RNA polymerase III is required for the repair of DNA double-strand breaks by homologous recombination

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The S‐phase checkpoint: targeting the replication fork

The S‐phase checkpoint is a surveillance mechanism, mediated by the protein kinases Mec1 and Rad53 in the budding yeast Saccharomyces cerevisiae (ATR and Chk2 in human cells, respectively) that responds to DNA damage and replication perturbations by co‐ordinating a global cellular response necessary to maintain genome integrity. A key aspect of this response is the stabilization of DNA replication forks, which is critical for cell survival. A defective checkpoint causes irreversible replication‐fork collapse and leads to genomic instability, a hallmark of cancer cells. Although the precise mechanisms by which Mec1/Rad53 maintain functional replication forks are currently unclear, our knowledge about this checkpoint function has significantly increased during the last years. Focusing mainly on the advances obtained in S. cerevisiae, the present review will summarize our understanding of how the S‐phase checkpoint preserves the integrity of DNA replication forks and discuss the most recent findings on this topic.     

SCFDia2 regulates DNA replication forks during S‐phase in budding yeast

Dia2 is an F‐box protein, which is involved in the regulation of DNA replication in the budding yeast Saccharomyces cerevisiae. The function of Dia2, however, remains largely unknown. In this study, we report that Dia2 is associated with the replication fork and regulates replication fork progression. Using modified yeast two‐hybrid screening, we have identified components of the replisome (Mrc1, Ctf4 and Mcm2), as Dia2‐binding proteins. Mrc1 and Ctf4 were ubiquitinated by SCF^Dia2 both in vivo and in vitro. Domain analysis of Dia2 revealed that the leucine‐rich repeat motif was indispensable for the regulation of replisome progression, whereas the tetratricopeptide repeat (TPR) motif was involved in the interaction with replisome components. In addition, the TPR motif was shown to be involved in Dia2 stability; deleting the TPR stabilized Dia2, mimicking the effect of DNA damage. ChIP‐on‐chip analysis illustrated that Dia2 localizes to the replication fork and regulates fork progression on hydroxyurea treatment. These results demonstrate that Dia2 is involved in the regulation of replisome activity through a direct interaction with replisome components.     

PARP is activated at stalled forks to mediate Mre11‐dependent replication restart and recombination

If replication forks are perturbed, a multifaceted response including several DNA repair and cell cycle checkpoint pathways is activated to ensure faithful DNA replication. Here, we show that poly(ADP‐ribose) polymerase 1 (PARP1) binds to and is activated by stalled replication forks that contain small gaps. PARP1 collaborates with Mre11 to promote replication fork restart after release from replication blocks, most likely by recruiting Mre11 to the replication fork to promote resection of DNA. Both PARP1 and PARP2 are required for hydroxyurea‐induced homologous recombination to promote cell survival after replication blocks. Together, our data suggest that PARP1 and PARP2 detect disrupted replication forks and attract Mre11 for end processing that is required for subsequent recombination repair and restart of replication forks.     

Rad52 recruitment is DNA replication independent and regulated by Cdc28 and the Mec1 kinase

Recruitment of the homologous recombination machinery to sites of double‐strand breaks is a cell cycle‐regulated event requiring entry into S phase and CDK1 activity. Here, we demonstrate that the central recombination protein, Rad52, forms foci independent of DNA replication, and its recruitment requires B‐type cyclin/CDK1 activity. Induction of the intra‐S‐phase checkpoint by hydroxyurea (HU) inhibits Rad52 focus formation in response to ionizing radiation. This inhibition is dependent upon Mec1/Tel1 kinase activity, as HU‐treated cells form Rad52 foci in the presence of the PI3 kinase inhibitor caffeine. These Rad52 foci colocalize with foci formed by the replication clamp PCNA. These results indicate that Mec1 activity inhibits the recruitment of Rad52 to both sites of DNA damage and stalled replication forks during the intra‐S‐phase checkpoint. We propose that B‐type cyclins promote the recruitment of Rad52 to sites of DNA damage, whereas Mec1 inhibits spurious recombination at stalled replication forks.     

