Histone chaperone Anp32e removes H2A.Z from DNA double-strand breaks and promotes nucleosome reorganization and DNA repair

The repair of DNA double-strand breaks (DSBs) requires open, flexible chromatin domains. The NuA4–Tip60 complex creates these flexible chromatin structures by exchanging histone H2A.Z onto nucleosomes and promoting acetylation of histone H4. Here, we demonstrate that the accumulation of H2A.Z on nucleosomes at DSBs is transient, and that rapid eviction of H2A.Z is required for DSB repair. Anp32e, an H2A.Z chaperone that interacts with the C-terminal docking domain of H2A.Z, is rapidly recruited to DSBs. Anp32e functions to remove H2A.Z from nucleosomes, so that H2A.Z levels return to basal within 10 min of DNA damage. Further, H2A.Z removal by Anp32e disrupts inhibitory interactions between the histone H4 tail and the nucleosome surface, facilitating increased acetylation of histone H4 following DNA damage. When H2A.Z removal by Anp32e is blocked, nucleosomes at DSBs retain elevated levels of H2A.Z, and assume a more stable, hypoacetylated conformation. Further, loss of Anp32e leads to increased CtIP-dependent end resection, accumulation of single-stranded DNA, and an increase in repair by the alternative nonhomologous end joining pathway. Exchange of H2A.Z onto the chromatin and subsequent rapid removal by Anp32e are therefore critical for creating open, acetylated nucleosome structures and for controlling end resection by CtIP. Dynamic modulation of H2A.Z exchange and removal by Anp32e reveals the importance of the nucleosome surface and nucleosome dynamics in processing the damaged chromatin template during DSB repair.The repair of DNA double-strand breaks (DSBs), which cleave the DNA backbone, requires remodeling of the local chromatin architecture. This reorganization of the chromatin is important for promoting access to the site of damage, for creating a template for the repair machinery, and for repackaging the chromatin and resetting the epigenetic landscape following repair. Chromatin remodeling at DSBs is linked to changes in posttranslational modification of histones. DSBs activate the ataxia-telangiectasia mutated (ATM) and DNA–PKcs kinases, which phosphorylate multiple DNA repair proteins, including histone H2AX. Phosphorylated H2AX (γH2AX) provides a binding site for mdc1, which promotes spreading of γH2AX for hundreds of kilobases either side of the break (1–3). DSBs also promote complex patterns of chromatin ubiquitination, including ubiquitination of H2A/H2AX by the RNF8/RNF168 ubiquitin ligases, which, in turn, creates binding sites for repair proteins such as 53BP1 and brca1 (4–7). DSBs also lead to increased methylation of histone H3 on lysine 36, which can regulate DNA repair pathway choice (8, 9) and methylation of H3 on lysine 9 (10), which drives activation of the Tip60 acetyltransferase and the ATM kinase (11, 12). Further, the NuA4–Tip60 complex (5, 13–15) promotes acetylation of histone H4 at DSBs and drives the formation of open, flexible chromatin domains at DSBs (5, 13, 14). The repair of DSBs is therefore fundamentally a chromatin-driven process requiring dynamic changes in histone modification and chromatin reorganization, which directly promote recruitment of DSB repair proteins (16).The NuA4–Tip60 remodeling complex plays a central role in nucleosome reorganization at DSBs (16). NuA4–Tip60 is a 16 subunit complex containing 2 key subunits—the p400 SWI/SNF ATPase and the Tip60 acetyltransferase. The p400 ATPase promotes exchange of H2A for the histone variant H2A.Z (13, 17). This increase in H2A.Z at DSBs then promotes acetylation of histone H4 by the Tip60, creating open, flexible chromatin at sites of DNA damage (5, 11, 14). Inactivation of NuA4–Tip60 blocks both H2A.Z exchange and H4 acetylation, leading to a reduction in chromatin mobility at DSBs. Consequently, loss of H2A.Z exchange leads to defective DSB repair, increased sensitivity to DNA damage, and genomic instability (13, 18, 19).Here, we demonstrate that H2A.Z exchange at DSBs is dynamic, with H2A.Z accumulating at DSBs within minutes of damage, followed by rapid H2A.Z eviction. Further, we show that Anp32e, an H2A.Z-specific histone chaperone, binds specifically to the docking domain of H2A.Z and is required to remove H2A.Z from the damaged chromatin template. Failure to remove H2A.Z leads to defects in DSB repair, including a loss of H4 acetylation, defects in nonhomologous end joining (NHEJ), and increased end resection of DSBs.     

DNA double-strand breaks promote methylation of histone H3 on lysine 9 and transient formation of repressive chromatin

Dynamic changes in histone modification are critical for regulating DNA double-strand break (DSB) repair. Activation of the Tip60 acetyltransferase by DSBs requires interaction of Tip60 with histone H3 methylated on lysine 9 (H3K9me3). However, how H3K9 methylation is regulated during DSB repair is not known. Here, we demonstrate that a complex containing kap-1, HP1, and the H3K9 methyltransferase suv39h1 is rapidly loaded onto the chromatin at DSBs. Suv39h1 methylates H3K9, facilitating loading of additional kap-1/HP1/suv39h1 through binding of HP1’s chromodomain to the nascent H3K9me3. This process initiates cycles of kap-1/HP1/suv39h1 loading and H3K9 methylation that facilitate spreading of H3K9me3 and kap-1/HP1/suv39h1 complexes for tens of kilobases away from the DSB. These domains of H3K9me3 function to activate the Tip60 acetyltransferase, allowing Tip60 to acetylate both ataxia telangiectasia-mutated (ATM) kinase and histone H4. Consequently, cells lacking suv39h1 display defective activation of Tip60 and ATM, decreased DSB repair, and increased radiosensitivity. Importantly, activated ATM rapidly phosphorylates kap-1, leading to release of the repressive kap-1/HP1/suv39h1 complex from the chromatin. ATM activation therefore functions as a negative feedback loop to remove repressive suv39h1 complexes at DSBs, which may limit DSB repair. Recruitment of kap-1/HP1/suv39h1 to DSBs therefore provides a mechanism for transiently increasing the levels of H3K9me3 in open chromatin domains that lack H3K9me3 and thereby promoting efficient activation of Tip60 and ATM in these regions. Further, transient formation of repressive chromatin may be critical for stabilizing the damaged chromatin and for remodeling the chromatin to create an efficient template for the DNA repair machinery.DNA double-strand breaks (DSBs) are toxic and must be repaired to maintain genomic stability. Detection of DSBs requires recruitment of the mre11–rad50–nbs1 (MRN) complex to the DNA ends (1). MRN then recruits and activates the ataxia telangiectasia-mutated (ATM) kinase (2, 3) through a mechanism that also requires the Tip60 acetyltransferase (3). Tip60 directly acetylates and activates ATM’s kinase activity (4–6) and functions, in combination with MRN, to promote ATM-dependent phosphorylation of DSB repair proteins (3), including histone H2AX. This process creates domains of phosphorylated H2AX (γH2AX) extending for hundreds of kilobases along the chromatin (7, 8). Mdc1 then binds to γH2AX, providing a landing pad for other DSB repair proteins, including the RNF8/RNF168 ubiquitin ligases (1, 3, 9, 10). Tip60 also plays a critical role in regulating chromatin structure at DSBs as part of the NuA4–Tip60 complex (11). NuA4-Tip60 catalyzes histone exchange (via the p400 ATPase subunit) and acetylation of histone H4 (by Tip60) at DSBs (12–15), leading to the formation of open, flexible chromatin domains adjacent to the break (12, 13). These open chromatin structures then facilitate histone ubiquitination, the loading of brca1 and 53BP1, and repair of the DSB (13, 16). The ordered acetylation and ubiquitination of the chromatin and loading of DNA repair proteins is therefore critical for DSB repair.Activation of Tip60’s acetyltransferase activity requires interaction between Tip60’s chromodomain and histone H3 methylated on lysine 9 (H3K9me3) on nucleosomes at the break (4, 6). This interaction, in combination with tyrosine phosphorylation of Tip60 (17), increases Tip60’s acetyltransferase activity and promotes acetylation of both the ATM kinase and histone H4 (4–6, 17). Consequently, loss of H3K9me2/3 leads to failure to activate the ATM signaling pathway, loss of H4 acetylation during DSB repair, disruption of heterochromatin, genomic instability, and defective DSB repair (4, 17–19). H3K9me3s therefore play a critical role in linking chromatin structure at DSBs to the activation of DSB signaling proteins such as Tip60 and ATM.How Tip60 gains access to H3K9me3 and how H3K9me3 levels at DSBs are regulated is not known. H3K9me3 is concentrated in heterochromatin domains, where it recruits HP1, kap-1, and H3K9 methyltransferases (20, 21) to maintain the silent, compact conformation of heterochromatin (20). This implies that Tip60’s acetyltransferase activity can only be activated at DSBs in regions of high H3K9me3 density, such as heterochromatin. Alternatively, H3K9 methylation may be actively increased at DSBs in regions of low H3K9me3 density to allow for Tip60 activation and efficient DSB repair in euchromatin. Understanding the dynamics of H3K9 methylation at DSBs is therefore critical to understanding how Tip60 activity is regulated by the local chromatin architecture. Here, we show that the suv39h1 methyltransferase is recruited to DSBs in euchromatin as part of a larger kap-1/HP1/suv39h1 complex. Suv39h1 increases H3K9me3 at DSBs, activating Tip60’s acetyltransferase activity and promoting the subsequent acetylation and activation of ATM. Further, loss of inducible H3K9me3 at DSBs leads to defective repair and increased radiosensitivity. Finally, loading of the kap-1/HP1/suv39h1 complex is transient, and the complex is rapidly released from the chromatin through a negative feedback loop driven by ATM-dependent phosphorylation of the kap-1 protein.     

