Cockayne syndrome group B protein regulates fork restart,fork progression and MRE11-dependent fork degradation in BRCA1/2-deficient cells

Cockayne syndrome group B (CSB) protein has been implicated in the repair of a variety of DNA lesions that induce replication stress. However, little is known about its role at stalled replication forks. Here, we report that CSB is recruited to stalled forks in a manner dependent upon its T1031 phosphorylation by CDK. While dispensable for MRE11 association with stalled forks in wild-type cells, CSB is required for further accumulation of MRE11 at stalled forks in BRCA1/2-deficient cells. CSB promotes MRE11-mediated fork degradation in BRCA1/2-deficient cells. CSB possesses an intrinsic ATP-dependent fork reversal activity in vitro, which is activated upon removal of its N-terminal region that is known to autoinhibit CSB’s ATPase domain. CSB functions similarly to fork reversal factors SMARCAL1, ZRANB3 and HLTF to regulate slowdown in fork progression upon exposure to replication stress, indicative of a role of CSB in fork reversal in vivo. Furthermore, CSB not only acts epistatically with MRE11 to facilitate fork restart but also promotes RAD52-mediated break-induced replication repair of double-strand breaks arising from cleavage of stalled forks by MUS81 in BRCA1/2-deficient cells. Loss of CSB exacerbates chemosensitivity in BRCA1/2-deficient cells, underscoring an important role of CSB in the treatment of cancer lacking functional BRCA1/2.     

Functional analysis of DNA replication fork reversal catalyzed by Mycobacterium tuberculosis RuvAB proteins

Initially discovered in Escherichia coli, RuvAB proteins are ubiquitous in bacteria and play a dual role as molecular motor proteins responsible for branch migration of the Holliday junction(s) and reversal of stalled replication forks. Despite mounting genetic evidence for a crucial role of RuvA and RuvB proteins in reversal of stalled replication forks, the mechanistic aspects of this process are still not fully understood. Here, we elucidate the ability of Mycobacterium tuberculosis RuvAB (MtRuvAB) complex to catalyze the reversal of replication forks using a range of DNA replication fork substrates. Our studies show that MtRuvAB, unlike E. coli RuvAB, is able to drive replication fork reversal via the formation of Holliday junction intermediates, suggesting that RuvAB-catalyzed fork reversal involves concerted unwinding and annealing of nascent leading and lagging strands. We also demonstrate the reversal of replication forks carrying hemi-replicated DNA, indicating that MtRuvAB complex-catalyzed fork reversal is independent of symmetry at the fork junction. The fork reversal reaction catalyzed by MtRuvAB is coupled to ATP hydrolysis, is processive, and culminates in the formation of an extended reverse DNA arm. Notably, we found that sequence heterology failed to impede the fork reversal activity of MtRuvAB. We discuss the implications of these results in the context of recognition and processing of varied types of replication fork structures by RuvAB proteins.     

DNA2 drives processing and restart of reversed replication forks in human cells

Accurate processing of stalled or damaged DNA replication forks is paramount to genomic integrity and recent work points to replication fork reversal and restart as a central mechanism to ensuring high-fidelity DNA replication. Here, we identify a novel DNA2- and WRN-dependent mechanism of reversed replication fork processing and restart after prolonged genotoxic stress. The human DNA2 nuclease and WRN ATPase activities functionally interact to degrade reversed replication forks with a 5′-to-3′ polarity and promote replication restart, thus preventing aberrant processing of unresolved replication intermediates. Unexpectedly, EXO1, MRE11, and CtIP are not involved in the same mechanism of reversed fork processing, whereas human RECQ1 limits DNA2 activity by preventing extensive nascent strand degradation. RAD51 depletion antagonizes this mechanism, presumably by preventing reversed fork formation. These studies define a new mechanism for maintaining genome integrity tightly controlled by specific nucleolytic activities and central homologous recombination factors.     

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.     

