Recombination proteins and rescue of arrested replication forks

Recombination proteins play crucial roles in the rescue of inactivated replication forks in Escherichia coli. The enzymes that catalyze the repair of DNA double-strand breaks by a classical strand-exchange reaction (RecBCD, RecA) act in two well-characterized fork repair pathways. They repair the DNA double-strand end made when a replication fork runs into a single-strand interruption. They reset the DNA double-strand end generated by replication fork reversal when a component of the replication machinery is inactivated. In addition, recombination proteins also act at replication forks in ways other than the classical strand-exchange reaction. For example, the RuvAB enzyme that catalyzes Holliday junction branch-migration during homologous recombination is also able to catalyze replication fork reversal in certain replication mutants, i.e. to convert certain blocked replication forks into Holliday junctions. Finally, some of the actions of recombination proteins after replication impairment are still unclear, as for example in UV-irradiated cells, where RecFOR and RecA catalyze gap repair but also participate, in a yet undefined way, in "replisome reactivation".     

UvsX recombinase and Dda helicase rescue stalled bacteriophage T4 DNA replication forks in vitro

The rescue of stalled replication forks via a series of steps that include fork regression, template switching, and fork restoration often has been proposed as a major mechanism for accurately bypassing non-coding DNA lesions. Bacteriophage T4 encodes almost all of the proteins required for its own DNA replication, recombination, and repair. Both recombination and recombination repair in T4 rely on UvsX, a RecA-like recombinase. We show here that UvsX plus the T4-encoded helicase Dda suffice to rescue stalled T4 replication forks in vitro. This rescue is based on two sequential template-switching reactions that allow DNA replication to bypass a non-coding DNA lesion in a non-mutagenic manner.     

RecO and RecR Are Necessary for RecA Loading in Response to DNA Damage and Replication Fork Stress

RecA is central to maintaining genome integrity in bacterial cells. Despite the near-ubiquitous conservation of RecA in eubacteria, the pathways that facilitate RecA loading and repair center assembly have remained poorly understood in Bacillus subtilis. Here, we show that RecA rapidly colocalizes with the DNA polymerase complex (replisome) immediately following DNA damage or damage-independent replication fork arrest. In Escherichia coli, the RecFOR and RecBCD pathways serve to load RecA and the choice between these two pathways depends on the type of damage under repair. We found in B. subtilis that the rapid localization of RecA to repair centers is strictly dependent on RecO and RecR in response to all types of damage examined, including a site-specific double-stranded break and damage-independent replication fork arrest. Furthermore, we provide evidence that, although RecF is not required for RecA repair center formation in vivo, RecF does increase the efficiency of repair center assembly, suggesting that RecF may influence the initial stages of RecA nucleation or filament extension. We further identify single-stranded DNA binding protein (SSB) as an additional component important for RecA repair center assembly. Truncation of the SSB C terminus impairs the ability of B. subtilis to form repair centers in response to damage and damage-independent fork arrest. With these results, we conclude that the SSB-dependent recruitment of RecOR to the replisome is necessary for loading and organizing RecA into repair centers in response to DNA damage and replication fork arrest.     

PriC-mediated DNA Replication Restart Requires PriC Complex Formation with the Single-stranded DNA-binding Protein

Frequent collisions between cellular DNA replication complexes (replisomes) and obstacles such as damaged DNA or frozen protein complexes make DNA replication fork progression surprisingly sporadic. These collisions can lead to the ejection of replisomes prior to completion of replication, which, if left unrepaired, results in bacterial cell death. As such, bacteria have evolved DNA replication restart mechanisms that function to reload replisomes onto abandoned DNA replication forks. Here, we define a direct interaction between PriC, a key Escherichia coli DNA replication restart protein, and the single-stranded DNA-binding protein (SSB), a protein that is ubiquitously associated with DNA replication forks. PriC/SSB complex formation requires evolutionarily conserved residues from both proteins, including a pair of Arg residues from PriC and the C terminus of SSB. In vitro, disruption of the PriC/SSB interface by sequence changes in either protein blocks the first step of DNA replication restart, reloading of the replicative DnaB helicase onto an abandoned replication fork. Consistent with the critical role of PriC/SSB complex formation in DNA replication restart, PriC variants that cannot bind SSB are non-functional in vivo. Single-molecule experiments demonstrate that PriC binding to SSB alters SSB/DNA complexes, exposing single-stranded DNA and creating a platform for other proteins to bind. These data lead to a model in which PriC interaction with SSB remodels SSB/DNA structures at abandoned DNA replication forks to create a DNA structure that is competent for DnaB loading.     