UbcH7 regulates 53BP1 stability and DSB repair

DNA double-strand break (DSB) repair is not only key to genome stability but is also an important anticancer target. Through an shRNA library-based screening, we identified ubiquitin-conjugating enzyme H7 (UbcH7, also known as Ube2L3), a ubiquitin E2 enzyme, as a critical player in DSB repair. UbcH7 regulates both the steady-state and replicative stress-induced ubiquitination and proteasome-dependent degradation of the tumor suppressor p53-binding protein 1 (53BP1). Phosphorylation of 53BP1 at the N terminus is involved in the replicative stress-induced 53BP1 degradation. Depletion of UbcH7 stabilizes 53BP1, leading to inhibition of DSB end resection. Therefore, UbcH7-depleted cells display increased nonhomologous end-joining and reduced homologous recombination for DSB repair. Accordingly, UbcH7-depleted cells are sensitive to DNA damage likely because they mainly used the error-prone nonhomologous end-joining pathway to repair DSBs. Our studies reveal a novel layer of regulation of the DSB repair choice and propose an innovative approach to enhance the effect of radiotherapy or chemotherapy through stabilizing 53BP1.Prompt response to double-strand breaks (DSBs) caused by, for example, ionization radiation (IR), requires sequential and coordinated assembly of DNA damage response (DDR) proteins at damage sites (1). Recent research findings reveal key roles of the tumor suppressor p53-binding protein 1 (53BP1) and BRCA1 in the decision making of DSB repair. 53BP1, together with Rif1, suppress BRCA1-dependent homologous recombination (HR), thereby promoting nonhomologous end-joining (NHEJ) in G1 phase (2–6). Conversely, BRCA1 antagonizes 53BP1/Rif1, favoring HR in S and G2 phases (7, 8). In the absence of BRCA1 or with enhanced retention of 53BP1 at DSB sites, cells primarily use the error-prone NHEJ to repair DSBs throughout the cell cycle, which leads to gene rearrangement, cell death, and increased sensitivity to anticancer therapies (9–11). Consistently, BRCA1-null mice are early embryonic lethal (12, 13) and codepletion of TP53BP1 rescued the lethality phenotype of BRCA1-null mice (12–14).Low expression level of 53BP1 was found to be associated with poor clinical outcome in triple negative breast cancer patients with BRCA1 mutation (12, 15), as well as resistance to genotoxins and poly(ADP-ribose) polymerase inhibitors (12, 16, 17). This finding is probably because loss of 53BP1 restored HR and promoted cell survival (12–14). Reduced expression of 53BP1 was also observed in tumors from the brain (18), lymph node (19), and pancreas (20). These data indicate that loss of 53BP1 might be a common mechanism for advanced tumors to evade from radiotherapy or chemotherapy. However, molecular mechanisms controlling the protein level of 53BP1 remain less well understood.Here we show that UbcH7, an E2 enzyme involved in the ubiquitin (Ub) pathway, controls the protein stability of 53BP1, thereby determining the DSB repair choice. Loss of UbcH7 stabilizes 53BP1, forcing cells to choose NHEJ, but not HR, to repair DSBs, which poses a significant threat to cells treated with DNA damage, especially S-phase genotoxins, such as camptothecin (CPT), a topoisomerase 1 (Top1) inhibitor. The ternary CPT-Top1-DNA complex places a roadblock in the path of advancing DNA replication forks, leading to replication fork collapse and generation of one-ended DSBs. Such one-ended DSBs require HR, but not NHEJ, to repair (8). In contrast, repair of one-ended DSBs by NHEJ leads to radial chromosomes and cell death (12–14). Therefore, stabilization of 53BP1 by UbcH7 depletion increased the sensitivity of cancers cells to CPT and other DNA damaging agents. Our data suggest a novel strategy in enhancing the anticancer effect of radiotherapy or chemotherapy through stabilizing or increasing 53BP1.     

