Exo1 processes stalled replication forks and counteracts fork reversal in checkpoint-defective cells

The replication checkpoint coordinates the cell cycle with DNA replication and recombination, preventing genome instability and cancer. The budding yeast Rad53 checkpoint kinase stabilizes stalled forks and replisome-fork complexes, thus preventing the accumulation of ss-DNA regions and reversed forks at collapsed forks. We searched for factors involved in the processing of stalled forks in HU-treated rad53 cells. Using the neutral-neutral two-dimensional electrophoresis technique (2D gel) and psoralen crosslinking combined with electron microscopy (EM), we found that the Exo1 exonuclease is recruited to stalled forks and, in rad53 mutants, counteracts reversed fork accumulation by generating ss-DNA intermediates. Hence, Exo1-mediated fork processing resembles the action of E. coli RecJ nuclease at damaged forks. Fork stability and replication restart are influenced by both DNA polymerase-fork association and Exo1-mediated processing. We suggest that Exo1 counteracts fork reversal by resecting newly synthesized chains and resolving the sister chromatid junctions that cause regression of collapsed forks.     

The MMS22L-TONSL complex mediates recovery from replication stress and homologous recombination

Genome integrity is jeopardized each time DNA replication forks stall or collapse. Here we report the identification of a complex composed of MMS22L (C6ORF167) and TONSL (NFKBIL2) that participates in the recovery from replication stress. MMS22L and TONSL are homologous to yeast Mms22 and plant Tonsoku/Brushy1, respectively. MMS22L-TONSL accumulates at regions of ssDNA associated with distressed replication forks or at processed DNA breaks, and its depletion results in high levels of endogenous DNA double-strand breaks caused by an inability to complete DNA synthesis after replication fork collapse. Moreover, cells depleted of MMS22L are highly sensitive to camptothecin,?a topoisomerase I poison that impairs DNA replication progression. Finally, MMS22L and TONSL are necessary for the efficient formation of RAD51 foci after DNA damage, and their depletion impairs homologous recombination. These results indicate that MMS22L and TONSL are genome caretakers that stimulate the recombination-dependent repair of stalled or collapsed replication forks.     

