed to facilitate late origin licensing, was enriched in late replicating initiation zones and gene deserts of stochastic replication fork direction. We discuss potential mechanisms that specify when and where replication initiates in human cells.

The committed step of eukaryotic DNA replication occurs when the replicative Mcm2-7 helicase pairs that license each replication origin are activated. Helicase activation requires the recruitment of Cdc45 and GINS to Mcm2-7, forming Cdc45-Mcm2-7-GINS complexes (CMGs). Using single-molecule biochemical assays to monitor CMG formation, we found that Cdc45 and GINS are recruited to loaded Mcm2-7 in two stages. Initially, Cdc45 and GINS are individually recruited to unstructured Mcm2-7 N-terminal tails in a Dbf4-dependent kinase (DDK)-dependent manner, forming Cdc45-tail-GINS intermediates (CtGs). The multiple phosphorylation sites on the Mcm2-7 tails promote DDK-dependent modulation of the number of CtGs formed per Mcm2-7. In a second, inefficient event, a subset of CtGs transfer their Cdc45 and GINS components to form CMGs. Importantly, higher CtG multiplicity results in increased frequency of CMG formation. Our findings reveal molecular mechanisms sensitizing helicase activation to DDK levels with implications for the control of replication origin efficiency and timing.

The replication of chromosomes during S phase is critical for cellular and organismal function. Replicative stress can result in genome instability, which is a major driver of cancer. Yet how chromatin is made accessible during eukaryotic DNA synthesis is poorly understood.Here, we report the identification of a novel class of chromatin remodeling enzyme, entirely distinct from classical SNF2-ATPase family remodelers. Yta7 is a AAA+-ATPase that assembles into ~ 1 MDa hexameric complexes capable of segregating histones from DNA. Yta7 chromatin segregase promotes chromosome replication both in vivo and in vitro. Biochemical reconstitution experiments using purified proteins revealed that Yta7’s enzymatic activity is regulated by S phase-forms of Cyclin-Dependent Kinase (S-CDK). S-CDK phosphorylation stimulates ATP hydrolysis by Yta7, promoting nucleosome disassembly and chromatin replication.Our results present a novel mechanism of how cells orchestrate chromatin dynamics in co-ordination with the cell cycle machinery to promote genome duplication during S phase.

Significance The structure of the eukaryotic chromosomal replicase, DNA polymerase (Pol) δ, was determined in complex with its cognate proliferating cell nuclear antigen (PCNA) sliding clamp on primed DNA. The results show that the Pol3 catalytic subunit binds atop the PCNA ring, and the two regulatory subunits of Pol δ, Pol31, and Pol32, are positioned off to the side of the Pol3 clamp. The catalytic Pol3 binds DNA and PCNA such as to thread the DNA straight through the circular PCNA clamp. Considering the large diameter of the PCNA clamp, there is room for water between DNA and the inner walls of PCNA, indicating the clamp “waterskates” on DNA during function with polymerase., The DNA polymerase (Pol) δ of Saccharomyces cerevisiae (S.c.) is composed of the catalytic subunit Pol3 along with two regulatory subunits, Pol31 and Pol32. Pol δ binds to proliferating cell nuclear antigen (PCNA) and functions in genome replication, repair, and recombination. Unique among DNA polymerases, the Pol3 catalytic subunit contains a 4Fe-4S cluster that may sense the cellular redox state. Here we report the 3.2-Å cryo-EM structure of S.c. Pol δ in complex with primed DNA, an incoming ddTTP, and the PCNA clamp. Unexpectedly, Pol δ binds only one subunit of the PCNA trimer. This singular yet extensive interaction holds DNA such that the 2-nm-wide DNA threads through the center of the 3-nm interior channel of the clamp without directly contacting the protein. Thus, a water-mediated clamp and DNA interface enables the PCNA clamp to “waterskate” along the duplex with minimum drag. Pol31 and Pol32 are positioned off to the side of the catalytic Pol3-PCNA-DNA axis. We show here that Pol31-Pol32 binds single-stranded DNA that we propose underlies polymerase recycling during lagging strand synthesis, in analogy to Escherichia coli replicase. Interestingly, the 4Fe-4S cluster in the C-terminal CysB domain of Pol3 forms the central interface to Pol31-Pol32, and this strategic location may explain the regulation of the oxidation state on Pol δ activity, possibly useful during cellular oxidative stress. Importantly, human cancer and other disease mutations map to nearly every domain of Pol3, suggesting that all aspects of Pol δ replication are important to human health and disease.

