SLFN11 Blocks Stressed Replication Forks Independently of ATR.

SLFN11 sensitizes cancer cells to a broad range of DNA-targeted therapies. Here we show that, in response to replication stress induced by camptothecin, SLFN11 tightly binds chromatin at stressed replication foci via RPA1 together with the replication helicase subunit MCM3. Unlike ATR, SLFN11 neither interferes with the loading of CDC45 and PCNA nor inhibits the initiation of DNA replication but selectively blocks fork progression while inducing chromatin opening across replication initiation sites. The ATPase domain of SLFN11 is required for chromatin opening, replication block, and cell death but not for the tight binding of SLFN11 to chromatin. Replication stress by the CHK1 inhibitor Prexasertib also recruits SLFN11 to nascent replicating DNA together with CDC45 and PCNA. We conclude that SLFN11 is recruited to stressed replication forks carrying extended RPA filaments where it blocks replication by changing chromatin structure across replication sites.

Mechanism of Bidirectional Leading-Strand Synthesis Establishment at Eukaryotic DNA Replication Origins.

DNA replication commences at eukaryotic replication origins following assembly and activation of bidirectional CMG helicases. Once activated, CMG unwinds the parental DNA duplex and DNA polymerase α-primase initiates synthesis on both template strands. By utilizing an origin-dependent replication system using purified yeast proteins, we have mapped start sites for leading-strand replication. Synthesis is mostly initiated outside the origin sequence. Strikingly, rightward leading strands are primed left of the origin and vice versa. We show that each leading strand is established from a lagging-strand primer synthesized by the replisome on the opposite side of the origin. Preventing elongation of primers synthesized left of the origin blocked rightward leading strands, demonstrating that replisomes are interdependent for leading-strand synthesis establishment. The mechanism we reveal negates the need for dedicated leading-strand priming and necessitates a crucial role for the lagging-strand polymerase Pol δ in connecting the nascent leading strand with the advancing replisome.

How bacteria initiate DNA replication comes into focus.

The ability to initiate DNA replication is a critical step in the proliferation of all organisms. In bacteria, this process is mediated by an ATP-dependent replication initiator protein, DnaA, which recognizes and melts replication origin (oriC) elements. Despite decades of biochemical and structural work, a mechanistic understanding of how DnaA recognizes and unwinds oriC has remained enigmatic. A recent study by Pelliciari et al. provides important new structural insights into how DnaA from Bacillus subtilis recognizes and processes its cognate oriC, showing how DnaA uses sequence features encoded in the origin to engage melted DNA. Comparison of the DnaA-oriC structure with archaeal/eukaryl replication origin complexes based on Orc-family proteins reveals a high degree of similarity in origin engagement by initiators from di domains of life, despite fundamental differences in origin melting mechanisms. These findings provide valuable insights into bacterial replication initiation and highlight the intriguing evolutionary history of this fundamental biological process.

Chromatin Constrains the Initiation and Elongation of DNA Replication.

Eukaryotic chromosomal DNA is faithfully replicated in a complex series of cell-cycle-regulated events that are incompletely understood. Here we report the reconstitution of DNA replication free in solution with purified proteins from the budding yeast Saccharomyces cerevisiae. The system recapitulates regulated bidirectional origin activation; synthesis of leading and lagging strands by the three replicative DNA polymerases Pol α, Pol δ, and Pol ε; and canonical maturation of Okazaki fragments into continuous daughter strands. We uncover a dual regulatory role for chromatin during DNA replication: promoting origin dependence and determining Okazaki fragment length by restricting Pol δ progression. This system thus provides a functional platform for the detailed mechanistic analysis of eukaryotic chromosome replication.

Rpd3 regulates single-copy origins independently of the rDNA array by opposing Fkh1-mediated origin stimulation.