Human Primpol1: a novel guardian of stalled replication forks

Huang and colleagues identify a human primase-polymerase that is required for stalled replication fork restart and the maintenance of genome integrity.EMBO reports (2013) 14 12, 1104–1112 doi:10.1038/embor.2013.159The successful duplication of genomic DNA during S phase is essential for the proper transmission of genetic information to the next generation of cells. Perturbation of normal DNA replication by extrinsic stimuli or intrinsic stress can result in stalled replication forks, ultimately leading to abnormal chromatin structures and activation of the DNA damage response. On formation of stalled replication forks, many DNA repair and recombination pathway proteins are recruited to resolve the stalled fork and resume proper DNA synthesis. Initiation of replication at sites of stalled forks differs from traditional replication and, therefore, requires specialized proteins to reactivate DNA synthesis. In this issue of EMBO reports, Wan et al [1] introduce human primase-polymerase 1 (hPrimpol1)/CCDC111, a novel factor that is essential for the restart of stalled replication forks. This article is the first, to our knowledge, to ascertain the function of human Primpol enzymes, which were originally identified as members of the archaeao-eukaryotic primase (AEP) family [2].Single-stranded DNA (ssDNA) forms at stalled replication forks because of uncoupling of the DNA helicase from the polymerase, and is coated by replication protein A (RPA) for stabilization and recruitment of proteins involved in DNA repair and restart of replication. To identify novel factors playing important roles in the resolution of stalled replication forks, Wan and colleagues [1] used mass spectrometry to identify RPA-binding partners. Among the proteins identified were those already known to be located at replication forks, including SMARCAL1/HARP, BLM and TIMELESS. In addition they found a novel interactor, the 560aa protein CCDC111. This protein interacts with the carboxyl terminus of RPA1 through its own C-terminal region, and localizes with RPA foci in cells after hydroxyurea or DNA damage induced by ionizing irradiation. Owing to the presence of AEP and zinc-ribbon-like domains at the amino-terminal and C-terminal regions, respectively [2], CCDC111 was predicted to have both primase and polymerase enzymatic activities, which was confirmed with in vitro assays, leading to the name hPrimpol1 for this unique enzyme.The most outstanding discovery in this article is that hPrimpol1 is required for the restart of DNA synthesis from a stalled replication fork (Fig 1). With use of a single DNA fibre assay, knock down of hPrimpol1 had no effect on normal replication-fork progression or the firing of new origins in the presence of replication stress. After removal of replication stress, however, the restart of stalled forks was significantly impaired. Furthermore, the authors observed that hPrimpol1 depletion enhanced the toxicity of replication stress to human cells. Together, these data suggest that hPrimpol1 is a novel guardian protein that ensures the proper re-initiation of DNA replication by control of the repriming and repolymerization of newly synthesized DNA.Open in a separate windowFigure 1The role of hPrimpol1 in stalled replication fork restart. (A) Under normal conditions, the replicative helicase unwinds parental DNA, generating ssDNA that is coated by RPA and serves as a template for leading and lagging strand synthesis. Aside from interacting with RPA bound to the short stretches of ssDNA, the role of hPrimpol1 in normal progression of replication forks is unknown. (B) Following repair of a stalled replication fork, (1) hPrimpol1 rapidly resumes DNA synthesis of long stretches of RPA-coated ssDNA located at the stalled fork site. Later, the leading-strand polymerase (2) or lagging-strand primase and polymerase (3) replace hPrimpol1 to complete replication of genomic DNA. RPA, replication protein A; ssDNA, single-stranded DNA.Eukaryotic DNA replication is initiated at specific sites, called origins, through the help of various proteins, including ORC, CDC6, CDT1 and the MCM helicase complex [3]. On unwinding of the parental duplexed DNA, lagging strand ssDNA is coated by the RPA complex and used as a template for newly synthesized daughter DNA. DNA primase, a type of RNA polymerase, catalyses short RNA primers on the RPA-coated ssDNA that facilitate further DNA synthesis by DNA polymerase. While the use of a short RNA primer is occasionally necessary to restart leading-strand replication, such as in the case of a stalled DNA polymerase, it is primarily utilized in lagging-strand synthesis for the continuous production of Okazaki fragments. The lagging-strand DNA polymerase must efficiently coordinate its action with DNA primase and other replication factors, including DNA helicase and RPA [4]. Cooperation between DNA polymerase and primase is disturbed after DNA damage, ultimately resulting in the collapse of stalled replication forks. Until now, it was believed that DNA primase and DNA polymerase performed separate and catalytically unique functions in replication-fork progression in human cells, but this report provides the first example, to our knowledge, of a single enzyme performing both primase and polymerase functions to restart DNA synthesis at stalled replication forks after DNA damage (Fig 1).… this report provides the first example of a single enzyme performing both primase and polymerase function to restart DNA synthesis at stalled replication forksA stalled replication fork, if not properly resolved, can be extremely detrimental to a cell, causing permanent cell-cycle arrest and, ultimately, death. Therefore, eukaryotic cells have developed many pathways for the identification, repair and restart of stalled forks [5]. RPA recognizes ssDNA at stalled forks and activates the intra-S-phase checkpoint pathway, which involves various signalling proteins, including ATR, ATRIP and CHK1 [6]. This checkpoint pathway halts cell-cycle progression until the stalled forks are properly repaired and restarted. Compared with the recognition and repair of stalled forks, the mechanism of fork restart is relatively elusive. Studies have, however, begun to shed light on this process. For instance, RPA-directed SMARCAL1 has been discovered to be important for restart of DNA replication in bacteria and humans [7]. Together with the identification of hPrimpol1, these findings have helped to expand the knowledge of the mechanism of restarting DNA replication. Furthermore, both reports raise many questions regarding the cooperative mechanism of hPrimpol1 and SMARCAL1 with RPA at stalled forks to ensure genomic stability and proper fork restart [7].First, these findings raise the question of why cells need the specialized hPrimpol1 to restart DNA replication at stalled forks rather than using the already present DNA primase and polymerase. One possibility is that other DNA polymerases are functionally inhibited due to the response of the cell to DNA damage. Although the cells are ready to restart replication, the impaired polymerases might require additional time to recover after DNA damage, necessitating the use of hPrimpol1. In support of this idea, we found that the p12 subunit of DNA polymerase δ is degraded by CRL4^CDT2 E3 ligase after ultraviolet damage [8]. As a result, alternative polymerases, such as hPrimpol1, could compensate for temporarily non-functioning traditional polymerases. A second explanation is that the polymerase and helicase uncoupling after stalling of a fork results in long stretches of ssDNA that are coated with RPA. To restart DNA synthesis, cells must quickly reprime and polymerize large stretches of ssDNA to prevent renewed fork collapse. By its constant interaction with RPA1, hPrimpol1 is present on the ssDNA and can rapidly synthesize the new strand of DNA after the recovery of stalled forks. Third, the authors found that the association of hPrimpol1 with RPA1 is independent of its functional AEP and zinc-ribbon-like domains and occurs in the absence of DNA damage. These results might indicate a role for hPrimpol1 in normal replication fork progression, but further work is necessary to determine whether that is true.The discovery of hPrimpol1 is also important in an evolutionary contextSeveral questions remain. First, what is the fidelity of the polymerase activity? Other specialized polymerases that act at DNA damage sites sometimes have the ability to misincorporate a nucleotide across from a site of damage, for example pol-eta and -zeta [9]. It will be interesting to know whether hPrimpol1 is a high-fidelity polymerase or an error-prone polymerase. Second, is the polymerase only brought into action after fork stalling? If hPrimpol1 is an error-prone polymerase, one could envision other types of DNA damage that can be bypassed by hPrimpol1. Third, is the primase selective for ribonucleotides, or can it also incorporate deoxynucleotides? The requirement of the same domain—AEP—for primase and polymerase activities raises the possibility that NTPs or dNTPs could be used for primase or polymerase activities.The discovery of hPrimpol1 is also important in an evolutionary context. In 2003, an enzyme with catalytic activities like that of hPrimpol1 was discovered in a thermophilic archeaon and in Gram-positive bacteria [10]. This protein had several catalytic activities in vitro, including ATPase, primase and polymerase. In contrast to these Primpol enzymes, those capable of primase and polymerase functions had not been found in higher eukaryotes, which suggested that evolutionary pressures forced a split of these dual-function enzymes. Huang et al''s report suggests, however, that human cells do in fact retain enzymes similar to Primpol. In summary, the role of hPrimpol1 at stalled forks broadens our knowledge of the restart of DNA replication in human cells after fork stalling, allowing for proper duplication of genomic DNA, and provides insight into the evolution of primases in eukaryotes.     

