Replication Fork Pause Research Articles

TOF1 and RRM3 reveal a link between gene silencing and the pausing of replication forks.

Eukaryotic DNA replication is accompanied by the disassembly and reassembly of nucleosomes and the transmission of epigenetic marks to the newly assembled chromatids. Several histone chaperones, including CAF-1 and Asf1p, are central to these processes. On the other hand, replication forks pause at numerous positions throughout the genome, but it is not known if and how this pausing affects the reassembly and maintenance of chromatin structures. Here, we applied drug-free gene silencing assays to analyze the genetic interactions between CAC1, ASF1, and two genes that regulate the stability of the paused replisome (TOF1) and the resumption of elongation (RRM3). Our results show that TOF1 and RRM3 differentially interact with CAF-1 and ASF1 and that the deletions of TOF1 and RRM3 lead to reduced silencing and increased frequency of epigenetic conversions at three loci in the genome of S. cerevisiae. Our study adds details to the known activities of CAF-1 and Asf1p and suggests that the pausing of the replication fork can lead to epigenetic instability.

The in vivo measurement of replication fork velocity and pausing by lag-time analysis

An important step towards understanding the mechanistic basis of the central dogma is the quantitative characterization of the dynamics of nucleic-acid-bound molecular motors in the context of the living cell. To capture these dynamics, we develop lag-time analysis, a method for measuring in vivo dynamics. Using this approach, we provide quantitative locus-specific measurements of fork velocity, in units of kilobases per second, as well as replisome pause durations, some with the precision of seconds. The measured fork velocity is observed to be both locus and time dependent, even in wild-type cells. In this work, we quantitatively characterize known phenomena, detect brief, locus-specific pauses at ribosomal DNA loci in wild-type cells, and observe temporal fork velocity oscillations in three highly-divergent bacterial species.

Cryo-EM Structure of the Fork Protection Complex Bound to CMG at a Replication Fork

SummaryThe eukaryotic replisome, organized around the Cdc45-MCM-GINS (CMG) helicase, orchestrates chromosome replication. Multiple factors associate directly with CMG, including Ctf4 and the heterotrimeric fork protection complex (Csm3/Tof1 and Mrc1), which has important roles including aiding normal replication rates and stabilizing stalled forks. How these proteins interface with CMG to execute these functions is poorly understood. Here we present 3 to 3.5 Å resolution electron cryomicroscopy (cryo-EM) structures comprising CMG, Ctf4, and the fork protection complex at a replication fork. The structures provide high-resolution views of CMG-DNA interactions, revealing a mechanism for strand separation, and show Csm3/Tof1 “grip” duplex DNA ahead of CMG via a network of interactions important for efficient replication fork pausing. Although Mrc1 was not resolved in our structures, we determine its topology in the replisome by cross-linking mass spectrometry. Collectively, our work reveals how four highly conserved replisome components collaborate with CMG to facilitate replisome progression and maintain genome stability.

Too Much of a Good Thing: How Ectopic DNA Replication Affects Bacterial Replication Dynamics.

Each cell division requires the complete and accurate duplication of the entire genome. In bacteria, the duplication process of the often-circular chromosomes is initiated at a single origin per chromosome, resulting in two replication forks that traverse the chromosome in opposite directions. DNA synthesis is completed once the two forks fuse in a region diametrically opposite the origin. In some bacteria, such as Escherichia coli, the region where forks fuse forms a specialized termination area. Polar replication fork pause sites flanking this area can pause the progression of replication forks, thereby allowing forks to enter but not to leave. Transcription of all required genes has to take place simultaneously with genome duplication. As both of these genome trafficking processes share the same template, conflicts are unavoidable. In this review, we focus on recent attempts to add additional origins into various ectopic chromosomal locations of the E. coli chromosome. As ectopic origins disturb the native replichore arrangements, the problems resulting from such perturbations can give important insights into how genome trafficking processes are coordinated and the problems that arise if this coordination is disturbed. The data from these studies highlight that head-on replication–transcription conflicts are indeed highly problematic and multiple repair pathways are required to restart replication forks arrested at obstacles. In addition, the existing data also demonstrate that the replication fork trap in E. coli imposes significant constraints to genome duplication if ectopic origins are active. We describe the current models of how replication fork fusion events can cause serious problems for genome duplication, as well as models of how such problems might be alleviated both by a number of repair pathways as well as the replication fork trap system. Considering the problems associated both with head-on replication-transcription conflicts as well as head-on replication fork fusion events might provide clues of how these genome trafficking issues have contributed to shape the distinct architecture of bacterial chromosomes.