DNA polymerases δ and λ cooperate in repairing double-strand breaks by microhomology-mediated end-joining in Saccharomyces cerevisiae

Maintenance of genome stability is carried out by a suite of DNA repair pathways that ensure the repair of damaged DNA and faithful replication of the genome. Of particular importance are the repair pathways, which respond to DNA double-strand breaks (DSBs), and how the efficiency of repair is influenced by sequence homology. In this study, we developed a genetic assay in diploid Saccharomyces cerevisiae cells to analyze DSBs requiring microhomologies for repair, known as microhomology-mediated end-joining (MMEJ). MMEJ repair efficiency increased concomitant with microhomology length and decreased upon introduction of mismatches. The central proteins in homologous recombination (HR), Rad52 and Rad51, suppressed MMEJ in this system, suggesting a competition between HR and MMEJ for the repair of a DSB. Importantly, we found that DNA polymerase delta (Pol δ) is critical for MMEJ, independent of microhomology length and base-pairing continuity. MMEJ recombinants showed evidence that Pol δ proofreading function is active during MMEJ-mediated DSB repair. Furthermore, mutations in Pol δ and DNA polymerase 4 (Pol λ), the DNA polymerase previously implicated in MMEJ, cause a synergistic decrease in MMEJ repair. Pol λ showed faster kinetics associating with MMEJ substrates following DSB induction than Pol δ. The association of Pol δ depended on RAD1, which encodes the flap endonuclease needed to cleave MMEJ intermediates before DNA synthesis. Moreover, Pol δ recruitment was diminished in cells lacking Pol λ. These data suggest cooperative involvement of both polymerases in MMEJ.DNA double-strand breaks (DSBs) are toxic lesions that can be repaired by two major pathways in eukaryotes: nonhomologous end-joining (NHEJ) and homologous recombination (HR) (1). Although HR repairs DSBs in a template-dependent, high-fidelity manner, NHEJ functions to ligate DSB ends together using no or very short (1–4 bp) homology. Recently, a new pathway was identified in eukaryotes, which uses microhomologies (MHs) to repair a DSB and does not require the central proteins used in HR (Rad51, Rad52) or NHEJ (Ku70–Ku80) (2–5). In mammalian cells, this pathway of repair is known as alternative end-joining (Alt-EJ) and is often but not always associated with MHs, whereas in budding yeast, the commensurate pathway, MH-mediated end-joining (MMEJ), will typically use 5–25 bp of MH (6, 7). These pathways are associated with genomic rearrangements, and cancer genomes show evidence of MH-mediated rearrangements (8–12). In addition, eukaryotic genomes contain many dispersed repetitive elements that can lead to genome rearrangements when recombination occurs between them (13–16). Therefore, controlling DSB repair in the human genome, which features a variety of repeats, is especially important given the fact that recombination between repetitive elements has been implicated in genomic instability associated with disease (17–20).The original characterization of Alt-EJ in mammalian cells suggested it did not represent a significant DNA repair pathway and only operated in the absence of functional HR and NHEJ pathways. More recent analyses demonstrate a physiological role of Alt-EJ during DNA repair in the presence of active HR and NHEJ pathways (2, 12, 21, 22). Furthermore, examination of I-SceI–induced translocation junctions in mammalian cells revealed the frequent presence of MHs (23, 24). NHEJ-deficient and p53-null mice develop pro–B-cell lymphomas, and nonreciprocal translocations characterized by small MHs are found at their break point junctions (25–28). Similarly, in human cancers, many translocation break point junctions contain MHs, suggesting a role for Alt-EJ in cancer development (29–31) and resistance to chemotherapy and genetic disease (32–36). Hence, the presence of many short repetitive sequences in the human genome is likely to increase rearrangements mediated by MHs following the creation of a DSB.MMEJ is a distinct DSB repair pathway that operates in the presence of functional NHEJ and HR pathways (10, 37). The genetic requirements of MMEJ are being studied in the model eukaryote Saccharomyces cerevisiae and involve components traditionally considered specific to the NHEJ (Pol λ) and HR (Rad1–Rad10, Rad59, and Mre11–Rad50–Xrs2) pathways (4, 5, 10, 38). Although being clearly independent of the central NHEJ factor Ku70–Ku80 heterodimer (10, 37), the involvement of the key HR factor Rad52 in MMEJ remains uncertain. It has been reported that Rad52 is required for MMEJ repair (4, 10, 38), whereas in another assay system Rad52 suppresses MMEJ repair (37). More recently, it has been proposed that the replication protein A (RPA) regulates pathway choice between HR and MMEJ (37). In addition, several models have been proposed that identify specific pathways that may use MHs for the repair of DNA damage (39–41). Despite current advancements in our understanding of MMEJ, the precise involvement of DNA polymerases in supporting the repair of DSBs using MHs remains poorly understood. DNA polymerase λ (also called Pol4 in budding yeast) and its human homolog Pol λ are considered to be the primary candidates for the DNA polymerases working in NHEJ and MMEJ (4, 5, 42–46). Both genetic and biochemical evidence shows that Pol δ is recruited during HR to extend Rad51-dependent recombination intermediates (47–50). Recent analysis using pol32 mutants (5, 10) implicated the Pol32 subunit of Pol δ in MMEJ. Pol32 and Pol31 were also identified as subunits of the DNA polymerase zeta complex (Pol ζ) (51, 52), but previous analysis showed no effect of rev3 mutants in MMEJ (10). REV3 encodes the catalytic subunit of Pol ζ. However, an involvement of Pol δ had not been demonstrated directly before, and it is possible that Pol32 could act in conjunction with yet another DNA polymerase.Here, we report the development of a series of interchromosomal MMEJ assays in diploid S. cerevisiae to assess the mechanisms underlying the repair of DSBs using varying MHs. We focus on diploid cells, as they represent the natural state of budding yeast, which is a diplontic organism (53). The yeast mating-type switching system represents a mechanism to return haploid yeast as efficiently as possible to diploidy (54). Using a combination of genetic, molecular, and in vivo chromatin immunoprecipitation (ChIP) experiments, we provide compelling evidence for a direct involvement of Pol δ in coordinating with Pol λ in MMEJ in budding yeast.     

Organization and dynamics of the nonhomologous end-joining machinery during DNA double-strand break repair