FEN1 Ensures Telomere Stability by Facilitating Replication Fork Re-initiation

Telomeres are terminal repetitive DNA sequences whose stability requires the coordinated actions of telomere-binding proteins and the DNA replication and repair machinery. Recently, we demonstrated that the DNA replication and repair protein Flap endonuclease 1 (FEN1) is required for replication of lagging strand telomeres. Here, we demonstrate for the first time that FEN1 is required for efficient re-initiation of stalled replication forks. At the telomere, we find that FEN1 depletion results in replicative stress as evidenced by fragile telomere expression and sister telomere loss. We show that FEN1 participation in Okazaki fragment processing is not required for efficient telomere replication. Instead we find that FEN1 gap endonuclease activity, which processes DNA structures resembling stalled replication forks, and the FEN1 interaction with the RecQ helicases are vital for telomere stability. Finally, we find that FEN1 depletion neither impacts cell cycle progression nor in vitro DNA replication through non-telomeric sequences. Our finding that FEN1 is required for efficient replication fork re-initiation strongly suggests that the fragile telomere expression and sister telomere losses observed upon FEN1 depletion are the direct result of replication fork collapse. Together, these findings suggest that other nucleases compensate for FEN1 loss throughout the genome during DNA replication but fail to do so at the telomere. We propose that FEN1 maintains stable telomeres by facilitating replication through the G-rich lagging strand telomere, thereby ensuring high fidelity telomere replication.     

Protein--protein interactions in the eubacterial replisome

Replication of genomic DNA is a universal process that proceeds in distinct stages, from initiation to elongation and finally to termination. Each stage involves multiple stable or transient interactions between protein subunits with functions that are more or less conserved in all organisms. In Escherichia coli, initiation of bidirectional replication at the origin (oriC) occurs through the concerted actions of the DnaA replication initiator protein, the hexameric DnaB helicase, the DnaC?helicase loading partner and the DnaG primase, leading to establishment of two replication forks. Elongation of RNA primers at each fork proceeds simultaneously on both strands by actions of the multimeric replicase, DNA polymerase III holoenzyme. The fork that arrives first in the terminus region is halted by its encounter with a correctly-oriented complex of the Tus replication terminator protein bound at one of several Ter sites, where it is trapped until the other fork arrives. We summarize current understanding of interactions among the various proteins that act in the different stages of replication of the chromosome of E. coli, and make some comparisons with the analogous proteins in Bacillus subtilis and the coliphages T4 and T7.     

RecBCD and RecJ/RecQ initiate DNA degradation on distinct substrates in UV-irradiated Escherichia coli

After UV irradiation, recA mutants fail to recover replication, and a dramatic and nearly complete degradation of the genomic DNA occurs. Although the RecBCD helicase/nuclease complex is known to mediate this catastrophic DNA degradation, it is not known how or where this degradation is initiated. Previous studies have speculated that RecBCD targets and initiates degradation from the nascent DNA at replication forks arrested by DNA damage. To test this question, we examined which enzymes were responsible for the degradation of genomic DNA and the nascent DNA in UV-irradiated recA cells. We show here that, although RecBCD degrades the genomic DNA after UV irradiation, it does not target the nascent DNA at arrested replication forks. Instead, we observed that the nascent DNA at arrested replication forks in recA cultures is degraded by RecJ/RecQ, similar to what occurs in wild-type cultures. These findings indicate that the genomic DNA degradation and nascent DNA degradation in UV-irradiated recA mutants are mediated separately through RecBCD and RecJ/RecQ, respectively. In addition, they demonstrate that RecBCD initiates degradation at a site(s) other than the arrested replication fork directly.     

sbcB sbcC null mutations allow RecF-mediated repair of arrested replication forks in rep recBC mutants