Anticipating chromosomal replication fork arrest: SSB targets repair DNA helicases to active forks

In bacteria, several salvage responses to DNA replication arrest culminate in reassembly of the replisome on inactivated forks to resume replication. The PriA DNA helicase is a prominent trigger of this replication restart process, preceded in many cases by a repair and/or remodeling of the arrested fork, which can be performed by many specific proteins. The mechanisms that target these rescue effectors to damaged forks in the cell are unknown. We report that the single-stranded DNA binding (SSB) protein is the key factor that links PriA to active chromosomal replication forks in vivo. This targeting mechanism determines the efficiency by which PriA reaches its specific DNA-binding site in vitro and directs replication restart in vivo. The RecG and RecQ DNA helicases, which are involved in intricate replication reactivation pathways, also associate with the chromosomal replication forks by similarly interacting with SSB. These results identify SSB as a platform for linking a 'repair toolbox' with active replication forks, providing a first line of rescue responses to accidental arrest.     

DNA replication: Pif1 pulls the plug on stalled replication forks

The conserved PIF helicase family appears to function in replication to ensure termination and passage through regions that slow or arrest replication fork movement. Findings in fission yeast extend evidence from budding yeast, and argue for universal mechanisms that ensure replication integrity.     

Novel function of the flap endonuclease 1 complex in processing stalled DNA replication forks

Restarting stalled replication forks partly depends on the break-induced recombination pathway, in which a DNA double-stranded break (DSB) is created on the stalled replication fork to initiate the downstream recombination cascades. Single-stranded DNA gaps accumulating on stalled replication forks are potential targets for endonucleases to generate DSBs. However, it is unclear how this process is executed and which nucleases are involved in eukaryotic cells. Here, we identify a novel gap endonuclease (GEN) activity of human flap endonuclease 1 (FEN-1), critical in resolving stalled replication fork. In response to replication arrest, FEN-1 interacts specifically with Werner syndrome protein for efficient fork cleavage. Replication protein A facilitates FEN-1 interaction with DNA bubble structures. Human FEN-1, but not the GEN-deficient mutant, E178A, was shown to rescue the defect in resistance to UV and camptothecin in a yeast FEN-1 null mutant.     