Eukaryotic chromosomes are organized into structural and functional domains with characteristic replication timings, which are thought to contribute to epigenetic programming and genome stability. Differential replication timing results from epigenetic mechanisms that positively and negatively regulate the competition for limiting replication initiation factors. Histone deacetylase Sir2 negatively regulates initiation of the multicopy (∼150) rDNA origins, while Rpd3 histone deacetylase negatively regulates firing of single-copy origins. However, Rpd3's effect on single-copy origins might derive indirectly from a positive function for Rpd3 in rDNA origin firing shifting the competitive balance. Our quantitative experiments support the idea that origins compete for limiting factors; however, our results show that Rpd3's effect on single-copy origin is independent of rDNA copy-number and of Sir2's effects on rDNA origin firing. Whereas RPD3 deletion and SIR2 deletion alter the early S phase dynamics of single-copy and rDNA origin firings in opposite fashion, unexpectedly only RPD3 deletion suppresses overall rDNA origin efficiency across S phase. Increased origin activation in rpd3Δ requires Fkh1/2, suggesting that Rpd3 opposes Fkh1/2-origin stimulation, which involves recruitment of Dbf4-dependent kinase (DDK). Indeed, Fkh1 binding increases at Rpd3-regulated origins in rpd3Δ cells in G1, supporting a mechanism whereby Rpd3 influences initiation timing of single-copy origins directly through modulation of Fkh1-origin binding. Genetic suppression of a DBF4 hypomorphic mutation by RPD3 deletion further supports the conclusion that Rpd3 impedes DDK recruitment by Fkh1, revealing a mechanism of Rpd3 in origin regulation.

Interactions between Fkh1 monomers stabilize its binding to DNA replication origins.

Eukaryotic DNA replication is initiated from multiple genomic origins, which can be broadly categorized as firing early or late in the S phase. Several factors can influence the temporal usage of origins to determine the timing of their firing. In budding yeast, the Forkhead family proteins Fkh1 and Fkh2 bind to a subset of replication origins and activate them at the beginning of the S phase. In these origins, the Fkh1/2 binding sites are arranged in a strict configuration, suggesting that Forkhead factors must bind the origins in a specific manner. To explore these binding mechanisms in more detail, we mapped the domains of Fkh1 that were required for its role in DNA replication regulation. We found that a short region of Fkh1 near its DNA binding domain was essential for the protein to bind and activate replication origins. Analysis of purified Fkh1 proteins revealed that this region mediates dimerization of Fkh1, suggesting that intramolecular contacts of Fkh1 are required for efficient binding and regulation of DNA replication origins. We also show that the Sld3-Sld7-Cdc45 complex is recruited to Forkhead-regulated origins already in the G1 phase and that Fkh1 is constantly required to keep these factors bound on origins before the onset of the S phase. Together, our results suggest that dimerization-mediated stabilization of DNA binding by Fkh1 is crucial for its ability to activate DNA replication origins.

Clustering of strong replicators associated with active promoters is sufficient to establish an early-replicating domain.

Vertebrate genomes replicate according to a precise temporal program strongly correlated with their organization into A/B compartments. Until now, the molecular mechanisms underlying the establishment of early-replicating domains remain largely unknown. We defined two minimal cis-element modules containing a strong replication origin and chromatin modifier binding sites capable of shifting a targeted mid-late-replicating region for earlier replication. The two origins overlap with a constitutive or a silent tissue-specific promoter. When inserted side-by-side, these modules advance replication timing over a 250 kb region through the cooperation with one endogenous origin located 30 kb away. Moreover, when inserted at two chromosomal sites separated by 30 kb, these two modules come into close physical proximity and form an early-replicating domain establishing more contacts with active A compartments. The synergy depends on the presence of the active promoter/origin. Our results show that clustering of strong origins located at active promoters can establish early-replicating domains.

DNA replication timing: Biochemical mechanisms and biological significance.