We have proposed previously that, in Escherichia coli, blockage of replication forks can lead to the reversal of the fork. Annealing of the newly synthesized strands creates a double-stranded end adjacent to a Holliday junction. The junction is migrated away from the DNA end by RuvAB and can be cleaved by RuvC, while RecBCD is required for the repair of the double-stranded tail. Consequently, the rep mutant, in which replication arrests are frequent and fork reversal occurs, requires RecBCD for growth. We show here that the combination of sbcB sbcCD null mutations restores the viability to rep recBC mutants by activation of the RecF pathway of recombination. This shows that the proteins belonging to the RecF pathway are able to process the DNA ends made by the replication fork reversal into a structure that allows recombination-dependent replication restart. However, we confirm that, unlike sbcB null mutations, sbcB15, which suppresses all other recBC mutant defects, does not restore the viability of rep recBC sbcCD strains. We also show that ruvAB inactivation suppresses the lethality and the formation of double-stranded breaks (DSBs) in a rep recBC recF strain, totally deficient for homologous recombination, as well as in rep recBC mutants. This confirms that RuvAB processing of arrested replication forks is independent of the presence of recombination intermediates.     

真核细胞染色体DNA复制起始及复制叉稳定性维持的机制

在真核生物中,DNA复制在染色体上特定的多位点起始．当细胞处在晚M及G1期,多个复制起始蛋白依次结合到DNA复制源,组装形成复制前复合体．pre．RC在Gl-S的转折期得到激活,随后,多个直接参与DNA复制又形成的蛋白结合到DNA复制源,启动DNA的复制,形成两个双向的DNA复制又．在染色体上,移动的DNA复制又经常会碰到复制障碍（二级DNA结构、一些蛋白的结合位点、损伤的碱基等）而暂停下来,此时,需要细胞周期检验点的调控来稳定复制叉,否则,会导致复制又垮塌及基因组不稳定．本文就真核细胞染色体DNA复制起始的机制,以及复制又稳定性的维持机制进行简要综述．     

Local and global functions of Timeless and Tipin in replication fork protection

The eukaryotic cell replicates its chromosomal DNA with almost absolute fidelity in the course of every cell cycle. This accomplishment is remarkable considering that the conditions for DNA replication are rarely ideal. The replication machinery encounters a variety of obstacles on the chromosome, including damaged template DNA. In addition, a number of chromosome regions are considered to be difficult to replicate owing to DNA secondary structures and DNA binding proteins required for various transactions on the chromosome. Under these conditions, replication forks stall or break, posing grave threats to genomic integrity. How does the cell combat such stressful conditions during DNA replication? The replication fork protection complex (FPC) may help answer this question. Recent studies have demonstrated that the FPC is required for the smooth passage of replication forks at difficult-to-replicate genomic regions and plays a critical role in coordinating multiple genome maintenance processes at the replication fork.     

Local and global functions of Timeless and Tipin in replication fork protection

The eukaryotic cell replicates its chromosomal DNA with almost absolute fidelity in the course of every cell cycle. This accomplishment is remarkable considering that the conditions for DNA replication are rarely ideal. The replication machinery encounters a variety of obstacles on the chromosome, including damaged template DNA. In addition, a number of chromosome regions are considered to be difficult to replicate owing to DNA secondary structures and DNA binding proteins required for various transactions on the chromosome. Under these conditions, replication forks stall or break, posing grave threats to genomic integrity. How does the cell combat such stressful conditions during DNA replication? The replication fork protection complex (FPC) may help answer this question. Recent studies have demonstrated that the FPC is required for the smooth passage of replication forks at difficult-to-replicate genomic regions and plays a critical role in coordinating multiple genome maintenance processes at the replication fork.     

RecBCD is required to complete chromosomal replication: Implications for double-strand break frequencies and repair mechanisms

Several aspects of the mechanism of homologous double-strand break repair remain unclear. Although intensive efforts have focused on how recombination reactions initiate, far less is known about the molecular events that follow. Based upon biochemical studies, current models propose that RecBCD processes double-strand ends and loads RecA to initiate recombinational repair. However, recent studies have shown that RecBCD plays a critical role in completing replication events on the chromosome through a mechanism that does not involve RecA or recombination. Here, we examine several studies, both early and recent, that suggest RecBCD also operates late in the recombination process – after initiation, strand invasion, and crossover resolution have occurred. Similar to its role in completing replication, we propose a model in which RecBCD is required to resect and resolve the DNA synthesis associated with homologous recombination at the point where the missing sequences on the broken molecule have been restored. We explain how the impaired ability to complete chromosome replication in recBC and recD mutants is likely to account for the loss of viability and genome instability in these mutants, and conclude that spontaneous double-strand breaks and replication fork collapse occur far less frequently than previously speculated.     