Replication fork pausing at protein barriers on chromosomes.

When a cell divides prior to completion of DNA replication, serious DNA damage may occur. Thus, in addition to accuracy, the processivity of the replication forks is important. DNA synthesis at replication forks should be completed in time, and forks overcome aberrant structures on the template DNA, including damaged sites, using trans-lesion synthesis, occasionally introducing mutations. By contrast, the protein barrier built on the DNA is known to block the progression of replication forks at specific chromosomal loci. Such protein barriers avert any collision of replication and transcription machineries, or control the recombination of specific loci. The components and the mechanisms of action of protein barriers have been revealed mainly using genetic and biochemical techniques. In addition to proteins involved in replication fork pausing, the interaction of the replicative helicase and DNA polymerase is also essential for replication fork pausing. Here, we provide an overview of replication fork pausing at protein barriers.

Two Pif1 Family DNA Helicases Cooperate in Centromere Replication and Segregation in Saccharomyces cerevisiae.

Pif1 family helicases are found in virtually all eukaryotes. Saccharomyces cerevisiae (Sc) encodes two Pif1 family helicases, ScPif1 and Rrm3. ScPif1 is multifunctional, required not only for maintenance of mitochondrial DNA but also for multiple distinct nuclear functions. Rrm3 moves with the replication fork and promotes movement of the fork through ∼1400 hard-to-replicate sites, including centromeres. Here we show that ScPif1, like Rrm3, bound robustly to yeast centromeres but only if the centromere was active. While Rrm3 binding to centromeres occurred in early to mid S phase, about the same time as centromere replication, ScPif1 binding occurred later in the cell cycle when replication of most centromeres is complete. However, the timing of Rrm3 and ScPif1 centromere binding was altered by the absence of the other helicase, such that Rrm3 centromere binding occurred later in pif1-m2 cells and ScPif1 centromere binding occurred earlier in rrm3Δ cells. As shown previously, the modest pausing of replication forks at centromeres seen in wild-type cells was increased in the absence of Rrm3. While a lack of ScPif1 did not result in increased fork pausing at centromeres, pausing was even higher in rrm3Δ pif1Δ cells than in rrm3Δ cells. Likewise, centromere function as monitored by the loss rate of a centromere plasmid was increased in rrm3Δ but not pif1Δ cells, and was even higher in rrm3Δ pif1Δ cells than in rrm3Δ cells. Thus, ScPif1 promotes centromere replication and segregation, but only in the absence of Rrm3. These data also hint at a potential post-S phase function for ScPif1 at centromeres. These studies add to the growing list of ScPif1 functions that promote chromosome stability.

DNA polymerase ε-dependent modulation of the pausing property of the CMG helicase at the barrier.

The proper pausing of replication forks at barriers on chromosomes is important for genome integrity. However, the detailed mechanism underlying this process has not been well elucidated. Here, we successfully reconstituted fork-pausing reactions from purified yeast proteins on templates that had binding sites for the LacI, LexA, and/or Fob1 proteins; the forks paused specifically at the protein-bound sites. Moreover, although the replicative helicase Cdc45-Mcm2-7-GINS (CMG) complex alone unwound the protein-bound templates, the unwinding of the LacI-bound site was impeded by the presence of a main leading strand DNA polymerase: polymerase ε (Polε). This suggests that Polε modulates CMG to pause at these sites.

Origins Left, Right, and Centre: Increasing the Number of Initiation Sites in the Escherichia coli Chromosome.

The bacterium Escherichia coli contains a single circular chromosome with a defined architecture. DNA replication initiates at a single origin called oriC. Two replication forks are assembled and proceed in opposite directions until they fuse in a specialised zone opposite the origin. This termination area is flanked by polar replication fork pause sites that allow forks to enter, but not to leave. Thus, the chromosome is divided into two replichores, each replicated by a single replication fork. Recently, we analysed the replication parameters in E. coli cells, in which an ectopic origin termed oriZ was integrated in the right-hand replichore. Two major obstacles to replication were identified: (1) head-on replication–transcription conflicts at highly transcribed rrn operons, and (2) the replication fork trap. Here, we describe replication parameters in cells with ectopic origins, termed oriX and oriY, integrated into the left-hand replichore, and a triple origin construct with oriX integrated in the left-hand and oriZ in the right-hand replichore. Our data again highlight both replication–transcription conflicts and the replication fork trap as important obstacles to DNA replication, and we describe a number of spontaneous large genomic rearrangements which successfully alleviate some of the problems arising from having an additional origin in an ectopic location. However, our data reveal additional factors that impact efficient chromosome duplication, highlighting the complexity of chromosomal architecture.