Nonhomologous end-joining (NHEJ) is a major repair pathway for DNA double-strand breaks (DSBs), involving synapsis and ligation of the broken strands. We describe the use of in vivo and in vitro single-molecule methods to define the organization and interaction of NHEJ repair proteins at DSB ends. Super-resolution fluorescence microscopy allowed the precise visualization of XRCC4, XLF, and DNA ligase IV filaments adjacent to DSBs, which bridge the broken chromosome and direct rejoining. We show, by single-molecule FRET analysis of the Ku/XRCC4/XLF/DNA ligase IV NHEJ ligation complex, that end-to-end synapsis involves a dynamic positioning of the two ends relative to one another. Our observations form the basis of a new model for NHEJ that describes the mechanism whereby filament-forming proteins bridge DNA DSBs in vivo. In this scheme, the filaments at either end of the DSB interact dynamically to achieve optimal configuration and end-to-end positioning and ligation.Chromosomal double-strand breaks (DSBs), considered the most cytotoxic form of DNA damage, occur as a result of normal cellular processes (1, 2) as well as cancer therapies (3–5). The cellular DNA damage response (DDR) and repair pathways responsible for maintaining genomic integrity are highly regulated and synchronized processes, both temporally and spatially, involving the coordinated recruitment, assembly, and disassembly of numerous macromolecular complexes (6, 7). In mammalian cells, nonhomologous end-joining (NHEJ) is the primary DSB repair pathway; it is active throughout the cell cycle and is crucial for viability. Dysfunctional NHEJ is associated with several clinical conditions, including LIG4 syndrome and severe combined immunodeficiency (1, 8). Despite its importance, however, the details of how the NHEJ complex assembles at DSBs, brings together a pair of breaks, and organizes subsequent catalytic repair steps remain unknown.In NHEJ, DSBs are initially recognized by the Ku 70/80 heterodimer (Ku), which encircles dsDNA ends (Ku:DNA) and serves as a molecular scaffold for recruitment of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), XRCC4 (X-ray repair cross-complementing protein 4), XLF (XRCC4 like factor), and DNA ligase IV (LigIV) (1, 9–14). Previous NHEJ models suggested that after binding of Ku to DNA ends, DNA-PKcs binds Ku:DNA to form a DNA-PK holoenzyme and bridges the broken ends (15–18); however, recent experiments indicate that DNA-PKcs may have different roles in NHEJ, such as the stabilization of core NHEJ factors, recruitment and retention of accessory factors, involvement in the DDR signaling cascade, and repair of complex and clustered DSBs (19–25). In addition, recent structural studies have shown that XRCC4 and XLF form filamentous structures in vitro (26–28). Whether such filaments mediate repair in vivo has not yet been determined.Our present understanding of the cellular NHEJ response to DSBs is based primarily on in vitro biochemical and structural studies done with purified proteins, together with cellular observations in which a radiologic or pharmacologic stimulus damages DNA, allowing observation of the repair process (29–31). Typically, cellular assays rely on a microscope to read out the response, looking for colocalization of DSB repair proteins with large DDR foci; however, conventional microscopy methods allow for only an inferential approach, given that the resolution limit of light is two orders of magnitude greater than the length scale of proteins. Here we used super-resolution (SR) localization microscopy and single-molecule FRET (smFRET) to analyze the cellular organization of NHEJ proteins and define the dynamics associated with end joining in vitro. SR microscopy circumvents the conventional resolution limit of light microscopy by temporally separating emitting fluorophores and computationally fitting the location of each below the diffraction limit. Reconstructing thousands of points in this manner generates an image with a resolution typically an order of magnitude better that that of conventional microscopy (32–34). In addition to this approach, we used smFRET, a powerful method capable of monitoring the dynamics of individual nucleoprotein complexes in real time (35).Using SR microscopy, we identified previously uncharacterized repair intermediates formed at DSBs in which Ku resides at the break site and XRCC4, XLF, and LigIV form long filamentous structures around and over DSB sites. We categorized these intermediates into two different structural subtypes, and defined their kinetics and the structural transitions that occur during the progression of repair. We further verified the formation of these structures using SR imaging analysis of NHEJ reactions carried out in vitro with recombinant proteins. Finally, we used smFRET to characterize the dynamics of end-joining in vitro, revealing that XRCC4/XLF/LigIV mediates end synapsis, and that after initial pairing, the DNA ends undergo dynamic interactions. Our findings identify XRCC4/XLF/LigIV filaments forming on either side of the break and then merging as a key step in DSB repair via NHEJ, and provide a detailed mechanism that is radically different from current models of NHEJ.     

Single-molecule imaging reveals the mechanism of Exo1 regulation by single-stranded DNA binding proteins

Exonuclease 1 (Exo1) is a 5′→3′ exonuclease and 5′-flap endonuclease that plays a critical role in multiple eukaryotic DNA repair pathways. Exo1 processing at DNA nicks and double-strand breaks creates long stretches of single-stranded DNA, which are rapidly bound by replication protein A (RPA) and other single-stranded DNA binding proteins (SSBs). Here, we use single-molecule fluorescence imaging and quantitative cell biology approaches to reveal the interplay between Exo1 and SSBs. Both human and yeast Exo1 are processive nucleases on their own. RPA rapidly strips Exo1 from DNA, and this activity is dependent on at least three RPA-encoded single-stranded DNA binding domains. Furthermore, we show that ablation of RPA in human cells increases Exo1 recruitment to damage sites. In contrast, the sensor of single-stranded DNA complex 1—a recently identified human SSB that promotes DNA resection during homologous recombination—supports processive resection by Exo1. Although RPA rapidly turns over Exo1, multiple cycles of nuclease rebinding at the same DNA site can still support limited DNA processing. These results reveal the role of single-stranded DNA binding proteins in controlling Exo1-catalyzed resection with implications for how Exo1 is regulated during DNA repair in eukaryotic cells.All DNA maintenance processes require nucleases, which enzymatically cleave the phosphodiester bonds in nucleic acids. Exo1, a member of the Rad2 family of nucleases, participates in DNA mismatch repair (MMR), double-strand break (DSB) repair, nucleotide excision repair (NER), and telomere maintenance (1–3). Exo1 is the only nuclease implicated in MMR, where its 5ʹ to 3ʹ exonuclease activity is used to remove long tracts of mismatch-containing single-stranded DNA (ssDNA) (2, 4–7). In addition, functionally deficient Exo1 variants have been identified in familial colorectal cancers, and Exo1-null mice exhibit a significant increase in tumor development, decreased lifespan, and sterility (8, 9). Exo1 also promotes DSB repair via homologous recombination (HR) by processing the free DNA ends to generate kilobase-length ssDNA resection products (1, 10–12). The resulting ssDNA is paired with a homologous DNA sequence located on a sister chromatid, and the missing genetic information is then restored via DNA synthesis. The central role of Exo1 in DNA repair is highlighted by the large set of genetic interactions between Exo1 and nearly all other DNA maintenance and metabolism pathways (13).Exo1 generates long tracts of ssDNA in both MMR and DSB repair (3). This ssDNA is rapidly bound by replication protein A (RPA), a ubiquitous heterotrimeric protein that participates in all DNA transactions that generate ssDNA intermediates (14). RPA protects the ssDNA from degradation, participates in DNA damage response signaling, and acts as a loading platform for downstream DSB repair proteins (15–17). RPA also coordinates DNA resection by removing secondary ssDNA structures and by modulating the Bloom syndrome, RecQ helicase-like (BLM)/DNA2- and Exo1-dependent DNA resection pathways (18–21). Reconstitution of both the yeast and human BLM (Sgs1 in yeast)/DNA2-dependent resection reactions established that RPA stimulates DNA unwinding by BLM/Sgs1 and enforces a 5′-endonuclease polarity on DNA2 (20, 22). However, the effect of RPA on Exo1 remains unresolved. Independent studies using reconstituted yeast proteins reported that RPA could both inhibit (23) and stimulate yeast Exo1 (yExo1) (18). Similarly, human RPA has variously been reported to stimulate (19) or inhibit human Exo1 (hExo1) (4, 5, 21).In addition to RPA, human cells also encode SOSS1, a heterotrimeric ssDNA-binding complex that is essential for HR (24). SOSS1 consists of INTS3 (SOSSA), hSSB1 (SOSSB1), and C9orf80 (SOSSC) (24–26). SOSSB1 encodes a single ssDNA-binding domain that bears structural homology to Escherichia coli ssDNA-binding protein (SSB) (24). SOSS1 foci form rapidly after induction of DNA breaks, and ablation of SOSS1 severely reduces DNA resection, γH2AX foci formation, and HR at both ionizing radiation- and restriction endonuclease-induced DSBs (12, 24, 25, 27). In vitro, SOSS1 stimulates hExo1-mediated DNA resection and may help to load hExo1 at ss/dsDNA junctions (21). However, the functional relationship between SOSS1 and RPA during hExo1 resection remains unresolved.Here, we use high-throughput single-molecule DNA curtains and quantitative cell biology to reveal the interplay between human and yeast Exo1 and SSBs during DNA resection. We show that both human and yeast Exo1s are processive nucleases, but are rapidly stripped from DNA by RPA. RPA inhibition is dependent on its multiple DNA binding domains. Remarkably, SOSS1 and other SSBs with fewer than three DNA binding domains support long-range resection by hExo1. In human cells, depletion of RPA increases the rate of hExo1 recruitment to laser-induced DNA damage but reduces the extent of resection. In the presence of RPA, both human and yeast Exo1 can resect DNA using a distributive, multiple-turnover mechanism, potentially reconciling prior conflicting in vitro observations. Together, our work reveals the mechanistic basis for how RPA and SOSS1 differentially modulate hExo1 activity and highlights an additional, unexpected role for these SSBs in DNA resection. We anticipate that these findings will shed light on how Exo1 is regulated in multiple genome maintenance pathways.     