UV stalled replication forks restart by re-priming in human fibroblasts

Restarting stalled replication forks is vital to avoid fatal replication errors. Previously, it was demonstrated that hydroxyurea-stalled replication forks rescue replication either by an active restart mechanism or by new origin firing. To our surprise, using the DNA fibre assay, we only detect a slightly reduced fork speed on a UV-damaged template during the first hour after UV exposure, and no evidence for persistent replication fork arrest. Interestingly, no evidence for persistent UV-induced fork stalling was observed even in translesion synthesis defective, Polη(mut) cells. In contrast, using an assay to measure DNA molecule elongation at the fork, we observe that continuous DNA elongation is severely blocked by UV irradiation, particularly in UV-damaged Polη(mut) cells. In conclusion, our data suggest that UV-blocked replication forks restart effectively through re-priming past the lesion, leaving only a small gap opposite the lesion. This allows continuation of replication on damaged DNA. If left unfilled, the gaps may collapse into DNA double-strand breaks that are repaired by a recombination pathway, similar to the fate of replication forks collapsed after hydroxyurea treatment.     

Viral transport of DNA damage that mimics a stalled replication fork

Adeno-associated virus type 2 (AAV2) infection incites cells to arrest with 4N DNA content or die if the p53 pathway is defective. This arrest depends on AAV2 DNA, which is single stranded with inverted terminal repeats that serve as primers during viral DNA replication. Here, we show that AAV2 DNA triggers damage signaling that resembles the response to an aberrant cellular DNA replication fork. UV treatment of AAV2 enhances the G2 arrest by generating intrastrand DNA cross-links which persist in infected cells, disrupting viral DNA replication and maintaining the viral DNA in the single-stranded form. In cells, such DNA accumulates into nuclear foci with a signaling apparatus that involves DNA polymerase delta, ATR, TopBP1, RPA, and the Rad9/Rad1/Hus1 complex but not ATM or NBS1. Focus formation and damage signaling strictly depend on ATR and Chk1 functions. Activation of the Chk1 effector kinase leads to the virus-induced G2 arrest. AAV2 provides a novel way to study the cellular response to abnormal DNA replication without damaging cellular DNA. By using the AAV2 system, we show that in human cells activation of phosphorylation of Chk1 depends on TopBP1 and that it is a prerequisite for the appearance of DNA damage foci.     

A step backward in advancing DNA replication: rescue of stalled replication forks by RecG.

In the October 5 issue of Cell, Singleton et al. report the crystal structure of RecG protein bound to an analog of a stalled DNA replication fork. This structure shows how RecG can recognize branched DNA structures and suggests a mechanism for fork reversal, an early event in recombination-dependent reinitiation of DNA replication.     

Differential roles of XRCC2 in homologous recombinational repair of stalled replication forks

Homologous recombination is an important mechanism in DNA replication to ensure faithful DNA synthesis and genomic stability. In this study, we investigated the role of XRCC2, a member of the RAD51 paralog family, in cellular recovery from replication arrest via homologous recombination. The protein expression of XRCC2, as well as its binding partner RAD51D, is dramatically increased in S- and G2-phases, suggesting that these proteins function during and after DNA synthesis. XRCC2 mutant irs1 cells exhibit hypersensitivity to hydroxyurea (HU) and are defective in the induction of RAD51 foci after HU treatment. In addition, the HU-induced chromatin association of RAD51 is deficient in irs1 mutant. Interestingly, irs1 cells are only slightly sensitive to thymidine and able to form intact RAD51 foci in S-phase cells arrested with thymidine. Irs1 cells showed increased level of chromatin-bound RAD51 as well as the wild type cells after thymidine treatment. Both HU and thymidine induce gamma-H2AX foci in arrested S-phase nuclei. These results suggest that XRCC2 is involved in repair of HU-induced damage, but not thymidine-induced damage, at the stalled replication forks. Our data suggest that there are at least two sub-pathways in homologous recombination, XRCC2-dependent and -independent, for repair of stalled replication forks and assembly of RAD51 foci following replication arrest in S-phase.     

Error-prone repair of stalled replication forks drives mutagenesis and loss of heterozygosity in haploinsufficient BRCA1 cells

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Posttranslational modifications of repair factors and histones in the cellular response to stalled replication forks

DNA damage during replication requires an integration of checkpoint response with replication itself and distinct repair pathways, such as replication pausing, recombination and translesion synthesis. Here we focus on recent advances in our understanding of how protein posttranslational modifications contribute to the maintenance of fork integrity. In particular, we examine the role of histone modifications and chromatin remodeling complexes in this process.     