Effects of the bacteriophage T4 dda protein on DNA synthesis catalyzed by purified T4 replication proteins

The T4 bacteriophage dda protein is a DNA-dependent ATPase and DNA helicase that is the product of an apparently nonessential T4 gene. We have examined its effects on in vitro DNA synthesis catalyzed by a purified, multienzyme T4 DNA replication system. When DNA synthesis is catalyzed by the T4 DNA polymerase on a single-stranded DNA template, the addition of the dda protein is without effect whether or not other replication proteins are present. In contrast, on a double-stranded DNA template, where a mixture of the DNA polymerase, its accessory proteins, and the gene 32 protein is required, the dda protein greatly stimulates DNA synthesis. The dda protein exerts this effect by speeding up the rate of replication fork movement; in this respect, it acts identically with the other DNA helicase in the T4 replication system, the T4 gene 41 protein. However, whereas a 41 protein molecule remains bound to the same replication fork for a prolonged period, the dda protein seems to be continually dissociating from the replication fork and rebinding to it as the fork moves. Some gene 32 protein is required to observe DNA synthesis on a double-stranded DNA template, even in the presence of the dda protein. However, there is a direct competition between this helix-destabilizing protein and the dda protein for binding to single-stranded DNA, causing the rate of replication fork movement to decrease at a high ratio of gene 32 protein to dda protein. As shown elsewhere, the dda protein becomes absolutely required for in vitro DNA synthesis when E. coli RNA polymerase molecules are bound to the DNA template, because these molecules otherwise stop fork movement (Bedinger, P., Hochstrasser, M., Jongeneel, C.V., and Alberts, B. M. (1983) Cell 34, 115-123).     

Separable functions of Tof1/Timeless in intra-S-checkpoint signalling,replisome stability and DNA topological stress

The highly conserved Tof1/Timeless proteins minimise replication stress and promote normal DNA replication. They are required to mediate the DNA replication checkpoint (DRC), the stable pausing of forks at protein fork blocks, the coupling of DNA helicase and polymerase functions during replication stress (RS) and the preferential resolution of DNA topological stress ahead of the fork. Here we demonstrate that the roles of the Saccharomyces cerevisiae Timeless protein Tof1 in DRC signalling and resolution of DNA topological stress require distinct N and C terminal regions of the protein, whereas the other functions of Tof1 are closely linked to the stable interaction between Tof1 and its constitutive binding partner Csm3/Tipin. By separating the role of Tof1 in DRC from fork stabilisation and coupling, we show that Tof1 has distinct activities in checkpoint activation and replisome stability to ensure the viable completion of DNA replication following replication stress.     

RecD2 Helicase Limits Replication Fork Stress in Bacillus subtilis

DNA helicases have important roles in genome maintenance. The RecD helicase has been well studied as a component of the heterotrimeric RecBCD helicase-nuclease enzyme important for double-strand break repair in Escherichia coli. Interestingly, many bacteria lack RecBC and instead contain a RecD2 helicase, which is not known to function as part of a larger complex. Depending on the organism studied, RecD2 has been shown to provide resistance to a broad range of DNA-damaging agents while also contributing to mismatch repair (MMR). Here we investigated the importance of Bacillus subtilis RecD2 helicase to genome integrity. We show that deletion of recD2 confers a modest increase in the spontaneous mutation rate and that the mutational signature in ΔrecD2 cells is not consistent with an MMR defect, indicating a new function for RecD2 in B. subtilis. To further characterize the role of RecD2, we tested the deletion strain for sensitivity to DNA-damaging agents. We found that loss of RecD2 in B. subtilis sensitized cells to several DNA-damaging agents that can block or impair replication fork movement. Measurement of replication fork progression in vivo showed that forks collapse more frequently in ΔrecD2 cells, supporting the hypothesis that RecD2 is important for normal replication fork progression. Biochemical characterization of B. subtilis RecD2 showed that it is a 5′-3′ helicase and that it directly binds single-stranded DNA binding protein. Together, our results highlight novel roles for RecD2 in DNA replication which help to maintain replication fork integrity during normal growth and when forks encounter DNA damage.     