RecQ helicase and RecJ nuclease provide complementary functions to resect DNA for homologous recombination

Recombinational DNA repair by the RecF pathway of Escherichia coli requires the coordinated activities of RecA, RecFOR, RecQ, RecJ, and single-strand DNA binding (SSB) proteins. These proteins facilitate formation of homologously paired joint molecules between linear double-stranded (dsDNA) and supercoiled DNA. Repair starts with resection of the broken dsDNA by RecQ, a 3′→5′ helicase, RecJ, a 5′→3′ exonuclease, and SSB protein. The ends of a dsDNA break can be blunt-ended, or they may possess either 5′- or 3′-single-stranded DNA (ssDNA) overhangs of undefined length. Here we show that RecJ nuclease alone can initiate nucleolytic resection of DNA with 5′-ssDNA overhangs, and that RecQ helicase can initiate resection of DNA with blunt-ends or 3′-ssDNA overhangs by DNA unwinding. We establish that in addition to its well-known ssDNA exonuclease activity, RecJ can display dsDNA exonuclease activity, degrading 100–200 nucleotides of the strand terminating with a 5′-ssDNA overhang. The dsDNA product, with a 3′-ssDNA overhang, is an optimal substrate for RecQ, which unwinds this intermediate to reveal the complementary DNA strand with a 5′-end that is degraded iteratively by RecJ. On the other hand, RecJ cannot resect duplex DNA that is either blunt-ended or terminated with 3′-ssDNA; however, such DNA is unwound by RecQ to create ssDNA for RecJ exonuclease. RecJ requires interaction with SSB for exonucleolytic degradation of ssDNA but not dsDNA. Thus, complementary action by RecJ and RecQ permits initiation of recombinational repair from all dsDNA ends: 5′-overhangs, blunt, or 3′-overhangs. Such helicase–nuclease coordination is a common mechanism underlying resection in all organisms.Homologous recombination is a relatively error-free mechanism to repair double-stranded DNA (dsDNA) breaks (DSBs) and single-stranded DNA (ssDNA) gaps, which are produced by UV light, γ-irradiation, and chemical mutagens (1). In wild-type Escherichia coli, the labor of recombinational repair is divided between the RecBCD and RecF pathways of recombination, which are responsible for the repair of DSBs and ssDNA gaps, respectively (2–5). However, the proteins of the RecF pathway are capable of DSB repair, as well as ssDNA gap repair: in recBC mutant cells containing the suppressor mutations, sbcB and sbcC (suppressors of recBC), the proteins of the RecF pathways provide the needed recombinational DNA repair functions (2, 6).The RecF pathway in E. coli involves the functions of RecA, RecF, RecG, RecJ, RecN, RecO, RecQ, RecR, RuvA, RuvB, RuvC, and single-strand DNA binding (SSB) proteins (1, 7). The RecF pathway of recombination is evolutionarily conserved across Bacteria, with most of components present in all bacteria (8). In addition, orthologs of RecF pathway proteins are found in Eukarya. RecA promotes DNA strand invasion and exchange (9–11), as does eukaryotic Rad51 (12, 13). RecO can both anneal SSB–ssDNA complexes (14, 15) and, in conjunction with RecR (and RecF), mediate loading of RecA onto SSB–ssDNA complexes (16–18). Saccharomyces cerevisiae Rad52 is a functional homolog of RecO in that it also displays both DNA-annealing and Rad51-loading activities (19–22). The RecFOR complex promotes the loading of RecA onto SSB-coated gapped DNA at ssDNA–dsDNA junctions (17, 18) and, when mutated, is suppressed by hyperactive alleles of recA (23), a property that is shared with the yeast Rad55/57 proteins (24). Furthermore, human BRCA2 protein and a fungal analog, Brh2, are partial functional analogs of the RecFOR proteins (25–27).RecQ helicase plays several roles in both early and late steps of recombination (28, 29), as do the RecQ-family helicases in Eukarya [e.g., Sgs1 and Bloom Syndrome helicase (BLM)] (30–32). In addition, eukaryotic Exonuclease 1 (Exo1) and Dna2 helicase/nuclease function somewhat analogously, although not identically, to RecJ nuclease (33–36). The in vitro reconstitution of DSB repair in E. coli, yeast, and human have shown that resection involves specific pairs of a helicase and nuclease for DNA end resection: RecQ/RecJ, Sgs1/Dna2, BLM/DNA2, and BLM/EXO1 (28, 37–39).A comparison of DSB repair by the RecBCD and RecF pathways shows that repair starts with the processing a DSB into resected dsDNA with a 3′-ssDNA overhang (7). RecJ has a 5′ to 3′ exonuclease activity on ssDNA and the action of RecJ is facilitated by RecQ, which has a 3′ to 5′ helicase activity (40, 41). The resulting processed DNA has a 3′-ssDNA overhang. The RecFOR complex binds to the 5′-end at the junction between ssDNA and dsDNA, and loads RecA protein onto the adjacent ssDNA (17, 18). Finally, the RecA nucleoprotein filament promotes pairing with homologous dsDNA (9). These steps have been reconstituted in vitro in a coordinated reaction using RecAFORQJ and SSB proteins (28).Despite progress, most studies have used DNA substrates with simple blunt-ends. However, in vivo, there are many potential structures at the end of a DSB. When the DSB is created by a replication fork encountering nicked DNA, the break can be blunt-ended (5). However, related mechanisms can produce dsDNA with either 5′- or 3′-ssDNA overhangs. Similarly, the actual intermediates of DNA processing may result in dsDNA with either 5′- or 3′-ssDNA ends. Clearly, a DNA repair pathway must be capable of dealing with such a variety of DNA end structures. In this study, we investigated the processing of DSBs by RecJ and RecQ, both individually and together. We found that a DNA with a 5′-ssDNA overhang end was degraded by RecJ nuclease and converted into an intermediate with a 3′-ssDNA overhang. Although this intermediate was no longer a substrate for RecJ, RecQ could bind to this intermediate and initiate unwinding, thereby supplying 5′-tailed ssDNA for further resection by RecJ. In addition, we established that RecQ allows RecJ to initiate nucleolytic resection on otherwise poor substrates (e.g., blunt-end DNA or DNA with 3′-ssDNA overhangs). Thus, RecQ and RecJ cooperate biochemically to create DNA intermediates for one another that enable resection of all types of broken DNA molecules.     