SUMOylation mediates CtIP’s functions in DNA end resection and replication fork protection

Double-strand breaks and stalled replication forks are a significant threat to genomic stability that can lead to chromosomal rearrangements or cell death. The protein CtIP promotes DNA end resection, an early step in homologous recombination repair, and has been found to protect perturbed forks from excessive nucleolytic degradation. However, it remains unknown how CtIP’s function in fork protection is regulated. Here, we show that CtIP recruitment to sites of DNA damage and replication stress is impaired upon global inhibition of SUMOylation. We demonstrate that CtIP is a target for modification by SUMO-2 and that this occurs constitutively during S phase. The modification is dependent on the activities of cyclin-dependent kinases and the PI-3-kinase-related kinase ATR on CtIP’s carboxyl-terminal region, an interaction with the replication factor PCNA, and the E3 SUMO ligase PIAS4. We also identify residue K578 as a key residue that contributes to CtIP SUMOylation. Functionally, a CtIP mutant where K578 is substituted with a non-SUMOylatable arginine residue is defective in promoting DNA end resection, homologous recombination, and in protecting stalled replication forks from excessive nucleolytic degradation. Our results shed further light on the tightly coordinated regulation of CtIP by SUMOylation in the maintenance of genome stability.     

The Replisome-Coupled E3 Ubiquitin Ligase Rtt101Mms22 Counteracts Mrc1 Function to Tolerate Genotoxic Stress

Faithful DNA replication and repair requires the activity of cullin 4-based E3 ubiquitin ligases (CRL4), but the underlying mechanisms remain poorly understood. The budding yeast Cul4 homologue, Rtt101, in complex with the linker Mms1 and the putative substrate adaptor Mms22 promotes progression of replication forks through damaged DNA. Here we characterized the interactome of Mms22 and found that the Rtt101^Mms22 ligase associates with the replisome progression complex during S-phase via the amino-terminal WD40 domain of Ctf4. Moreover, genetic screening for suppressors of the genotoxic sensitivity of rtt101Δ cells identified a cluster of replication proteins, among them a component of the fork protection complex, Mrc1. In contrast to rtt101Δ and mms22Δ cells, mrc1Δ rtt101Δ and mrc1Δ mms22Δ double mutants complete DNA replication upon replication stress by facilitating the repair/restart of stalled replication forks using a Rad52-dependent mechanism. Our results suggest that the Rtt101^Mms22 E3 ligase does not induce Mrc1 degradation, but specifically counteracts Mrc1’s replicative function, possibly by modulating its interaction with the CMG (Cdc45-MCM-GINS) complex at stalled forks.     

Mms1 and Mms22 stabilize the replisome during replication stress

Mms1 and Mms22 form a Cul4(Ddb1)-like E3 ubiquitin ligase with the cullin Rtt101. In this complex, Rtt101 is bound to the substrate-specific adaptor Mms22 through a linker protein, Mms1. Although the Rtt101(Mms1/Mms22) ubiquitin ligase is important in promoting replication through damaged templates, how it does so has yet to be determined. Here we show that mms1Δ and mms22Δ cells fail to properly regulate DNA replication fork progression when replication stress is present and are defective in recovery from replication fork stress. Consistent with a role in promoting DNA replication, we find that Mms1 is enriched at sites where replication forks have stalled and that this localization requires the known binding partners of Mms1-Rtt101 and Mms22. Mms1 and Mms22 stabilize the replisome during replication stress, as binding of the fork-pausing complex components Mrc1 and Csm3, and DNA polymerase ε, at stalled replication forks is decreased in mms1Δ and mms22Δ. Taken together, these data indicate that Mms1 and Mms22 are important for maintaining the integrity of the replisome when DNA replication forks are slowed by hydroxyurea and thereby promote efficient recovery from replication stress.     

The emerging determinants of replication fork stability

A universal response to replication stress is replication fork reversal, where the nascent complementary DNA strands are annealed to form a protective four-way junction allowing forks to avert DNA damage while replication stress is resolved. However, reversed forks are in turn susceptible to nucleolytic digestion of the regressed nascent DNA arms and rely on dedicated mechanisms to protect their integrity. The most well studied fork protection mechanism involves the BRCA pathway and its ability to catalyze RAD51 nucleofilament formation on the reversed arms of stalled replication forks. Importantly, the inability to prevent the degradation of reversed forks has emerged as a hallmark of BRCA deficiency and underlies genome instability and chemosensitivity in BRCA-deficient cells. In the past decade, multiple factors underlying fork stability have been discovered. These factors either cooperate with the BRCA pathway, operate independently from it to augment fork stability in its absence, or act as enablers of fork degradation. In this review, we examine these novel determinants of fork stability, explore the emergent conceptual underpinnings underlying fork protection, as well as the impact of fork protection on cellular viability and cancer therapy.     