Origins of eukaryotic DNA replication are ‘licensed’ during G1 phase of the cell cycle by loading the six related minichromosome maintenance (MCM) proteins into a double hexameric ring around double-stranded DNA. In S phase, some double hexamers (MCM DHs) are converted into active CMG (Cdc45-MCM-GINS) helicases which nucleate assembly of bidirectional replication forks. The remaining unfired MCM DHs act as ‘dormant’ origins to provide backup replisomes in the event of replication fork stalling. The fate of unfired MCM DHs during replication is unknown. Here we show that active replisomes cannot remove unfired MCM DHs. Instead, they are pushed ahead of the replisome where they prevent fork convergence during replication termination and replisome progression through nucleosomes. Pif1 helicase, together with the replisome, can remove unfired MCM DHs specifically from replicating DNA, allowing efficient replication and termination. Our results provide an explanation for how excess replication license is removed during S phase.

Interaction of PCNA with DNA polymerase is vital to efficient and processive DNA synthesis. PCNA being a homotrimeric ring possesses three hydrophobic pockets mostly involved in an interaction with its binding partners. PCNA interacting proteins contain a short sequence of eight amino acids, popularly coined as PIP motif, which snuggly fits into the hydrophobic pocket of PCNA to stabilize the interaction. In the last two decades, several PIP motifs have been mapped or predicted in eukaryotic DNA polymerases. In this review, we summarize our understandings of DNA polymerase-PCNA interaction, the function of such interaction during DNA synthesis, and emphasize the lacunae that persist. Because of the presence of multiple ligands in the replisome complex and due to many interaction sites in DNA polymerases, we also propose two modes of DNA polymerase positioning on PCNA required for DNA synthesis to rationalize the tool-belt model of DNA replication.

During translesion synthesis, eukaryotic DNA polymerase ζ (zeta) carries out extension from a wide range of DNA lesions. In this issue, Malik et al. (2020) (1) present the cryo-EM structure of polymerase ζ and show how it catalyzes the extension step of translesion synthesis.

The pre-initiation complex (pre-IC) has been proposed for two decades as an intermediate right before the maturation of the eukaryotic DNA replication fork. However, its existence and biochemical nature remain enigmatic. Here, through combining several enrichment strategies, we are able to isolate an endogenous dimeric CMG-containing complex (designated as d-CMG) distinct from traditional single CMG (s-CMG) and in vitro reconstituted dimeric CMG. D-CMG is assembled upon entry into the S phase and shortly matures into s-CMG/replisome, leading to the fact that only ~ 5% of the total CMG-containing complexes can be detected as d-CMG in vivo. Mass spectra reveal that RPA and DNA Pol α/primase co-purify with s-CMG, but not with d-CMG. Consistently, the former fraction is able to catalyze DNA unwinding and de novo synthesis, while the latter catalyzes neither. The two CMGs in d-CMG display flexibly orientated conformations under an electronic microscope. When DNA Pol α-primase is inactivated, d-CMG % rose up to 29%, indicating an incomplete pre-IC/fork transition. These findings reveal biochemical properties of the d-CMG/pre-IC and provide in vivo evidence to support the pre-IC/fork transition as a bona fide step in replication initiation.

Eukaryotic DNA replication is a highly dynamic and tightly regulated process. Replication involves several dozens of replication proteins, including the initiators ORC and Cdc6, replicative CMG helicase, DNA polymerase α-primase, leading-strand DNA polymerase ε, and lagging-strand DNA polymerase δ. These proteins work together in a spatially and temporally controlled manner to synthesize new DNA from the parental DNA templates. During DNA replication, epigenetic information imprinted on DNA and histone proteins is also copied to the daughter DNA to maintain the chromatin status. DNA methyltransferase 1 is primarily responsible for copying the parental DNA methylation pattern into the nascent DNA. Epigenetic information encoded in histones is transferred via a more complex and less well-understood process termed replication-couple nucleosome assembly. Here, we summarize the most recent structural and biochemical insights into DNA replication initiation, replication fork elongation, chromatin assembly and maintenance, and related regulatory mechanisms.