Molecular signature of the imprintosome complex at the mating-type locus in fission yeast.

Genetic and molecular studies have indicated that an epigenetic imprint at mat1, the sexual locus of fission yeast, initiates mating type switching. The polar DNA replication of mat1 generates an imprint on the Watson strand. The process by which the imprint is formed and maintained through the cell cycle remains unclear. To understand better the mechanism of imprint formation and stability, we characterized the recruitment of early players of mating type switching at the mat1 region. We found that the switch activating protein 1 (Sap1) is preferentially recruited inside the mat1M allele on a sequence (SS13) that enhances the imprint. The lysine specific demethylases, Lsd1/2, that control the replication fork pause at MPS1 and the formation of the imprint are specifically drafted inside of mat1, regardless of the allele. The CENP-B homolog, Abp1, is highly enriched next to mat1 but it is not required in the process. Additionally, we established the computational signature of the imprint. Using this signature, we show that both sides of the imprinted molecule are bound by Lsd1/2 and Sap1, suggesting a nucleoprotein protective structure defined as imprintosome.

Ribosomal DNA stability is supported by many 'buffer genes'-introduction to the Yeast rDNA Stability Database.

The ribosomal RNA gene (rDNA) is the most abundant gene in yeast and other eukaryotic organisms. Due to its heavy transcription, repetitive structure and programmed replication fork pauses, the rDNA is one of the most unstable regions in the genome. Thus, the rDNA is the best region to study the mechanisms responsible for maintaining genome integrity. Recently, we screened a library of ∼4800 budding yeast gene knockout strains to identify mutants defective in the maintenance of rDNA stability. The results of this screen are summarized in the Yeast rDNA Stability (YRS) Database, in which the stability and copy number of rDNA in each mutant are presented. From this screen, we identified ∼700 genes that may contribute to the maintenance of rDNA stability. In addition, ∼50 mutants had abnormally high or low rDNA copy numbers. Moreover, some mutants with unstable rDNA displayed abnormalities in another chromosome. In this review, we introduce the YRS Database and discuss the roles of newly identified genes that contribute to rDNA maintenance and genome integrity.

Replication Termination: Containing Fork Fusion-Mediated Pathologies in Escherichia coli.

Duplication of bacterial chromosomes is initiated via the assembly of two replication forks at a single defined origin. Forks proceed bi-directionally until they fuse in a specialised termination area opposite the origin. This area is flanked by polar replication fork pause sites that allow forks to enter but not to leave. The precise function of this replication fork trap has remained enigmatic, as no obvious phenotypes have been associated with its inactivation. However, the fork trap becomes a serious problem to cells if the second fork is stalled at an impediment, as replication cannot be completed, suggesting that a significant evolutionary advantage for maintaining this chromosomal arrangement must exist. Recently, we demonstrated that head-on fusion of replication forks can trigger over-replication of the chromosome. This over-replication is normally prevented by a number of proteins including RecG helicase and 3’ exonucleases. However, even in the absence of these proteins it can be safely contained within the replication fork trap, highlighting that multiple systems might be involved in coordinating replication fork fusions. Here, we discuss whether considering the problems associated with head-on replication fork fusion events helps us to better understand the important role of the replication fork trap in cellular metabolism.

Replication stalling and heteroduplex formation within CAG/CTG trinucleotide repeats by mismatch repair.