The intra-S phase checkpoint targets Dna2 to prevent stalled replication forks from reversing

When replication forks stall at damaged bases or upon nucleotide depletion, the intra-S phase checkpoint ensures they are stabilized and can restart. In intra-S checkpoint-deficient budding yeast, stalling forks collapse, and ～10% form pathogenic chicken foot structures, contributing to incomplete replication and cell death (Lopes et al., 2001; Sogo et al., 2002; Tercero and Diffley, 2001). Using fission yeast, we report that the Cds1(Chk2) effector kinase targets Dna2 on S220 to regulate, both in vivo and in vitro, Dna2 association with stalled replication forks in chromatin. We demonstrate that Dna2-S220 phosphorylation and the nuclease activity of Dna2 are required to prevent fork reversal. Consistent with this, Dna2 can efficiently cleave obligate precursors of fork regression-regressed leading or lagging strands-on model replication forks. We propose that Dna2 cleavage of regressed nascent strands prevents fork reversal and thus stabilizes stalled forks to maintain genome stability during replication stress.     

The role of HERC2 and RNF8 ubiquitin E3 ligases in the promotion of translesion DNA synthesis in the chicken DT40 cell line

The replicative DNA polymerases are generally blocked by template DNA damage. The resulting replication arrest can be released by one of two post-replication repair (PRR) pathways, translesion DNA synthesis (TLS) and template switching by homologous recombination (HR). The HERC2 ubiquitin ligase plays a role in homologous recombination by facilitating the assembly of the Ubc13 ubiquitin-conjugating enzyme with the RNF8 ubiquitin ligase. To explore the role of HERC2 and RNF8 in PRR, we examined immunoglobulin diversification in chicken DT40 cells deficient in HERC2 and RNF8. Unexpectedly, the HERC2^−/− and RNF8^−/− cells and HERC2^−/−/RNF8^−/− double mutant cells exhibit a significant reduction in the rate of immunoglobulin (Ig) hypermutation, compared to wild-type cells. Further, the HERC2^−/− and RNF8^−/− mutants exhibit defective maintenance of replication fork progression immediately after exposure to UV while retaining proficient post-replicative gap filling. These mutants are both proficient in mono-ubiquitination of PCNA. Taken together, these results suggest that HERC2 and RNF8 promote TLS past abasic sites and UV-lesions at or very close to stalled replication forks.     

Lamin A/C recruits ssDNA protective proteins RPA and RAD51 to stalled replication forks to maintain fork stability

Lamin A/C provides a nuclear scaffold for compartmentalization of genome function that is important for genome integrity. Lamin A/C dysfunction is associated with cancer, aging, and degenerative diseases. The mechanisms whereby lamin A/C regulates genome stability remain poorly understood. We demonstrate a crucial role for lamin A/C in DNA replication. Lamin A/C binds to nascent DNA, especially during replication stress (RS), ensuring the recruitment of replication fork protective factors RPA and RAD51. These ssDNA-binding proteins, considered the first and second responders to RS respectively, function in the stabilization, remodeling, and repair of the stalled fork to ensure proper restart and genome stability. Reduced recruitment of RPA and RAD51 upon lamin A/C depletion elicits replication fork instability (RFI) characterized by MRE11 nuclease–mediated degradation of nascent DNA, RS-induced DNA damage, and sensitivity to replication inhibitors. Importantly, unlike homologous recombination–deficient cells, RFI in lamin A/C-depleted cells is not linked to replication fork reversal. Thus, the point of entry of nucleases is not the reversed fork but regions of ssDNA generated during RS that are not protected by RPA and RAD51. Consistently, RFI in lamin A/C-depleted cells is rescued by exogenous overexpression of RPA or RAD51. These data unveil involvement of structural nuclear proteins in the protection of ssDNA from nucleases during RS by promoting recruitment of RPA and RAD51 to stalled forks. Supporting this model, we show physical interaction between RPA and lamin A/C. We suggest that RS is a major source of genomic instability in laminopathies and lamin A/C-deficient tumors.     