The regulation of DNA replication is a fascinating biological problem both from a mechanistic angle-How is replication timing regulated?-and from an evolutionary one-Why is replication timing regulated? Recent work has provided significant insight into the first question. Detailed biochemical understanding of the mechanism and regulation of replication initiation has made possible robust hypotheses for how replication timing is regulated. Moreover, technical progress, including high-throughput, single-molecule mapping of replication initiation and single-cell assays of replication timing, has allowed for direct testing of these hypotheses in mammalian cells. This work has consolidated the conclusion that differential replication timing is a consequence of the varying probability of replication origin initiation. The second question is more difficult to directly address experimentally. Nonetheless, plausible hypotheses can be made and one-that replication timing contributes to the regulation of chromatin structure-has received new experimental support.

Computational prediction of species-specific yeast DNA replication origin via iterative feature representation.

Deoxyribonucleic acid replication is one of the most crucial tasks taking place in the cell, and it has to be precisely regulated. This process is initiated in the replication origins (ORIs), and thus it is essential to identify such sites for a deeper understanding of the cellular processes and functions related to the regulation of gene expression. Considering the important tasks performed by ORIs, several experimental and computational approaches have been developed in the prediction of such sites. However, existing computational predictors for ORIs have certain curbs, such as building only single-feature encoding models, limited systematic feature engineering efforts and failure to validate model robustness. Hence, we developed a novel species-specific yeast predictor called yORIpred that accurately identify ORIs in the yeast genomes. To develop yORIpred, we first constructed optimal 40 baseline models by exploring eight different sequence-based encodings and five different machine learning classifiers. Subsequently, the predicted probability of 40 models was considered as the novel feature vector and carried out iterative feature learning approach independently using five different classifiers. Our systematic analysis revealed that the feature representation learned by the support vector machine algorithm (yORIpred) could well discriminate the distribution characteristics between ORIs and non-ORIs when compared with the other four algorithms. Comprehensive benchmarking experiments showed that yORIpred achieved superior and stable performance when compared with the existing predictors on the same training datasets. Furthermore, independent evaluation showcased the best and accurate performance of yORIpred thus underscoring the significance of iterative feature representation. To facilitate the users in obtaining their desired results without undergoing any mathematical, statistical or computational hassles, we developed a web server for the yORIpred predictor, which is available at: http://thegleelab.org/yORIpred.

DeepYY1: a deep learning approach to identify YY1-mediated chromatin loops.

The protein Yin Yang 1 (YY1) could form dimers that facilitate the interaction between active enhancers and promoter-proximal elements. YY1-mediated enhancer-promoter interaction is the general feature of mammalian gene control. Recently, some computational methods have been developed to characterize the interactions between DNA elements by elucidating important features of chromatin folding; however, no computational methods have been developed for identifying the YY1-mediated chromatin loops. In this study, we developed a deep learning algorithm named DeepYY1 based on word2vec to determine whether a pair of YY1 motifs would form a loop. The proposed models showed a high prediction performance (AUCs$\ge$0.93) on both training datasets and testing datasets in different cell types, demonstrating that DeepYY1 has an excellent performance in the identification of the YY1-mediated chromatin loops. Our study also suggested that sequences play an important role in the formation of YY1-mediated chromatin loops. Furthermore, we briefly discussed the distribution of the replication origin site in the loops. Finally, a user-friendly web server was established, and it can be freely accessed at http://lin-group.cn/server/DeepYY1.

Promoters are key organizers of the duplication of vertebrate genomes.

In vertebrates, single cell analyses of replication timing patterns brought to light a very well controlled program suggesting a tight regulation on initiation sites. Mapping of replication origins with different methods has revealed discrete preferential sites, enriched in promoters and potential G-quadruplex motifs, which can aggregate into initiation zones spanning several tens of kilobases (kb). Another characteristic of replication origins is a nucleosome-free region (NFR). A modified yeast strain containing a humanized origin recognition complex (ORC) fires new origins at NFRs revealing their regulatory role. In cooperation with NFRs, the histone variant H2A.Z facilitates ORC loading through di-methylation of lysine 20 of histone H4. Recent studies using genome editing methods show that efficient initiation sites associated with transcriptional activity can synergize over several tens of kb by establishing physical contacts and lead to the formation of early domains of DNA replication demonstrating a co-regulation between replication initiation and transcription.