C-terminal region of activation-induced cytidine deaminase (AID) is required for efficient class switch recombination and gene conversion

Activation-induced cytidine deaminase (AID) introduces single-strand breaks (SSBs) to initiate class switch recombination (CSR), gene conversion (GC), and somatic hypermutation (SHM). CSR is mediated by double-strand breaks (DSBs) at donor and acceptor switch (S) regions, followed by pairing of DSB ends in two S regions and their joining. Because AID mutations at its C-terminal region drastically impair CSR but retain its DNA cleavage and SHM activity, the C-terminal region of AID likely is required for the recombination step after the DNA cleavage. To test this hypothesis, we analyzed the recombination junctions generated by AID C-terminal mutants and found that 0- to 3-bp microhomology junctions are relatively less abundant, possibly reflecting the defects of the classical nonhomologous end joining (C-NHEJ). Consistently, the accumulation of C-NHEJ factors such as Ku80 and XRCC4 was decreased at the cleaved S region. In contrast, an SSB-binding protein, poly (ADP)-ribose polymerase1, was recruited more abundantly, suggesting a defect in conversion from SSB to DSB. In addition, recruitment of critical DNA synapse factors such as 53BP1, DNA PKcs, and UNG at the S region was reduced during CSR. Furthermore, the chromosome conformation capture assay revealed that DNA synapse formation is impaired drastically in the AID C-terminal mutants. Interestingly, these mutants showed relative reduction in GC compared with SHM in chicken DT40 cells. Collectively, our data indicate that the C-terminal region of AID is required for efficient generation of DSB in CSR and GC and thus for the subsequent pairing of cleaved DNA ends during recombination in CSR.Activation-induced cytidine deaminase (AID) is essential for three different genetic events: class switch recombination (CSR), gene conversion (GC), and somatic hypermutation (SHM), which contribute to Ig gene diversification (1–5). Although AID generates single-strand breaks (SSBs) in the Ig genes, subsequent repair steps for CSR and GC are similar to each other but are distinct from SHM in their mechanistic properties, i.e, in (i) generation of the double-strand breaks (DSBs), (ii) recombination, and (iii) the requirement for uracil-DNA-glycosylase (UNG) for the pairing of the DSB ends (6–10). Despite the similarities between GC and CSR, their repair mechanisms have distinct features: CSR recombination requires nonhomologous end joining (NHEJ), and GC depends on homologous recombination (HR). During CSR, DSB ends normally are joined by classical NHEJ (C-NHEJ), which requires specific repair proteins such as Ku80, XRCC4, or DNA ligase IV (11, 12). In the absence of C-NHEJ, a back-up end-joining pathway called “alternative end joining” (A-EJ), which is reported to be slower and also more error prone than C-NHEJ, joins the broken DSBs ends (13). On the other hand, HR, the most common form of homology-directed repair, requires long sequence homology between donor and acceptor DNA to complete the recombination step by recruiting a distinct set of repair proteins such as RAD54, RAD52, and RAD51 to the break sites (14, 15).Various studies on AID mutations in the N-terminal or C-terminal regions (4, 8, 9, 16–19) have shown that N-terminal AID mutants are compromised for CSR and are defective in SHM, indicating that the N-terminal region of AID is required for DNA cleavage (9, 16, 19). On the other hand, the C-terminal region of AID, which contains a nuclear-export signal and is responsible for AID’s shuttling activity between the nucleus and cytoplasm, is required for CSR-specific activity but not for DNA cleavage activity and SHM (8, 16). Among the series of AID C-terminal mutants examined, two mutants show characteristic features: P20, which has an insertion of 34 amino acids at residue 182 and normal nuclear-cytoplasmic shuttling activity, and JP8Bdel, which has a 16-amino acid truncation at residue 183, accumulates in the nucleus, and shows higher DNA break activity at the donor switch (S) region (16, 17). Although several reports suggest that the C-terminal region of AID is involved in protein stability (20, 21), C-terminal mutants of AID stabilized by fusing the hormone-binding domain of estrogen receptor (ER) also show similar CSR-defective phenotypes (8). Taken together, these data suggest that the DNA cleavage activity and CSR-specific activity depend on different regions of AID (8, 19). In addition, the C-terminal region of AID is essential for the interaction of AID with poly (A)⁺ RNA via a specific cofactor (22). Because CSR requires de novo protein synthesis, we proposed that after DNA cleavage the C-terminal region of AID may be involved in the regulation of the recombination step through generation of a new protein (8, 16, 22).DSBs induced by AID during CSR ultimately are joined by the efficient DNA repair pathway that requires C-NHEJ factors such as Ku70/80 (12, 23). However, in the absence of C-NHEJ, the A-EJ pathway that relies on microhomology can join the broken DNA ends, although this pathway is associated with chromosomal translocations (11, 24). Previously, we reported that JP8Bdel enhances aberrant c-myc/IgH translocations and that it fails to carry out the efficient recombination between donor and acceptor S regions in the IgH locus (8). Therefore, it is important to examine whether the AID C-terminal mutants affect the S–S joining in CSR.In the current work we examined whether the C-terminal region of AID is involved in DNA synapse formation and recombination during CSR in CH12F3-2 and spleen B cells. We also examined the effect of AID C-terminal mutations on GC in chicken DT40 cells, which depends on HR between pseudo V genes and the downstream IgVλ region. Using these CSR- and GC-monitoring systems, we demonstrate that efficient CSR and GC require the C-terminal region of AID for the formation of DSB from SSB and subsequent end synapse. Considering these findings together, we conclude that, in addition to DNA cleavage, AID has a unique function in the generation of DSBs, which is required for S–S synapse formation and joining in CSR and recombination in GC.     

Ligase I and ligase III mediate the DNA double-strand break ligation in alternative end-joining