Oncogenes induce genotoxic stress by mitotic processing of unusual replication intermediates

Oncogene-induced DNA replication stress activates the DNA damage response (DDR), a crucial anticancer barrier. DDR inactivation in these conditions promotes genome instability and tumor progression, but the underlying molecular mechanisms are elusive. We found that overexpression of both Cyclin E and Cdc25A rapidly slowed down replication forks and induced fork reversal, suggestive of increased topological stress. Surprisingly, these phenotypes, per se, are neither associated with chromosomal breakage nor with significant DDR activation. Oncogene-induced DNA breakage and DDR activation instead occurred upon persistent G2/M arrest or, in a checkpoint-defective context, upon premature CDK1 activation. Depletion of MUS81, a cell cycle–regulated nuclease, markedly limited chromosomal breakage and led to further accumulation of reversed forks. We propose that nucleolytic processing of unusual replication intermediates mediates oncogene-induced genotoxicity and that limiting such processing to mitosis is a central anti-tumorigenic function of the DNA damage checkpoints.     

HUWE1 interacts with PCNA to alleviate replication stress

Defects in DNA replication, DNA damage response, and DNA repair compromise genomic stability and promote cancer development. In particular, unrepaired DNA lesions can arrest the progression of the DNA replication machinery during S‐phase, causing replication stress, mutations, and DNA breaks. HUWE1 is a HECT‐type ubiquitin ligase that targets proteins involved in cell fate, survival, and differentiation. Here, we report that HUWE1 is essential for genomic stability, by promoting replication of damaged DNA. We show that HUWE1‐knockout cells are unable to mitigate replication stress, resulting in replication defects and DNA breakage. Importantly, we find that this novel role of HUWE1 requires its interaction with the replication factor PCNA, a master regulator of replication fork restart, at stalled replication forks. Finally, we provide evidence that HUWE1 mono‐ubiquitinates H2AX to promote signaling at stalled forks. Altogether, our work identifies HUWE1 as a novel regulator of the replication stress response.     

EEPD1 Rescues Stressed Replication Forks and Maintains Genome Stability by Promoting End Resection and Homologous Recombination Repair

Replication fork stalling and collapse is a major source of genome instability leading to neoplastic transformation or cell death. Such stressed replication forks can be conservatively repaired and restarted using homologous recombination (HR) or non-conservatively repaired using micro-homology mediated end joining (MMEJ). HR repair of stressed forks is initiated by 5’ end resection near the fork junction, which permits 3’ single strand invasion of a homologous template for fork restart. This 5’ end resection also prevents classical non-homologous end-joining (cNHEJ), a competing pathway for DNA double-strand break (DSB) repair. Unopposed NHEJ can cause genome instability during replication stress by abnormally fusing free double strand ends that occur as unstable replication fork repair intermediates. We show here that the previously uncharacterized Exonuclease/Endonuclease/Phosphatase Domain-1 (EEPD1) protein is required for initiating repair and restart of stalled forks. EEPD1 is recruited to stalled forks, enhances 5’ DNA end resection, and promotes restart of stalled forks. Interestingly, EEPD1 directs DSB repair away from cNHEJ, and also away from MMEJ, which requires limited end resection for initiation. EEPD1 is also required for proper ATR and CHK1 phosphorylation, and formation of gamma-H2AX, RAD51 and phospho-RPA32 foci. Consistent with a direct role in stalled replication fork cleavage, EEPD1 is a 5’ overhang nuclease in an obligate complex with the end resection nuclease Exo1 and BLM. EEPD1 depletion causes nuclear and cytogenetic defects, which are made worse by replication stress. Depleting 53BP1, which slows cNHEJ, fully rescues the nuclear and cytogenetic abnormalities seen with EEPD1 depletion. These data demonstrate that genome stability during replication stress is maintained by EEPD1, which initiates HR and inhibits cNHEJ and MMEJ.     