Genome-Wide Profiling of Endogenous Single-Stranded DNA Using the SSiNGLe-P1 Method.

Endogenous single-stranded DNA (essDNA) can form in a mammalian genome as the result of a variety of molecular processes and can both play important roles inside the cell as well as have detrimental consequences to genome integrity, much of which remains to be fully understood. Here, we established the SSiNGLe-P1 approach based on limited digestion by P1 endonuclease for high-throughput genome-wide identification of essDNA regions. We applied this method to profile essDNA in both human mitochondrial and nuclear genomes. In the mitochondrial genome, the profiles of essDNA provide new evidence to support the strand-displacement model of mitochondrial DNA replication. In the nuclear genome, essDNA regions were found to be enriched in certain types of functional genomic elements, particularly, the origins of DNA replication, R-loops, and to a lesser degree, in promoters. Furthermore, interestingly, many of the essDNA regions identified by SSiNGLe-P1 have not been annotated and thus could represent yet unknown functional elements.

Anatomy and evolution of a DNA replication origin.

DNA amplification occurs at the DNA puff II/9A locus in the fungus fly Sciara coprophila. As a foundation to study the molecular mechanism for the initiating events of II/9A DNA re-replication, we have sequenced 14 kb spanning a DNase hypersensitive site (DHS) upstream of the 1 kb amplification origin and through transcription units II/9-1 and II/9-2 downstream of the origin. These elements are annotated as well as the ORC binding site at the origin and the transition point (TP) between continuous and discontinuous DNA syntheses that marks the origin of bidirectional replication at the nucleotide level. A 9 bp motif found at the TP is repeated near the other end of the 1 kb ORI and may identify a putative second TP. The steroid hormone ecdysone induces DNA amplification as well as transcription and puffing at locus II/9A. Within the 14 kb, several matches to the ecdysone response element (EcRE) consensus sequence were identified, including some in the amplification origin region. EcRE O-P is at a central axis of a remarkable symmetry, equidistant to the TPs that are themselves equidistant to EcRE O-1 and EcRE O-2. DNA sequence alterations have occurred throughout the II/9A region in a newly discovered polymorphism (#2). Polymorphism #2 is not specific to developmental stage, sex, or tissue, and it does not impair DNA amplification. The DHS, both 9 bp TP sequences, and EcREs O-1, O-P, and O-2 are conserved between the polymorphism #1 and #2 sequences, suggesting their functional importance and retention during evolutionary selection. Moreover, a 72 bp sequence in the Sciara DHS at DNA puff II/9A is conserved in DNA puff C-3 of Rhynchosciara americana. Comparisons are discussed between the Sciara II/9A amplicon and the chorion locus amplicon on the third chromosome of Drosophila.

The yeast Dbf4 Zn2+ finger domain suppresses single-stranded DNA at replication forks initiated from a subset of origins.

Dbf4 is the cyclin-like subunit for the Dbf4-dependent protein kinase (DDK), required for activating the replicative helicase at DNA replication origin that fire during S phase. Dbf4 also functions as an adaptor, targeting the DDK to different groups of origins and substrates. Here we report a genome-wide analysis of origin firing in a budding yeast mutant, dbf4-zn, lacking the Zn2+ finger domain within the C-terminus of Dbf4. At one group of origins, which we call dromedaries, we observe an unanticipated DNA replication phenotype: accumulation of single-stranded DNA spanning ± 5kbp from the center of the origins. A similar accumulation of single-stranded DNA at origins occurs more globally in pri1-m4 mutants defective for the catalytic subunit of DNA primase and rad53 mutants defective for the S phase checkpoint following DNA replication stress. We propose the Dbf4 Zn2+ finger suppresses single-stranded gaps at replication forks emanating from dromedary origins. Certain origins may impose an elevated requirement for the DDK to fully initiate DNA synthesis following origin activation. Alternatively, dbf4-zn may be defective for stabilizing/restarting replication forks emanating from dromedary origins during replication stress.