Eukaryotic DNA is organized in nucleosomes, which package DNA and regulate its accessibility to transcription, replication, recombination and repair. Here, we show that in living cells nucleosomes protect DNA from high-energy radiation and reactive oxygen species. We combined sequence-based methods (ATAC-seq and BLISS) to determine the position of both nucleosomes and double strand breaks (DSBs) in the genome of nucleosome-rich malignant mesothelioma cells, and of the same cells partially depleted of nucleosomes. The results were replicated in the human MCF-7 breast carcinoma cell line. We found that, for each genomic sequence, the probability of DSB formation is directly proportional to the fraction of time it is nucleosome-free; DSBs accumulate distal from the nucleosome dyad axis. Nucleosome free regions and promoters of actively transcribed genes are more sensitive to DSB formation, and consequently to mutation. We argue that this may be true for a variety of chemical and physical DNA damaging agents.

Hereditary disease prediction in eukaryotic DNA using signal processing approaches is an incredible work in bioinformatics. Researchers of various fields are trying to put forth a noninvasive approach to forecast the disease-related genes. As diseased genes are more random than the healthy ones, in this work, a comparison of the diseased gene is made against the healthy ones. An adaptive signal processing method like functional link artificial neural network-based Levenberg-Marquardt filter has been proposed in this regard. For parameter upgradation, the algorithm is modified using particle swarm optimization. Here, disease genes are discriminated from healthy ones based on the magnitude of mean square error (MSE), which is calculated through the adaptive filter. The performance of the algorithm is inspected by computing some evaluation parameters. Since accuracy is the prime concern, authors in this work have taken an attempt to improve the accuracy level compared to the existing methods. Taking the reference gene as healthy, the overall process is accomplished by categorizing the diseased and healthy targets with MSE value at a threshold of 0.012. The proposed technique predicts the test gene sets successfully.

DNA replication origins serve as sites of replicative helicase loading. In all eukaryotes, the six-subunit origin recognition complex (Orc1-6; ORC) recognizes the replication origin. During late M-phase of the cell-cycle, Cdc6 binds to ORC and the ORC–Cdc6 complex loads in a multistep reaction and, with the help of Cdt1, the core Mcm2-7 helicase onto DNA. A key intermediate is the ORC–Cdc6–Cdt1–Mcm2-7 (OCCM) complex in which DNA has been already inserted into the central channel of Mcm2-7. Until now, it has been unclear how the origin DNA is guided by ORC–Cdc6 and inserted into the Mcm2-7 hexamer. Here, we truncated the C-terminal winged-helix-domain (WHD) of Mcm6 to slow down the loading reaction, thereby capturing two loading intermediates prior to DNA insertion in budding yeast. In “semi-attached OCCM,” the Mcm3 and Mcm7 WHDs latch onto ORC–Cdc6 while the main body of the Mcm2-7 hexamer is not connected. In “pre-insertion OCCM,” the main body of Mcm2-7 docks onto ORC–Cdc6, and the origin DNA is bent and positioned adjacent to the open DNA entry gate, poised for insertion, at the Mcm2–Mcm5 interface. We used molecular simulations to reveal the dynamic transition from preloading conformers to the loaded conformers in which the loading of Mcm2-7 on DNA is complete and the DNA entry gate is fully closed. Our work provides multiple molecular insights into a key event of eukaryotic DNA replication.

Genomic DNA is constantly damaged by factors produced during natural metabolic processes as well as agents coming from the external environment. Considering such a wide array of damaging agents, eukaryotic cells have evolved a DNA damage response (DRR) that opposes the influence of deleterious factors. Despite the broad knowledge regarding DNA damage and repair, new areas of research are emerging. New players in the field of DDR are constantly being discovered. The aim of this study is to review current knowledge regarding the roles of sirtuins, heat shock proteins, long-noncoding RNAs and the circadian clock in DDR and distinguish new agents that may have a prominent role in DNA damage response and repair.