Ubiquitin-specific Protease 11 (USP11) Deubiquitinates Hybrid Small Ubiquitin-like Modifier (SUMO)-Ubiquitin Chains to Counteract RING Finger Protein 4 (RNF4)

Ring finger protein 4 (RNF4) is a SUMO-targeted ubiquitin E3 ligase with a pivotal function in the DNA damage response (DDR). SUMO interaction motifs (SIMs) in the N-terminal part of RNF4 tightly bind to SUMO polymers, and RNF4 can ubiquitinate these polymers in vitro. Using a proteomic approach, we identified the deubiquitinating enzyme ubiquitin-specific protease 11 (USP11), a known DDR-component, as a functional interactor of RNF4. USP11 can deubiquitinate hybrid SUMO-ubiquitin chains to counteract RNF4. SUMO-enriched nuclear bodies are stabilized by USP11, which functions downstream of RNF4 as a counterbalancing factor. In response to DNA damage induced by methyl methanesulfonate, USP11 could counteract RNF4 to inhibit the dissolution of nuclear bodies. Thus, we provide novel insight into cross-talk between ubiquitin and SUMO and uncover USP11 and RNF4 as a balanced SUMO-targeted ubiquitin ligase/protease pair with a role in the DDR.     

Structural analysis of DNA replication fork reversal by RecG

The stalling of DNA replication forks that occurs as a consequence of encountering DNA damage is a critical problem for cells. RecG protein is involved in the processing of stalled replication forks, and acts by reversing the fork past the damage to create a four-way junction that allows template switching and lesion bypass. We have determined the crystal structure of RecG bound to a DNA substrate that mimics a stalled replication fork. The structure not only reveals the elegant mechanism used by the protein to recognize junctions but has also trapped the protein in the initial stage of fork reversal. We propose a mechanism for how forks are processed by RecG to facilitate replication fork restart. In addition, this structure suggests that the mechanism and function of the two largest helicase superfamilies are distinct.     

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.     

Direct rescue of stalled DNA replication forks via the combined action of PriA and RecG helicase activities

The PriA protein of Escherichia coli plays a key role in the rescue of replication forks stalled on the template DNA. One attractive model of rescue relies on homologous recombination to establish a new fork via PriA-mediated loading of the DnaB replicative helicase at D loop intermediates. We provide genetic and biochemical evidence that PriA helicase activity can also rescue a stalled fork by an alternative mechanism that requires manipulation of the fork before loading of DnaB on the lagging strand template. This direct rescue depends on RecG, which unwinds forks and Holliday junctions and interconverts these structures. The combined action of PriA and RecG helicase activities may thus avoid the potential dangers of rescue pathways involving fork breakage and recombination.     

Dna2 offers support for stalled forks

The ATR and ATM checkpoint kinases preserve the integrity of replicating chromosomes by preventing the reversal of stalled and terminal replication forks. Hu et al. now show that the ATR pathway targets the Dna2 nuclease to process stalled forks and counteract fork reversal.     

Two redundant ubiquitin‐dependent pathways of BRCA1 localization to DNA damage sites

The tumor suppressor BRCA1 accumulates at sites of DNA damage in a ubiquitin‐dependent manner. In this work, we revisit the role of RAP80 in promoting BRCA1 recruitment to damaged chromatin. We find that RAP80 acts redundantly with the BRCA1 RING domain to promote BRCA1 recruitment to DNA damage sites. We show that that RNF8 E3 ligase acts upstream of both the RAP80‐ and RING‐dependent activities, whereas RNF168 acts uniquely upstream of the RING domain. BRCA1 RING mutations that do not impact BARD1 interaction, such as the E2 binding‐deficient I26A mutation, render BRCA1 unable to accumulate at DNA damage sites in the absence of RAP80. Cells that combine BRCA1 I26A and mutations that disable the RAP80–BRCA1 interaction are hypersensitive to PARP inhibition and are unable to form RAD51 foci. Our results suggest that in the absence of RAP80, the BRCA1 E3 ligase activity is necessary for recognition of histone H2A Lys13/Lys15 ubiquitylation by BARD1, although we cannot rule out the possibility that the BRCA1 RING facilitates ubiquitylated nucleosome recognition in other ways.