Checkpoint responses to replication stalling: inducing tolerance and preventing mutagenesis

Replication mutants often exhibit a mutator phenotype characterized by point mutations, single base frameshifts, and the deletion or duplication of sequences flanked by homologous repeats. Mutation in genes encoding checkpoint proteins can significantly affect the mutator phenotype. Here, we use fission yeast (Schizosaccharomyces pombe) as a model system to discuss the checkpoint responses to replication perturbations induced by replication mutants. Checkpoint activation induced by a DNA polymerase mutant, aside from delay of mitotic entry, up-regulates the translesion polymerase DinB (Polkappa). Checkpoint Rad9-Rad1-Hus1 (9-1-1) complex, which is loaded onto chromatin by the Rad17-Rfc2-5 checkpoint complex in response to replication perturbation, recruits DinB onto chromatin to generate the point mutations and single nucleotide frameshifts in the replication mutator. This chain of events reveals a novel checkpoint-induced tolerance mechanism that allows cells to cope with replication perturbation, presumably to make possible restarting stalled replication forks.Fission yeast Cds1 kinase plays an essential role in maintaining DNA replication fork stability in the face of DNA damage and replication fork stalling. Cds1 kinase is known to regulate three proteins that are implicated in maintaining replication fork stability: Mus81-Eme1, a hetero-dimeric structure-specific endonuclease complex; Rqh1, a RecQ-family helicase involved in suppressing inappropriate recombination during replication; and Rad60, a protein required for recombinational repair during replication. These Cds1-regulated proteins are thought to cooperatively prevent mutagenesis and maintain replication fork stability in cells under replication stress. These checkpoint-regulated processes allow cells to survive replication perturbation by preventing stalled replication forks from degenerating into deleterious DNA structures resulting in genomic instability and cancer development.     

Sld7, an Sld3-associated protein required for efficient chromosomal DNA replication in budding yeast

Genetic screening of yeast for sld (synthetic lethality with dpb11) mutations has identified replication proteins, including Sld2, -3, and -5, and clarified the molecular mechanisms underlying eukaryotic chromosomal DNA replication. Here, we report a new replication protein, Sld7, identified by rescreening of sld mutations. Throughout the cell cycle, Sld7 forms a complex with Sld3, which associates with replication origins in a complex with Cdc45, binds to Dpb11 when phosphorylated by cyclin-dependent kinase, and dissociates from origins once DNA replication starts. However, Sld7 does not move with the replication fork. Sld7 binds to the nonessential N-terminal portion of Sld3 and reduces its affinity for Cdc45, a component of the replication fork. Although Sld7 is not essential for cell growth, its absence reduces the level of cellular Sld3, delays the dissociation from origins of GINS, a component of the replication fork, and slows S-phase progression. These results suggest that Sld7 is required for the proper function of Sld3 at the initiation of DNA replication.     

Ub-family modifications at the replication fork: Regulating PCNA-interacting components

A vast array of proteins is recruited to the replication fork in a dynamic and coordinated manner through physical interactions with Proliferating Cell Nuclear Antigen, PCNA. How this complex exchange of PCNA binding partners is choreographed to ensure proper replication origin licensing, DNA synthesis during normal replication or repair of DNA damage, chromatin assembly, DNA methylation, histone modification, and sister chromatid cohesion is only beginning to be appreciated. In this review, several roles of ubiquitin-related modifications in the recruitment and turnover of PCNA-interacting proteins at the replication fork are considered.     

The SET Domain Is Essential for Metnase Functions in Replication Restart and the 5’ End of SS-Overhang Cleavage

Metnase (also known as SETMAR) is a chimeric SET-transposase protein that plays essential role(s) in non-homologous end joining (NHEJ) repair and replication fork restart. Although the SET domain possesses histone H3 lysine 36 dimethylation (H3K36me2) activity associated with an improved association of early repair components for NHEJ, its role in replication restart is less clear. Here we show that the SET domain is necessary for the recovery from DNA damage at the replication forks following hydroxyurea (HU) treatment. Cells overexpressing the SET deletion mutant caused a delay in fork restart after HU release. Our In vitro study revealed that the SET domain but not the H3K36me2 activity is required for the 5’ end of ss-overhang cleavage with fork and non-fork DNA without affecting the Metnase-DNA interaction. Together, our results suggest that the Metnase SET domain has a positive role in restart of replication fork and the 5’ end of ss-overhang cleavage, providing a new insight into the functional interaction of the SET and the transposase domains.