During eukaryotic DNA replication, DNA polymerase alpha/primase (Pol α) initiates synthesis on both the leading and lagging strands. It is unknown whether leading- and lagging-strand priming are mechanistically identical, and whether Pol α associates processively or distributively with the replisome. Here, we titrate cellular levels of Pol α in S. cerevisiae and analyze Okazaki fragments to study both replication initiation and ongoing lagging-strand synthesis in vivo. We observe that both Okazaki fragment initiation and the productive firing of replication origins are sensitive to Pol α abundance, and that both processes are disrupted at similar Pol α concentrations. When the replisome adaptor protein Ctf4 is absent or cannot interact with Pol α, lagging-strand initiation is impaired at Pol α concentrations that still support normal origin firing. Additionally, we observe that activation of the checkpoint becomes essential for viability upon severe depletion of Pol α. Using strains in which the Pol α-Ctf4 interaction is disrupted, we demonstrate that this checkpoint requirement is not solely caused by reduced lagging-strand priming. Our results suggest that Pol α recruitment for replication initiation and ongoing lagging-strand priming are distinctly sensitive to the presence of Ctf4. We propose that the global changes we observe in Okazaki fragment length and origin firing efficiency are consistent with distributive association of Pol α at the replication fork, at least when Pol α is limiting., Author summary Half of each eukaryotic genome is replicated continuously as the leading strand, while the other half is synthesized discontinuously as Okazaki fragments on the lagging strand. The bulk of DNA replication is completed by DNA polymerases ε and δ on the leading and lagging strand respectively, while synthesis on each strand is initiated by DNA polymerase α-primase (Pol α). Using the model eukaryote S. cerevisiae, we modulate cellular levels of Pol α and interrogate the impact of this perturbation on both replication initiation on DNA synthesis and cellular viability. We observe that Pol α can associate dynamically at the replication fork for initiation on both strands. Although the initiation of both strands is widely thought to be mechanistically similar, we determine that Ctf4, a hub that connects proteins to the replication fork, stimulates lagging-strand priming to a greater extent than leading-strand initiation. We also find that decreased leading-strand initiation results in a checkpoint response that is necessary for viability when Pol α is limiting. Because the DNA replication machinery is highly conserved from budding yeast to humans, this research provides insights into how DNA replication is accomplished throughout eukaryotes.

DNA replication protein Cdc45 is an integral part of the eukaryotic replicative helicase whose other components are the Mcm2-7 core, and GINS. We identified a PIP box motif in Leishmania donovani Cdc45. This motif is typically linked to interaction with the eukaryotic clamp proliferating cell nuclear antigen (PCNA). The homotrimeric PCNA can potentially bind upto three different proteins simultaneously via a loop region present in each monomer. Multiple binding partners have been identified from among the replication machinery in other eukaryotes, and the concerted /sequential binding of these partners are central to the fidelity of the replication process. Though conserved in Cdc45 across Leishmania species and Trypanosoma cruzi, the PIP box is absent in Trypanosoma brucei Cdc45. Here we investigate the possibility of Cdc45-PCNA interaction and the role of such an interaction in the in vivo context. Having confirmed the importance of Cdc45 in Leishmania DNA replication we establish that Cdc45 and PCNA interact stably in whole cell extracts, also interacting with each other directly in vitro. The interaction is mediated via the Cdc45 PIP box. This PIP box is essential for Leishmania survival. The importance of the Cdc45 PIP box is also examined in Schizosaccharomyces pombe, and it is found to be essential for cell survival here as well. Our results implicate a role for the Leishmania Cdc45 PIP box in recruiting or stabilizing PCNA on chromatin. The Cdc45-PCNA interaction might help tether PCNA and associated replicative DNA polymerase to the DNA template, thus facilitating replication fork elongation. Though multiple replication proteins that associate with PCNA have been identified in other eukaryotes, this is the first report demonstrating a direct interaction between Cdc45 and PCNA, and while our analysis suggests the interaction may not occur in human cells, it indicates that it may not be confined to trypanosomatids., Author summary Leishmaniases are manifested in three forms: cutaneous, sub-cutaneous and visceral. The prevalent form in the Indian subcontinent is visceral leishmaniasis (VL), which is fatal if not treated on time. While there are drugs for treatment, the hunt for additional drugs continues due to emerging drug resistance patterns. The parasite is transmitted by the bite of the sandfly, whereupon it establishes itself within cells of the host immune system (macrophages) and reproduces by binary fission. The replication of its genome is essential for parasite survival. Eukaryotic DNA replication is generally conserved across species. This study targets Cdc45, a protein that helps unwind the DNA double helix to enable copying of the two strands into two daughter strands. The new chains of DNA are synthesized by DNA polymerases, and a trimeric protein, proliferating cell nuclear antigen (PCNA), helps clamp the polymerases onto the template. In this study we find Cdc45 to interact with PCNA, and have identified the motif in Cdc45 via which it does so. Our results suggest this interaction may be used by other eukaryotes as well. Based on the results of our experiments we propose that Cdc45 may help moor PCNA-polymerase complexes to template DNA.