Trinucleotide repeat expansions are responsible for at least two dozen neurological disorders. Mechanisms leading to these large expansions of repeated DNA are still poorly understood. It was proposed that transient stalling of the replication fork by the repeat tract might trigger slippage of the newly-synthesized strand over its template, leading to expansions or contractions of the triplet repeat. However, such mechanism was never formally proven. Here we show that replication fork pausing and CAG/CTG trinucleotide repeat instability are not linked, stable and unstable repeats exhibiting the same propensity to stall replication forks when integrated in a yeast natural chromosome. We found that replication fork stalling was dependent on the integrity of the mismatch-repair system, especially the Msh2p-Msh6p complex, suggesting that direct interaction of MMR proteins with secondary structures formed by trinucleotide repeats in vivo, triggers replication fork pauses. We also show by chromatin immunoprecipitation that Msh2p is enriched at trinucleotide repeat tracts, in both stable and unstable orientations, this enrichment being dependent on MSH3 and MSH6. Finally, we show that overexpressing MSH2 favors the formation of heteroduplex regions, leading to an increase in contractions and expansions of CAG/CTG repeat tracts during replication, these heteroduplexes being dependent on both MSH3 and MSH6. These heteroduplex regions were not detected when a mutant msh2-E768A gene in which the ATPase domain was mutated was overexpressed. Our results unravel two new roles for mismatch-repair proteins: stabilization of heteroduplex regions and transient blocking of replication forks passing through such repeats. Both roles may involve direct interactions between MMR proteins and secondary structures formed by trinucleotide repeat tracts, although indirect interactions may not be formally excluded.

Genome-wide mapping of yeast histone chaperone anti-silencing function 1 reveals its role in condensin binding with chromatin.

Genome-wide participation and importance of the histone chaperone Asf1 (Anti-Silencing Function 1) in diverse DNA transactions like replication, repair, heterochromatic silencing and transcription are well documented. Yet its genome-wide targets have not been reported. Using ChIP-seq method, we found that yeast Asf1 associates with 590 unique targets including centromeres, telomeres and condensin-binding sites. It is found selectively on highly transcribed regions, which include replication fork pause sites. Asf1 preferentially associates with the genes transcribed by RNA polymerase (pol) III where its presence affects RNA production and replication-independent histone exchange. On pol II-transcribed genes, a negative correlation is found between Asf1 and nucleosome occupancy. It is not enriched on most of the reported sites of histone exchange or on the genes, which are misregulated in the asf1Δ cells. Interestingly, chromosome-wide distributions of Asf1 and one of the condensin subunits, Brn1 show a nearly identical pattern. Moreover, Brn1 shows reduced occupancy at various condensin-binding sites in asf1Δ cells. These results along with high association of Asf1 with heterochromatic centromeres and telomeres ascribe novel roles to Asf1 in condensin loading and chromatin dynamics.

Sgs1 and Sae2 promote telomere replication by limiting accumulation of ssDNA.

In budding yeast, DNA ends are processed by the consecutive action of MRX/Sae2 and two redundant pathways dependent on Sgs1/Dna2 and Exo1, and this processing is counteracted by Ku heterodimer. Here we show that DNA end resection by Sae2 and Sgs1 is dispensable for normal telomere maintenance by telomerase. Instead, these proteins facilitate telomere replication and limit the accumulation of single-strand DNA (ssDNA) at replication fork pause sites. Loss of Sae2 and Sgs1 drives selection for compensatory mutations, notably in Ku, which are responsible for abrupt telomere shortening in cells lacking Sae2 and Sgs1. In telomerase-negative cells, Sae2 and Sgs1 play non-overlapping roles in generating ssDNA at eroded telomeres and are required for the formation of type II survivors. Thus, although their primary function in telomerase-positive cells is to sustain DNA replication over the sites that are prone to fork pausing, Sae2 and Sgs1 contribute to telomere resection in telomerase-deficient cells.

Two-dimensional agarose gel electrophoresis for analysis of DNA replication.

The initiation, elongation, and termination of DNA replication are each associated with distinct, nonlinear DNA structures that can be resolved and identified by two-dimensional (2D) agarose gel electrophoresis. This method involves: isolation of genomic DNA while preserving fragile replication structures, digestion of the DNA with a restriction enzyme, separation of DNA by size and shape through two distinct stages of agarose gel electrophoresis, and Southern blotting to probe for the specific sequence(s) of interest. The method has been most commonly used to determine the activity level of putative replication origin-containing sequences, and has also been used to analyze replication timing, fork progression, fork pausing, fork stalling and collapse, termination, and recombinational repair.

Quantitative, Genome-Wide Analysis of Eukaryotic Replication Initiation and Termination

Many fundamental aspects of DNA replication, such as the exact locations where DNA synthesis is initiated and terminated, how frequently origins are used, and how fork progression is influenced by transcription, are poorly understood. Via the deep sequencing of Okazaki fragments, we comprehensively document replication fork directionality throughout the S. cerevisiae genome, which permits the systematic analysis of initiation, origin efficiency, fork progression, and termination. We show that leading-strand initiation preferentially occurs within a nucleosome-free region at replication origins. Using a strain in which late origins can be induced to fire early, we show that replication termination is a largely passive phenomenon that does not rely on cis-acting sequences or replication fork pausing. The replication profile is predominantly determined by the kinetics of origin firing, allowing us to reconstruct chromosome-wide timing profiles from an asynchronous culture.