The MMS22L–TONSL heterodimer directly promotes RAD51‐dependent recombination upon replication stress

Homologous recombination (HR) is a key pathway that repairs DNA double‐strand breaks (DSBs) and helps to restart stalled or collapsed replication forks. How HR supports replication upon genotoxic stress is not understood. Using in vivo and in vitro approaches, we show that the MMS22L–TONSL heterodimer localizes to replication forks under unperturbed conditions and its recruitment is increased during replication stress in human cells. MMS22L–TONSL associates with replication protein A (RPA)‐coated ssDNA, and the MMS22L subunit directly interacts with the strand exchange protein RAD51. MMS22L is required for proper RAD51 assembly at DNA damage sites in vivo, and HR‐mediated repair of stalled forks is abrogated in cells expressing a MMS22L mutant deficient in RAD51 interaction. Similar to the recombination mediator BRCA2, recombinant MMS22L–TONSL limits the assembly of RAD51 on dsDNA, which stimulates RAD51‐ssDNA nucleoprotein filament formation and RAD51‐dependent strand exchange activity in vitro. Thus, by specifically regulating RAD51 activity at uncoupled replication forks, MMS22L–TONSL stabilizes perturbed replication forks by promoting replication fork reversal and stimulating their HR‐mediated restart in vivo.     

ATM-dependent chromatin remodeler Rsf-1 facilitates DNA damage checkpoints and homologous recombination repair

As a member of imitation switch (ISWI) family in ATP-dependent chromatin remodeling factors, RSF complex consists of SNF2h ATPase and Rsf-1. Although it has been reported that SNF2h ATPase is recruited to DNA damage sites (DSBs) in a poly(ADP-ribosyl) polymerase 1 (PARP1)-dependent manner in DNA damage response (DDR), the function of Rsf-1 is still elusive. Here we show that Rsf-1 is recruited to DSBs confirmed by various cellular analyses. Moreover, the initial recruitment of Rsf-1 and SNF2h to DSBs shows faster kinetics than that of γH2AX after micro-irradiation. Signals of Rsf-1 and SNF2h are retained over 30 min after micro-irradiation, whereas γH2AX signals are gradually reduced at 10 min. In addition, Rsf-1 is accumulated at DSBs in ATM-dependent manner, and the putative pSQ motifs of Rsf-1 by ATM are required for its accumulation at DSBs. Furtheremore, depletion of Rsf-1 attenuates the activation of DNA damage checkpoint signals and cell survival upon DNA damage. Finally, we demonstrate that Rsf-1 promotes homologous recombination repair (HRR) by recruiting resection factors RPA32 and Rad51. Thus, these findings reveal a new function of chromatin remodeler Rsf-1 as a guard in DNA damage checkpoints and homologous recombination repair.     

DNA polymerase κ‐dependent DNA synthesis at stalled replication forks is important for CHK1 activation

Formation of primed single‐stranded DNA at stalled replication forks triggers activation of the replication checkpoint signalling cascade resulting in the ATR‐mediated phosphorylation of the Chk1 protein kinase, thus preventing genomic instability. By using siRNA‐mediated depletion in human cells and immunodepletion and reconstitution experiments in Xenopus egg extracts, we report that the Y‐family translesion (TLS) DNA polymerase kappa (Pol κ) contributes to the replication checkpoint response and is required for recovery after replication stress. We found that Pol κ is implicated in the synthesis of short DNA intermediates at stalled forks, facilitating the recruitment of the 9‐1‐1 checkpoint clamp. Furthermore, we show that Pol κ interacts with the Rad9 subunit of the 9‐1‐1 complex. Finally, we show that this novel checkpoint function of Pol κ is required for the maintenance of genomic stability and cell proliferation in unstressed human cells.     

Dynamic elements of replication protein A at the crossroads of DNA replication,recombination, and repair

Abstract

The heterotrimeric eukaryotic Replication protein A (RPA) is a master regulator of numerous DNA metabolic processes. For a long time, it has been viewed as an inert protector of ssDNA and a platform for assembly of various genome maintenance and signaling machines. Later, the modular organization of the RPA DNA binding domains suggested a possibility for dynamic interaction with ssDNA. This modular organization has inspired several models for the RPA-ssDNA interaction that aimed to explain how RPA, the high-affinity ssDNA binding protein, is replaced by the downstream players in DNA replication, recombination, and repair that bind ssDNA with much lower affinity. Recent studies, and in particular single-molecule observations of RPA-ssDNA interactions, led to the development of a new model for the ssDNA handoff from RPA to a specific downstream factor where not only stability and structural rearrangements but also RPA conformational dynamics guide the ssDNA handoff. Here we will review the current knowledge of the RPA structure, its dynamic interaction with ssDNA, and how RPA conformational dynamics may be influenced by posttranslational modification and proteins that interact with RPA, as well as how RPA dynamics may be harnessed in cellular decision making.     