Persistently stalled replication forks inhibit nucleotide excision repair in trans by sequestering Replication protein A

Rev3, the catalytic subunit of DNA polymerase ζ, is essential for translesion synthesis of cytotoxic DNA photolesions, whereas the Rev1 protein plays a noncatalytic role in translesion synthesis. Here, we reveal that mammalian Rev3^−/− and Rev1^−/− cell lines additionally display a nucleotide excision repair (NER) defect, specifically during S phase. This defect is correlated with the normal recruitment but protracted persistence at DNA damage sites of factors involved in an early stage of NER, while repair synthesis is affected. Remarkably, the NER defect becomes apparent only at 2 h post-irradiation indicating that Rev3 affects repair synthesis only indirectly, rather than performing an enzymatic role in NER. We provide evidence that the NER defect is caused by scarceness of Replication protein A (Rpa) available to NER, resulting from its sequestration at stalled replication forks. Also the induction of replicative stress using hydroxyurea precludes the accumulation of Rpa at photolesion sites, both in Rev3^−/− and in wild-type cells. These data support a model in which the limited Rpa pool coordinates replicative stress and NER, resulting in increased cytotoxicity of ultraviolet light when replicative stress exceeds a threshold.     

RNAi-based screening identifies the Mms22L-Nfkbil2 complex as a novel regulator of DNA replication in human cells

Cullin 4 (Cul4)-based ubiquitin ligases emerged as critical regulators of DNA replication and repair. Over 50 Cul4-specific adaptors (DNA damage-binding 1 (Ddb1)-Cul4-associated factors; DCAFs) have been identified and are thought to assemble functionally distinct Cul4 complexes. Using a live-cell imaging-based RNAi screen, we analysed the function of DCAFs and Cul4-linked proteins, and identified specific subsets required for progression through G1 and S phase. We discovered C6orf167/Mms22-like protein (Mms22L) as a putative human orthologue of budding yeast Mms22, which, together with cullin Rtt101, regulates genome stability by promoting DNA replication through natural pause sites and damaged templates. Loss of Mms22L function in human cells results in S phase-dependent genomic instability characterised by spontaneous double-strand breaks and DNA damage checkpoint activation. Unlike yeast Mms22, human Mms22L does not stably bind to Cul4, but is degraded in a Cul4-dependent manner and upon replication stress. Mms22L physically and functionally interacts with the scaffold-like protein Nfkbil2 that co-purifies with histones, several chromatin remodelling and DNA replication/repair factors. Together, our results strongly suggest that the Mms22L-Nfkbil2 complex contributes to genome stability by regulating the chromatin state at stalled replication forks.     

SSB and the RecG DNA helicase: an intimate association to rescue a stalled replication fork

In E. coli, the regression of stalled DNA replication forks is catalyzed by the DNA helicase RecG. One means of gaining access to the fork is by binding to the single strand binding protein or SSB. This interaction occurs via the wedge domain of RecG and the intrinsically disordered linker (IDL) of SSB, in a manner similar to that of SH3 domains binding to PXXP motif‐containing ligands in eukaryotic cells. During loading, SSB remodels the wedge domain so that the helicase domains bind to the parental, duplex DNA, permitting the helicase to translocate using thermal energy. This translocation may be used to clear the fork of obstacles, prior to the initiation of fork regression.     

DNA damage-induced accumulation of Rad18 protein at stalled replication forks in mammalian cells involves upstream protein phosphorylation

Rad18 protein is required for mono-ubiquitination of PCNA and trans-lesion synthesis during DNA lesion bypass in eukaryotic cells but it remains unknown how it is activated after DNA damage. We expressed GFP-tagged human (h)Rad18 in Chinese hamster cells and found that it can be completely extracted from undamaged nuclei by Triton X-100 and methanol. However, several hours after treatment with methyl methanesulfonate (MMS) Triton-insoluble form of GFP-hRad18 accumulates in S-phase nuclei where it colocalizes with PCNA. This accumulation is suppressed by inhibitors of protein kinases staurosporine and wortmannin but is not effected by roscovitine. We also found that methyl methanesulfonate induces phosphorylation of Ser-317 in protein kinase Chk1 and Ser-139 in histone H2AX and stimulates formation of single-stranded DNA at replication foci. Together, our results suggest that MMS-induced accumulation of hRad18 protein at stalled forks involves protein phosphorylation which may be performed by S-phase checkpoint kinases.     

DNA replication: making two forks from one prereplication complex

The copying of chromosomal DNA initiates from a single nucleoprotein assembly called the prereplication complex. New findings in a recent issue of Molecular Cell (Yardimci et?al., 2010) reveal that this complex dissolves into two independent replisomes that move away from each other as DNA synthesis ensues.     

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.