Yeast Stn1 promotes MCM to circumvent Rad53 control of the S phase checkpoint.

Treating yeast cells with the replication inhibitor hydroxyurea activates the S phase checkpoint kinase Rad53, eliciting responses that block DNA replication origin firing, stabilize replication forks, and prevent premature extension of the mitotic spindle. We previously found overproduction of Stn1, a subunit of the telomere-binding Cdc13-Stn1-Ten1 complex, circumvents Rad53 checkpoint functions in hydroxyurea, inducing late origin firing and premature spindle extension even though Rad53 is activated normally. Here, we show Stn1 overproduction acts through remarkably similar pathways compared to loss of RAD53, converging on the MCM complex that initiates origin firing and forms the catalytic core of the replicative DNA helicase. First, mutations affecting Mcm2 and Mcm5 block the ability of Stn1 overproduction to disrupt the S phase checkpoint. Second, loss of function stn1 mutations compensate rad53 S phase checkpoint defects. Third Stn1 overproduction suppresses a mutation in Mcm7. Fourth, stn1 mutants accumulate single-stranded DNA at non-telomeric genome locations, imposing a requirement for post-replication DNA repair. We discuss these interactions in terms of a model in which Stn1 acts as an accessory replication factor that facilitates MCM activation at ORIs and potentially also maintains MCM activity at replication forks advancing through challenging templates.

B Cell-Specific Transcription Activator PAX5 Recruits p300 To Support EBNA1-Driven Transcription.

The binding of Epstein-Barr Virus (EBV) nuclear antigen 1 (EBNA1) to the latent replication origin (oriP) triggers multiple downstream events to support virus-induced pathogenesis and tumorigenesis. Although EBV is widely recognized as a B-lymphotropic infectious agent, little is known about how tissue-specific factors are involved in the establishment of latency. Here, we showed that EBNA1 binds B cell activator PAX5 to promote EBNA1/oriP-dependent binding and transcription. In addition to showing that short hairpin RNA (shRNA)-mediated PAX5 knockdown substantially abrogated the above EBNA1-dependent functions, two mini-EBV reporter plasmids were used to perform nonlytic nano-luciferase (nLuc) activity and chromatin immunoprecipitation (ChIP) assays to show how EBNA1 cooperates with PAX5 to activate the transcription at the oriP site. The expression plasmids of two PAX5 mutants, V26G (EBNA1 binding mutant) and P80R (which remained EBNA1 associated), were used to assess their capability to restore the defects caused by PAX5 depletion in EBNA1/oriP-mediated binding, transcription, and maintenance of the genome copy number of the mini-EBV episome reporter in BJAB cells stably expressing EBNA1 or that of the EBV genome in EBV-infected BJAB cells. Since p300 is known to be associated with PAX5, we showed that the loss of function of the P80R mutant in support of EBNA1/oriP-mediated transcription under PAX5 depletion conditions was linked to its defective binding to p300. ChIP-quantitative PCR (qPCR) confirmed that P80R indeed failed to recruit p300 to the oriP DNA. Our discovery suggests that EBV has evolved an exquisite strategy to take advantage of tissue-specific factors to enable the establishment of viral latency.IMPORTANCE Although B cells are known to be the primary target for EBV infection, there is limited knowledge regarding the mechanism that determines this preferable tissue tropism. An in-depth understanding of the potential link of tissue-specific factors with the viral genes and their functioning is key to deciphering how EBV induces persistent infection in the distinct types of host cells. In this study, a substantial protein-protein interaction mediated by the B cell-specific activator PAX5 and EBNA1 was identified as the general requirement for the binding of EBNA1 to the latent replication origin and for downstream events. Of importance, the EBNA1-PAX5-p300 network is directly linked to EBNA1-dependent transcription. These findings suggest that targeting the viral gene-associated tissue-specific factors may lead to new therapeutic strategies for EBV-associated malignancies.