In eukaryotes, DNA double-strand breaks (DSBs), one of the most harmful types of DNA damage, are repaired by homologous repair (HR) and nonhomologous end-joining (NHEJ). Surprisingly, in cells deficient for core classic NHEJ factors such as DNA ligase IV (Lig4), substantial end-joining activities have been observed in various situations, suggesting the existence of alternative end-joining (A-EJ) activities. Several putative A-EJ factors have been proposed, although results are mostly controversial. By using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, we generated mouse CH12F3 cell lines in which, in addition to Lig4, either Lig1 or nuclear Lig3, representing the cells containing a single DNA ligase (Lig3 or Lig1, respectively) in their nucleus, was completely ablated. Surprisingly, we found that both Lig1- and Lig3-containing complexes could efficiently catalyze A-EJ for class switching recombination (CSR) in the IgH locus and chromosomal deletions between DSBs generated by CRISPR/Cas9 in cis-chromosomes. However, only deletion of nuclear Lig3, but not Lig1, could significantly reduce the interchromosomal translocations in Lig4^−/− cells, suggesting the unique role of Lig3 in catalyzing chromosome translocation. Additional sequence analysis of chromosome translocation junction microhomology revealed the specificity of different ligase-containing complexes. The data suggested the existence of multiple DNA ligase-containing complexes in A-EJ.Mammalian genomes are subjected substantial DNA damage from both endogenous processes [e.g., DNA replication, class switching recombination (CSR), etc.] and exogenous resources (e.g., ionic radiation, DNA-damaging chemicals, etc.). Evolutionarily conserved DNA repair pathways are essential to maintain both the structure integrity and the information accuracy of the genome (1). In eukaryotes, DNA double-strand breaks (DSBs), one of the most dangerous and severe types of DNA damage, are repaired mainly by two evolutionarily conserved repair pathways: homologous repair (HR) and nonhomologous end-joining (NHEJ) (2). NHEJ directly ligates two broken DSB ends, whereas HR uses a homologous template (in most of cases, the sister chromosome) for repair. Although NHEJ is simpler and faster than HR, repair by NHEJ often leads to change of sequences in the repair junctions via deletion, insertion, and mutations. Most importantly, NHEJ can also directly ligate two distant DSBs (both in cis- and trans-chromosomes), therefore leading to chromosomal deletions and translocations that are tightly linked to the genome evolution and carcinogenesis (3).The NHEJ in eukaryotes has been extensively studied in the last two decades (4–6). Mechanistically, NHEJ could be separated into several steps. First, in the DSB binding and tethering step, the DSB ends are recognized and bound by Ku70/Ku80 heterodimers that function as docking sites for other NHEJ factors. In the next end-processing step, nuclease and DNA polymerase activities are recruited to remove damaged or mismatched nucleotides and prepare the broken ends for ligation. Finally, DNA ligase IV (Lig4) complex, consisting of DNA Lig4 and its cofactor X-ray repair cross-complementing protein 4 (XRCC4), as well as the newly identified XRCC4-interacting factor (XLF), reseals the DSBs. Both V(D)J recombination and class switching recombination (CSR) have been used as important in vivo models to study the NHEJ (7). During the V(D)J recombination, DSBs generated by recombination-activating gene 1 (RAG1)/RAG2 endonuclease are joined exclusively by NHEJ. Deficiency of core NHEJ factors in mice, such as Lig4 and XRCC4, leads to complete abolishment of V(D)J recombination and lack of mature B and T cells (8, 9). When combined with a checkpoint-deficient genetic background (e.g., p53^−/−), unresolved RAG-generated DSB ends in B and T cells in core NHEJ factor-deficient mice could result in dramatic genomic instability. In particular, oncogenic chromosome translocations lead to the development of lymphoma and leukemia in those mice (10, 11). The fact that those chromosome translocations are formed by end-joining in the absence of core NHEJ factors suggested the existence of alternative end-joining pathway(s) (A-EJ). CSR, by which mammalian mature B cells change their production of Ig constant region type from one to the other, represents an excellent model to study classic NHEJ (c-NHEJ) and A-EJ (12, 13). DSBs in the IgH class switching (S) regions induced by activation-induced cytidine deaminase (AID) are repaired by NHEJ via a loop-out and deletion mechanism (14). In the absence of c-NHEJ core factors (such as Lig4, XRCC4, and Ku70/80), significant CSR activities, mediated by A-EJ, have been observed in both animals and cell lines (12, 13). There is no doubt that A-EJ contributes to all end-joining activities in the absence of c-NHEJ. However, the contribution of A-EJ in the presence of c-NHEJ is still debatable. For example, it has been suggested that A-EJ is the main end-joining activity to catalyze chromosomal translocations in murine (15) but not in human cells (16).Although A-EJ activities have been observed in many cell types and biological processes (12, 17–19), A-EJ’s exact components and mechanisms have been still not clearly revealed and sometimes are controversial (5, 20, 21). For example, whether A-EJ is a completely independent new pathway or an alternative c-NHEJ pathway in which alternative components could substitute the missing c-NHEJ factors is still debatable. Comparing with large numbers of factors and pathways involved in the early DSB repair steps, there are only three known DNA ligases (DNA Lig1, DNA Lig3, and DNA Lig4) in mammalian cells to finish the last ligation step (22). It has been proposed that those three DNA ligases function differently in various DNA metabolism processes. Although all three mammalian DNA ligases have highly homologous catalytic cores (including DBD, AdD, and OB-Fold domains), through their distinct N- and C-terminal regions, the DNA ligases may interact with different partners, which could confer functional specificity. In DSB repair, the role of Lig4 has been mostly restricted to c-NHEJ, whereas both Lig1 and Lig3 have been suggested to mediate the A-EJ in vitro and in vivo (23–28). Here, we used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) to generate cell lines in which Lig1 or Lig3 were completely depleted, and we tried to unequivocally reveal the ligases’ roles in A-EJ.     

Crossovers are associated with mutation and biased gene conversion at recombination hotspots

Meiosis is a potentially important source of germline mutations, as sites of meiotic recombination experience recurrent double-strand breaks (DSBs). However, evidence for a local mutagenic effect of recombination from population sequence data has been equivocal, likely because mutation is only one of several forces shaping sequence variation. By sequencing large numbers of single crossover molecules obtained from human sperm for two recombination hotspots, we find direct evidence that recombination is mutagenic: Crossovers carry more de novo mutations than nonrecombinant DNA molecules analyzed for the same donors and hotspots. The observed mutations were primarily CG to TA transitions, with a higher frequency of transitions at CpG than non-CpGs sites. This enrichment of mutations at CpG sites at hotspots could predominate in methylated regions involving frequent single-stranded DNA processing as part of DSB repair. In addition, our data set provides evidence that GC alleles are preferentially transmitted during crossing over, opposing mutation, and shows that GC-biased gene conversion (gBGC) predominates over mutation in the sequence evolution of hotspots. These findings are consistent with the idea that gBGC could be an adaptation to counteract the mutational load of recombination.Meiotic recombination, localized in recombination hotspots, not only increases genetic diversity via the formation of new haplotypes but is also an important driver of sequence evolution. The binding sites used by the human recombination machinery involving PRDM9 (PR domain containing 9) are more eroded in humans than the same sequences in chimps, given that PRDM9 in chimps uses different binding sites (1). Moreover, regions in close vicinity to these PRDM9 binding sites also showed a significant enrichment of polymorphisms in humans (2). In addition, within- and between-species sequence diversity positively correlates with regions of high recombination activity in humans (3–7) and other eukaryotes (reviewed in refs. 8–10).One process recognized as a major evolutionary force reshaping the genomic nucleotide landscape at recombination hotspots, as shown in humans (6), chimpanzees (6, 11), mice (12), yeast (13), and metazoans (14), is GC-biased gene conversion (gBGC). In gBGC, the repair of heteroduplex tracts formed during meiotic recombination leads to the non-Mendelian segregation of alleles favoring GC over AT variants. The precise molecular mechanisms leading to gBGC have yet to be unraveled, but experimental evidence has shown that in crossovers (COs) of fission yeast, GC alleles can be overtransmitted within ∼1–2 kb in length of the double-strand break (DSB) region (13), implicating mismatch repair (15).However, it is also plausible that the higher sequence variation observed at recombination hotspots is a result of a mutagenic effect of recombination: meiotic recombination is initiated by DSBs, which are associated with an increased mutation frequency. In the nonreducing division, mitosis, genetic experiments have shown that the repair of DSBs in homologous recombination involves error-prone translesion polymerases, increasing the mutation frequency at DSB sites in Drosophila (16) and yeast (17–19). In humans as well, an error-prone polymerase (DNA polymerase θ) carries out translesion synthesis in the repair of DSBs (20). In addition to mitotic DSB repair, it was recently shown that error-prone translesion synthesis polymerases (Rev1, PolZeta, and Rad30) are also involved in the repair of DSBs in meiosis in yeast (21) and could potentially contribute to a higher mutation rate in the germ cells at recombination hotspots, which are recurrent sites for DSBs.Although high mutation rates might be an important driver of the high genetic diversity and elevated divergence in regions of high recombination (refs. 5–7, 22, and 23 and reviewed by refs. 23 and 24), the mutagenic signature in population data may be obscured by a complex interplay of other factors, including selection, demographic history, and gBGC (8–10). Therefore, to detect and quantify any elevation in mutation rates arising during meiosis, we measured the frequency of de novo mutations in a large number of single COs. We provide, for the first time to our knowledge, experimental data showing that recombination associated with COs is mutagenic in humans. In our large survey of recombination products, we also find evidence that the transmission of GC alleles is favored during crossing over and that associated gBGC is acting in opposition to the introduced mutational bias.     