In eukaryotic DNA replication, DNA polymerase e (Pole) is responsible for leading strand synthesis, whereas DNA polymerases α and δ synthesize the lagging strand. The human Pole (hPole) holoenzyme is comprised of the catalytic p261 subunit and the noncatalytic p59, p17, and p12 small subunits. So far, the contribution of the noncatalytic subunits to hPole function is not well understood. Using pre-steady-state kinetic methods, we established a minimal kinetic mechanism for DNA polymerization and editing catalyzed by the hPole holoenzyme. Compared with the 140-kDa N-terminal catalytic fragment of p261 (p261N), which we kinetically characterized in our earlier studies, the presence of the p261 C-terminal domain (p261C) and the three small subunits increased the DNA binding affinity and the base substitution fidelity. Although the small subunits enhanced correct nucleotide incorporation efficiency, there was a wide range of rate constants when incorporating a correct nucleotide over a single-base mismatch. Surprisingly, the 3′→5′ exonuclease activity of the hPole holoenzyme was significantly slower than that of p261N when editing both matched and mismatched DNA substrates. This suggests that the presence of p261C and the three small subunits regulates the 3′→5′ exonuclease activity of the hPole holoenzyme. Together, the 3′→5′ exonuclease activity and the variable mismatch extension activity modulate the overall fidelity of the hPole holoenzyme by up to 3 orders of magnitude. Thus, the presence of p261C and the three noncatalytic subunits optimizes the dual enzymatic activities of the catalytic p261 subunit and makes the hPole holoenzyme an efficient and faithful replicative DNA polymerase.

Background Phytoplasma are obligate intracellular plant-pathogenic bacteria that infect a broad range of plant species and are transmitted by different insect species. Quantitative real-time PCR (qPCR) is one of the most commonly used techniques for pathogen detection, especially for pathogens that cannot be cultivated outside their host like phytoplasma. PCR analysis requires the purification of total DNA from the sample and subsequent amplification of pathogen DNA with specific primers. The purified DNA contains mainly host DNA and only a marginal proportion is of phytoplasmal origin. Therefore, detection of phytoplasma DNA in a host DNA background must be sensitive, specific and reliable and is highly dependent on the quality and concentration of the purified DNA. DNA quality and concentration and the presence of PCR-inhibitors therefore have a direct impact on pathogen detection. Thus, it is indispensable for PCR-based diagnostic tests to validate the DNA preparation and DNA integrity before interpreting diagnostic results, especially in case that no pathogen DNA is detected. The use of an internal control allows to evaluate DNA integrity and the detection of PCR-inhibiting substances. Internal controls are generally host-specific or limited to a defined group of related species. A control suitable for the broad range of phytoplasma hosts comprising different insect and plant species is still missing. Results We developed a primer and probe combination that allows amplification of a conserved stretch of the eukaryotic 28S rDNA gene. The developed endogenous qPCR control serves as a DNA quality control and allows the analysis of different eukaryotic host species, including plants, insects, fish, fungi, mammals and human with a single primer/probe set in single- or multiplex assays. Conclusions Quality and performance control is indispensable for pathogen detection by qPCR. Several plant pathogens are transmitted by insects and have a broad range of host species. The newly developed endogenous control can be used with all so far tested eukaryotic species and since multiplexing is possible, the described primer and probe set can be easily combined with other PCR-based pathogen detection systems.