Recent development of Ori-Finder system and DoriC database for microbial replication origins.

DNA replication begins at replication origins in all three domains of life. Identification and characterization of replication origins are important not only in providing insights into the structure and function of the replication origins but also in understanding the regulatory mechanisms of the initiation step in DNA replication. The Z-curve method has been used in the identification of replication origins in archaeal genomes successfully since 2002. Furthermore, the Web servers of Ori-Finder and Ori-Finder 2 have been developed to predict replication origins in both bacterial and archaeal genomes based on the Z-curve method, and the replication origins with manual curation have been collected into an online database, DoriC. Ori-Finder system and DoriC database are currently used in the research field of DNA replication origins in prokaryotes, including: (i) identification of oriC regions in bacterial and archaeal genomes; (ii) discovery and analysis of the conserved sequences within oriC regions; and (iii) strand-biased analysis of bacterial genomes. Up to now, more and more predicted results by Ori-Finder system were supported by subsequent experiments, and Ori-Finder system has been used to identify the replication origins in > 100 newly sequenced prokaryotes in their genome reports. In addition, the data in DoriC database have been widely used in the large-scale analyses of replication origins and strand bias in prokaryotic genomes. Here, we review the development of Ori-Finder system and DoriC database as well as their applications. Some future directions and aspects for extending the application of Ori-Finder and DoriC are also presented.

Genomic methods for measuring DNA replication dynamics.

Genomic DNA replicates according to a defined temporal program in which early-replicating loci are associated with open chromatin, higher gene density, and increased gene expression levels, while late-replicating loci tend to be heterochromatic and show higher rates of genomic instability. The ability to measure DNA replication dynamics at genome scale has proven crucial for understanding the mechanisms and cellular consequences of DNA replication timing. Several methods, such as quantification of nucleotide analog incorporation and DNA copy number analyses, can accurately reconstruct the genomic replication timing profiles of various species and cell types. More recent developments have expanded the DNA replication genomic toolkit to assays that directly measure the activity of replication origins, while single-cell replication timing assays are beginning to reveal a new level of replication timing regulation. The combination of these methods, applied on a genomic scale and in multiple biological systems, promises to resolve many open questions and lead to a holistic understanding of how eukaryotic cells replicate their genomes accurately and efficiently.

Molecular mechanisms of eukaryotic origin initiation, replication fork progression, and chromatin maintenance.

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.

Impaired replication elongation in Tetrahymena mutants deficient in histone H3 Lys 27 monomethylation.

Replication of nuclear DNA occurs in the context of chromatin and is influenced by histone modifications. In the ciliate Tetrahymena thermophila, we identified TXR1, encoding a histone methyltransferase. TXR1 deletion resulted in severe DNA replication stress, manifested by the accumulation of ssDNA, production of aberrant replication intermediates, and activation of robust DNA damage responses. Paired-end Illumina sequencing of ssDNA revealed intergenic regions, including replication origins, as hot spots for replication stress in ΔTXR1 cells. ΔTXR1 cells showed a deficiency in histone H3 Lys 27 monomethylation (H3K27me1), while ΔEZL2 cells, deleting a Drosophila E(z) homolog, were deficient in H3K27 di- and trimethylation, with no detectable replication stress. A point mutation in histone H3 at Lys 27 (H3 K27Q) mirrored the phenotype of ΔTXR1, corroborating H3K27me1 as a key player in DNA replication. Additionally, we demonstrated interactions between TXR1 and proliferating cell nuclear antigen (PCNA). These findings support a conserved pathway through which H3K27me1 facilitates replication elongation.