FANCM regulates repair pathway choice at stalled replication forks

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Sgs1 function in the repair of DNA replication intermediates is separable from its role in homologous recombinational repair

Mutations in human homologues of the bacterial RecQ helicase cause diseases leading to cancer predisposition and/or shortened lifespan (Werner, Bloom, and Rothmund–Thomson syndromes). The budding yeast Saccharomyces cerevisiae has one RecQ helicase, Sgs1, which functions with Top3 and Rmi1 in DNA repair. Here, we report separation‐of‐function alleles of SGS1 that suppress the slow growth of top3Δ and rmi1Δ cells similar to an SGS1 deletion, but are resistant to DNA damage similar to wild‐type SGS1. In one allele, the second acidic region is deleted, and in the other, only a single aspartic acid residue 664 is deleted. sgs1‐D664Δ, unlike sgs1Δ, neither disrupts DNA recombination nor has synthetic growth defects when combined with DNA repair mutants. However, during S phase, it accumulates replication‐associated X‐shaped structures at damaged replication forks. Furthermore, fluorescent microscopy reveals that the sgs1‐D664Δ allele exhibits increased spontaneous RPA foci, suggesting that the persistent X‐structures may contain single‐stranded DNA. Taken together, these results suggest that the Sgs1 function in repair of DNA replication intermediates can be uncoupled from its role in homologous recombinational repair.     

FANCM regulates DNA chain elongation and is stabilized by S‐phase checkpoint signalling

FANCM binds and remodels replication fork structures in vitro. We report that in vivo, FANCM controls DNA chain elongation in an ATPase‐dependent manner. In the presence of replication inhibitors that do not damage DNA, FANCM counteracts fork movement, possibly by remodelling fork structures. Conversely, through damaged DNA, FANCM promotes replication and recovers stalled forks. Hence, the impact of FANCM on fork progression depends on the underlying hindrance. We further report that signalling through the checkpoint effector kinase Chk1 prevents FANCM from degradation by the proteasome after exposure to DNA damage. FANCM also acts in a feedback loop to stabilize Chk1. We propose that FANCM is a ringmaster in the response to replication stress by physically altering replication fork structures and by providing a tight link to S‐phase checkpoint signalling.     

Extended DNA-binding interfaces beyond the canonical SAP domain contribute to the function of replication stress regulator SDE2 at DNA replication forks

Elevated DNA replication stress causes instability of the DNA replication fork and increased DNA mutations, which underlies tumorigenesis. The DNA replication stress regulator silencing-defective 2 (SDE2) is known to bind to TIMELESS (TIM), a protein of the fork protection complex, and enhances its stability, thereby supporting replisome activity at DNA replication forks. However, the DNA-binding activity of SDE2 is not well defined. Here, we structurally and functionally characterize a new conserved DNA-binding motif related to the SAP (SAF-A/B, Acinus, PIAS) domain in human SDE2 and establish its preference for ssDNA. Our NMR solution structure of the SDE2^SAP domain reveals a helix-extended loop-helix core with the helices aligned parallel to each other, consistent with known canonical SAP folds. Notably, we have shown that the DNA interaction of this SAP domain extends beyond the core SAP domain and is augmented by two lysine residues in the C-terminal tail, which is uniquely positioned adjacent to the SAP motif and conserved in the pre-mRNA splicing factor SF3A3. Furthermore, we found that mutation in the SAP domain and extended C terminus not only disrupts ssDNA binding but also impairs TIM localization at replication forks, thus inhibiting efficient fork progression. Taken together, our results establish SDE2^SAP as an essential element for SDE2 to exert its role in preserving replication fork integrity via fork protection complex regulation and highlight the structural diversity of the DNA–protein interactions achieved by a specialized DNA-binding motif.     

ATM and Artemis promote homologous recombination of radiation‐induced DNA double‐strand breaks in G2

Homologous recombination (HR) and non‐homologous end joining (NHEJ) represent distinct pathways for repairing DNA double‐strand breaks (DSBs). Previous work implicated Artemis and ATM in an NHEJ‐dependent process, which repairs a defined subset of radiation‐induced DSBs in G1‐phase. Here, we show that in G2, as in G1, NHEJ represents the major DSB‐repair pathway whereas HR is only essential for repair of ～15% of X‐ or γ‐ray‐induced DSBs. In addition to requiring the known HR proteins, Brca2, Rad51 and Rad54, repair of radiation‐induced DSBs by HR in G2 also involves Artemis and ATM suggesting that they promote NHEJ during G1 but HR during G2. The dependency for ATM for repair is relieved by depleting KAP‐1, providing evidence that HR in G2 repairs heterochromatin‐associated DSBs. Although not core HR proteins, ATM and Artemis are required for efficient formation of single‐stranded DNA and Rad51 foci at radiation‐induced DSBs in G2 with Artemis function requiring its endonuclease activity. We suggest that Artemis endonuclease removes lesions or secondary structures, which inhibit end resection and preclude the completion of HR or NHEJ.     