Background DNA transposons are ubiquitous components of eukaryotic genomes. Academ superfamily of DNA transposons is one of the least characterized DNA transposon superfamilies in eukaryotes. DNA transposons belonging to the Academ superfamily have been reported from various animals, one red algal species Chondrus crispus, and one fungal species Puccinia graminis. Six Academ families from P. graminis encode a helicase in addition to putative transposase, while some other families encode a single protein which contains a putative transposase and an XPG nuclease. Results Systematic searches on Repbase and BLAST searches against publicly available genome sequences revealed that several species of fungi and animals contain multiple Academ transposon families encoding a helicase. These AcademH families generate 9 or 10-bp target site duplications (TSDs) while Academ families lacking helicase generate 3 or 4-bp TSDs. Phylogenetic analysis clearly shows two lineages inside of Academ, designated here as AcademH and AcademX for encoding helicase or XPG nuclease, respectively. One sublineage of AcademH in animals encodes plant homeodomain (PHD) finger in its transposase, and its remnants are found in several fish genomes. Conclusions The AcademH lineage of TEs is widely distributed in animals and fungi, and originated early in the evolution of Academ DNA transposons. This analysis highlights the structural diversity in one less studied superfamily of eukaryotic DNA transposons.

Sixteen eukaryotic DNA polymerases have been identified and studied so far. Based on the sequence similarity of the catalytic subunits of DNA polymerases, these have been classified into four A, B, X and Y families except PrimPol, which belongs to the AEP family. The quaternary structure of these polymerases also varies depending upon whether they are composed of one or more subunits. Therefore, in this review, we used a quaternary structure-based classification approach to group DNA polymerases as either monomeric or multimeric and highlighted functional significance of their accessory subunits. Additionally, we have briefly summarized various DNA polymerase discoveries from a historical perspective, emphasized unique catalytic mechanism of each DNA polymerase and highlighted recent advances in understanding their cellular functions.

This review discusses recent advances in single-particle cryo-EM and single-molecule approaches used to visualise eukaryotic DNA replication reactions reconstituted in vitro. We comment on the new challenges facing structural biologists, as they turn to describing the dynamic cascade of events that lead to replication origin activation and fork progression.

periodicities within the genomic sequences play an important role in understanding the structural and functional behavior of DNA signals. The three-base periodicity (TBP) inherent to protein-coding regions is used as a discriminative feature for locating exons in eukaryotic DNA sequences. DSP tools such as Short time discrete Fourier transform (STDFT) and anti-notch filter (ANF) based techniques exploit three base periodicity property in spectral domain to distinguish exons and introns within a gene. In the last two decades, several other approaches have been evolved that are also based on this particular feature. However, the performance of these techniques is still not satisfactory. In order to enhance the prediction performance of these techniques, it is important to understand the underlying concepts of tree base periodicity and its impact on exon prediction. In this work, rather than proposing any new feature extraction technique, we have discussed the concept of the 3-base periodicity and analyzed its impact on DSP based exon prediction techniques. It is obtained that the period-3 feature is less prominent in the short length exons and therefore it is particularly difficult to predict. Moreover, the comparative study of STDFT and ANF technique is also presented.

About three-quarters of eukaryotic DNA is wrapped into nucleosomes; DNA spools with a protein core. The affinity of a given DNA stretch to be incorporated into a nucleosome is known to depend on the base-pair sequence-dependent geometry and elasticity of the DNA double helix. This causes the rotational and translational positioning of nucleosomes. In this study we ask the question whether the latter can be predicted by a simple coarse-grained DNA model with sequence-dependent elasticity, the rigid base-pair model. Whereas this model is known to be rather robust in predicting rotational nucleosome positioning, we show that the translational positioning is a rather subtle effect that is dominated by the guanine-cytosine content dependence of entropy rather than energy. A correct qualitative prediction within the rigid base-pair framework can only be achieved by assuming that DNA elasticity effectively changes on complexation into the nucleosome complex. With that extra assumption we arrive at a model which gives an excellent quantitative agreement to experimental in vitro nucleosome maps, under the additional assumption that nucleosomes equilibrate their positions only locally.