Lsd1 and Lsd2 Control Programmed Replication Fork Pauses and Imprinting in Fission Yeast

In the fission yeast Schizosaccharomyces pombe, a chromosomal imprinting event controls the asymmetric pattern of mating-type switching. The orientation of DNA replication at the mating-type locus is instrumental in this process. However, the factors leading to imprinting are not fully identified and the mechanism is poorly understood. Here, we show that the replication fork pause at the mat1 locus (MPS1), essential for imprint formation, depends on the lysine-specific demethylase Lsd1. We demonstrate that either Lsd1 or Lsd2 amine oxidase activity is required for these processes, working upstream of the imprinting factors Swi1 and Swi3 (homologs of mammalian Timeless and Tipin, respectively). We also show that the Lsd1/2 complex controls the replication fork terminators, within the rDNA repeats. These findings reveal a role for the Lsd1/2 demethylases in controlling polar replication fork progression, imprint formation, and subsequent asymmetric cell divisions.

English

Division of the ancestral prokaryotic genome into two circular double-stranded deoxyribonucleic acid (DNA) molecules by genetic recombination, is a basis for the future separate evolution of the nuclear and mitochondrial gene compartment. This suggests monophyletic origin of both mitochondrion and nucleus. Presumed organism which genome undergoes genetic recombination has to be searched among an aerobic, oxygen non-producing archaeon with no rigid cell wall, but a plasma membrane, probably an crenarchaeota containing functional bacteriochlorophyll a synthase gene and histones. In this proposal, origin of eukaryotes occur by a three-steps. First, replication fork pauses and collapses generating a breakage in the genome of archaeal ancestor of eukaryotes. Second, the double-strand break can be repaired intergenomically by complementary strands invasion. Third, this duplicated genome can be fissioned into two compartments by reciprocal genetic recombination. Scenario is accomplished by aberrant fission of the inner membane surrounding separately those two compartments. Key words: Origin of nucleus, origin of mitochondrion.

The Replisome Pausing Factor Timeless Is Required for Episomal Maintenance of Latent Epstein-Barr Virus

The Epstein-Barr virus (EBV) genome is maintained as an extrachromosomal episome during latent infection of B lymphocytes. Episomal maintenance is conferred by the interaction of the EBV-encoded nuclear antigen 1 (EBNA1) with a tandem array of high-affinity binding sites, referred to as the family of repeats (FR), located within the viral origin of plasmid replication (OriP). How this nucleoprotein array confers episomal maintenance is not completely understood. Previous studies have shown that DNA replication forks pause and terminate with high frequency at OriP. We now show that cellular DNA replication fork pausing and protection factors Timeless (Tim) and Tipin (Timeless-interacting protein) accumulate at OriP during S phase of the cell cycle. Depletion of Tim inhibits OriP-dependent DNA replication and causes a complete loss of the closed-circular form of EBV episomes in latently infected B lymphocytes. Tim depletion also led to the accumulation of double-strand breaks at the OriP region. These findings demonstrate that Tim is essential for sustaining the episomal forms of EBV DNA in latently infected cells and suggest that DNA replication fork protection is integrally linked to the mechanism of plasmid maintenance.

Identification of a Novel Type of Spacer Element Required for Imprinting in Fission Yeast

Asymmetrical segregation of differentiated sister chromatids is thought to be important for cellular differentiation in higher eukaryotes. Similarly, in fission yeast, cellular differentiation involves the asymmetrical segregation of a chromosomal imprint. This imprint has been shown to consist of two ribonucleotides that are incorporated into the DNA during lagging-strand synthesis in response to a replication pause, but the underlying mechanism remains unknown. Here we present key novel discoveries important for unravelling this process. Our data show that cis-acting sequences within the mat1 cassette mediate pausing of replication forks at the proximity of the imprinting site, and the results suggest that this pause dictates specific priming at the position of imprinting in a sequence-independent manner. Also, we identify a novel type of cis-acting spacer region important for the imprinting process that affects where subsequent primers are put down after the replication fork is released from the pause. Thus, our data suggest that the imprint is formed by ligation of a not-fully-processed Okazaki fragment to the subsequent fragment. The presented work addresses how differentiated sister chromatids are established during DNA replication through the involvement of replication barriers.