CDK targeting of NBS1 promotes DNA-end resection, replication restart and homologous recombination

The conserved MRE11–RAD50–NBS1 (MRN) complex is an important sensor of DNA double-strand breaks (DSBs) and facilitates DNA repair by homologous recombination (HR) and end joining. Here, we identify NBS1 as a target of cyclin-dependent kinase (CDK) phosphorylation. We show that NBS1 serine 432 phosphorylation occurs in the S, G2 and M phases of the cell cycle and requires CDK activity. This modification stimulates MRN-dependent conversion of DSBs into structures that are substrates for repair by HR. Impairment of NBS1 phosphorylation not only negatively affects DSB repair by HR, but also prevents resumption of DNA replication after replication-fork stalling. Thus, CDK-mediated NBS1 phosphorylation defines a molecular switch that controls the choice of repair mode for DSBs.     

Uncoupling of unwinding from DNA synthesis implies regulation of MCM helicase by Tof1/Mrc1/Csm3 checkpoint complex

The replicative DNA helicases can unwind DNA in the absence of polymerase activity in vitro. In contrast, replicative unwinding is coupled with DNA synthesis in vivo. The temperature-sensitive yeast polymerase alpha/primase mutants cdc17-1, pri2-1 and pri1-m4, which fail to execute the early step of DNA replication, have been used to investigate the interaction between replicative unwinding and DNA synthesis in vivo. We report that some of the plasmid molecules in these mutant strains became extensively negatively supercoiled when DNA synthesis is prevented. In contrast, additional negative supercoiling was not detected during formation of DNA initiation complex or hydroxyurea replication fork arrest. Together, these results indicate that the extensive negative supercoiling of DNA is a result of replicative unwinding, which is not followed by DNA synthesis. The limited number of unwound plasmid molecules and synthetic lethality of polymerase alpha or primase with checkpoint mutants suggest a checkpoint regulation of the replicative unwinding. In concordance with this suggestion, we found that the Tof1/Csm3/Mrc1 checkpoint complex interacts directly with the MCM helicase during both replication fork progression and when the replication fork is stalled.     

Structural basis for inhibition of homologous recombination by the RecX protein

The RecA/RAD51 nucleoprotein filament is central to the reaction of homologous recombination (HR). Filament activity must be tightly regulated in vivo as unrestrained HR can cause genomic instability. Our mechanistic understanding of HR is restricted by lack of structural information about the regulatory proteins that control filament activity. Here, we describe a structural and functional analysis of the HR inhibitor protein RecX and its mode of interaction with the RecA filament. RecX is a modular protein assembled of repeated three-helix motifs. The relative arrangement of the repeats generates an elongated and curved shape that is well suited for binding within the helical groove of the RecA filament. Structure-based mutagenesis confirms that conserved basic residues on the concave side of RecX are important for repression of RecA activity. Analysis of RecA filament dynamics in the presence of RecX shows that RecX actively promotes filament disassembly. Collectively, our data support a model in which RecX binding to the helical groove of the filament causes local dissociation of RecA protomers, leading to filament destabilisation and HR inhibition.     

Parental histone distribution and location of the replication obstacle at nascent strands control homologous recombination

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Sensing, signaling, and responding to DNA damage: organization of the checkpoint pathways in mammalian cells

The DNA damage and replication checkpoints are signaling mechanisms that regulate and coordinate cellular responses to genotoxic conditions. Unlike typical signal transduction mechanisms that respond to one or a few stimuli, checkpoints can be activated by a broad spectrum of extrinsically or intrinsically derived DNA damage or replication interference. Recent investigations have shed light on how the damage and replication checkpoints are able to respond to such diverse stimuli. The activation of checkpoints not only attenuates cell cycle progression but also facilitates DNA repair and recovery of faltered replication forks, thereby preventing DNA lesions from being converted to inheritable mutations. Recently, more checkpoint targets from the cell cycle and DNA replication apparatus have been identified, revealing the increasing complexity of the checkpoint control of the cell cycle. In this article, we discuss current models of the DNA damage and replication checkpoints and highlight recent advances in the field.