DNA synthesis is required by the cell for the processes of replication, repair, and lesion bypass. The present chapter looks into the structural-functional features of the molecular machinery, in the form of an enzyme complex called DNA polymerase, involved in DNA synthesis. In spite of their structural diversity, the DNA polymerases belonging to different families share a common architectural framework and adopt a common strategy for nucleotide incorporation. The structural basis for interactions between the DNA, the polymerase, and nucleotides has been elucidated. Special attention has been given to the initiation step where the polymerase is aided by the replicator and initiator. Furthermore, the example of Escherichia coli replicase has been elaborated to appreciate the remarkable processivity and fidelity of the DNA synthesizing machinery. Finally, the chapter explains how, in contrast to bacterial DNA replication where a single DNA polymerase is at work, eukaryotic DNA replication is undertaken by the coordinated action of three different polymerases.

International audience; Within the cell nucleus, the organization of the eukaryotic DNA into chromatin uses histones as components of its building block, the nucleosome. This chromatin organization contributes to the regulation of all DNA template-based reactions impacting genome function, stability and plasticity. Histones and their variants endow chromatin with unique properties and show a distinct distribution into the genome that is regulated by dedicated deposition machineries. The histone variants have important roles during early development, cell differentiation and chromosome segregation. Recent progress has also shed light on how mutations and transcriptional deregulation of these variants participate in tumorigenesis. In this chapter we introduce the organization of the genome in chromatin with a focus on the basic unit, the nucleosome, which contains histones as the major protein component. Then we review our current knowledge on the histone H3 family and its variants-in particular H3.3 and CenH3 CENP-Afocusing on their deposition pathways and their dedicated histone chaperones that are key players in histone dynamics.; Dans le noyau de la cellule, l'organisation de l'ADN eucaryote en chromatine utilise les histones comme composants de son élément constitutif, le nucléosome. Cette organisation de la chromatine contribue à la régulation de toutes les réactions basées sur les modèles d'ADN qui ont un impact sur la fonction, la stabilité et la plasticité du génome. Les histones et leurs variantes confèrent à la chromatine des propriétés uniques et présentent une distribution distincte dans le génome qui est régulée par des mécanismes de dépôt dédiés. Les variants d'histones jouent un rôle important dans le développement précoce, la différenciation des cellules et la ségrégation des chromosomes. Les progrès récents ont également mis en lumière la manière dont les mutations et la dérégulation transcriptionnelle de ces variants participent à la tumorigenèse. Dans ce chapitre, nous présentons l'organisation du génome dans la chromatine en nous concentrant sur l'unité de base, le nucléosome, qui contient des histones comme principal composant protéique. Ensuite, nous passons en revue nos connaissances actuelles sur la famille des histones H3 et ses variants - en particulier H3.3 et CenH3CENP-A - en nous concentrant sur leurs voies de dépôt et leurs chaperons d'histones dédiés qui sont des acteurs clés dans la dynamique des histones.

Eukaryotic DNA is packaged with histones and nonhistone proteins to form a heterogeneous protein-DNA complex called chromatin. Regulation of chromatin structure plays a central role in nearly all DNA-based processes in the nucleus—including transcription, DNA replication, DNA repair, and chromosome segregation. Regulation of chromatin structure is achieved through multiple interrelated mechanisms including modification of DNA and histones, binding of nonhistone proteins, ATP-dependent nucleosome remodeling, and higher-order compaction and folding of the chromatin fiber. These mechanisms work in concert to partition the genome into structurally and functionally distinct chromatin environments. Euchromatin is generally decondensed and transcriptionally active, whereas heterochromatin is highly condensed and transcriptionally silent. Work in the model fungus Neurospora crassa has been of particular importance for elucidating relationships between chromatin structure and function, serving as a model for other fungi and for eukaryotes in general. This chapter summarizes our current understanding of chromatin structure and function in N. crassa.