Suppression of DNA-damage checkpoint signaling by Rsk-mediated phosphorylation of Mre11

Ataxia telangiectasia mutant (ATM) is an S/T-Q–directed kinase that is critical for the cellular response to double-stranded breaks (DSBs) in DNA. Following DNA damage, ATM is activated and recruited by the MRN protein complex [meiotic recombination 11 (Mre11)/DNA repair protein Rad50/Nijmegen breakage syndrome 1 proteins] to sites of DNA damage where ATM phosphorylates multiple substrates to trigger cell-cycle arrest. In cancer cells, this regulation may be faulty, and cell division may proceed even in the presence of damaged DNA. We show here that the ribosomal s6 kinase (Rsk), often elevated in cancers, can suppress DSB-induced ATM activation in both Xenopus egg extracts and human tumor cell lines. In analyzing each step in ATM activation, we have found that Rsk targets loading of MRN complex components onto DNA at DSB sites. Rsk can phosphorylate the Mre11 protein directly at S676 both in vitro and in intact cells and thereby can inhibit the binding of Mre11 to DNA with DSBs. Accordingly, mutation of S676 to Ala can reverse inhibition of the response to DSBs by Rsk. Collectively, these data point to Mre11 as an important locus of Rsk-mediated checkpoint inhibition acting upstream of ATM activation.Cells have evolved multiple pathways signaling DNA damage that trigger DNA repair, cell-cycle arrest and, in the event of irreparable damage, cell death. Among the various forms of DNA damage, double-stranded breaks (DSBs) in DNA, generated by exposure to ionizing radiation and radiomimetic chemicals such as neocarzinostatin (NCS), are the most lethal for cells (1).DSBs often are repaired by homologous recombination during the S and G2/M phases of the cell cycle, which involves the meiotic recombination 11 (Mre11)/DNA repair protein Rad50/Nijmegen breakage syndrome 1 (Nbs1) (MRN) complex and the ataxia telangiectasia mutated (ATM) kinase (2, 3). The MRN complex first recognizes sites of DNA damage and then promotes binding of ATM to the DSB site. ATM, activated by monomerization and autophosphorylation, phosphorylates downstream proteins including p53, checkpoint kinase 2 (Chk2), and breast cancer 1, early onset (BRCA1) (4–7). These factors then convey the signal to induce cycle arrest, apoptosis, or DNA repair (8). Failure of this process results in genome instability, increasing the risk of cancer, neurodegeneration, and other pathologies (9).Ribosomal S6 kinase (Rsk), which functions downstream of mitogen-activated protein kinase kinase (MEK) and ERK, is frequently activated in cancer cells (10, 11). Rsk activation can be promoted by multiple signaling pathways in cancer cells, including those triggered by steroids, insulin, EGF, and estrogen (10, 12–15). Additionally, Rsk activation can be triggered by PKC signaling [via the PKC/rapidly accelerated fibrosarcoma (RAF)/mitogen-activated protein kinases (MAPK) pathway], which is activated by phorbol12-myristate13-acetate (PMA) (16, 17). Previous studies have found that Rsk2 is overexpressed in 50% of breast cancers and prostate tumors (18, 19), and Rsk signaling has been implicated in the regulation of survival, anchorage-independent growth, and transformation of breast cancer cells in culture (20). Rsk-specific inhibition (with BI-D1870 or SL0101) significantly reduced proliferation of MCF7, PC3, or LnCaP cancer cells (18, 19). Rsk also inhibits apoptosis in PC3 prostate cancer cells (21).A hallmark of cancer cells is their ability to override cell-cycle checkpoints, including the DSB checkpoint, which arrests the cell cycle to allow adequate time for damage repair. Previous studies have implicated the MAPK pathway in inhibition of DNA-damage signaling: PKC suppresses DSB-induced G2/M checkpoint signaling following ionizing radiation via activation of ERK1/2 (22); activation of RAF kinase, leading to activation of MEK/ERK/Rsk, also can suppress G2/M checkpoint signaling (23).Given its prominent role in multiple cancers, the MAPK pathway is an attractive therapeutic target. Indeed, treatment of melanoma using the RAF inhibitor vemurafenib has shown some clinical success, as has treatment of nonsmall cell lung carcinoma with MEK inhibitors (24). However, targeting components at the apex of a signaling pathway may induce side effects caused by the plethora of downstream effectors (25, 26). As a terminal kinase in the MAPK pathway, Rsk may avoid these complications as a potential target. Thus, there has been interest in targeting Rsk for cancers with notable Rsk elevation (e.g., prostate cancers) (27). Several Rsk-specific inhibitors have been described, including SL0101 and BI-D1870 (18, 28, 29). Whether these or derivative drugs will be clinically successful remains unclear. However, if Rsk inhibition can reinstate DSB-induced checkpoint function, then combination therapy of Rsk inhibitors with DNA-damaging agents may be effective in inducing tumor cell arrest or death.The precise mechanism underlying checkpoint inhibition downstream of MEK/ERK/Rsk signaling is not yet clear. One recent paper has shown that, after doxorubicin-induced DNA damage (both DSB and ssDNA breaks), Rsk can silence the G2/M checkpoint by phosphorylating (and inhibiting) the Chk1 kinase on S280 (the same site that can be targeted by protein kinase B/Akt kinase) (30). However, another group has reported that phosphorylation of this site on Chk1 enhanced its ability to enforce the checkpoint by promoting its nuclear translocation (31).We show here that Rsk signaling silences the DSB-induced G2/M checkpoint by preventing activation of the ATM kinase. Specifically, we have found that Rsk targets the Mre11 component of the MRN complex by phosphorylating S676, thereby preventing Mre11 binding to DNA. Mutation of S676 restored ATM activation, even in the face of high Rsk activity, and rendered cells refractory to the checkpoint-inhibitory effects of Rsk. This finding is consistent with a previous study showing that phosphorylation on Mre11 is mostly inhibitory (32). Taken together, these findings identify a locus of checkpoint regulation by Rsk and support the idea of targeting Rsk for therapeutic benefit.     

Rad51 recombinase prevents Mre11 nuclease-dependent degradation and excessive PrimPol-mediated elongation of nascent DNA after UV irradiation

After UV irradiation, DNA polymerases specialized in translesion DNA synthesis (TLS) aid DNA replication. However, it is unclear whether other mechanisms also facilitate the elongation of UV-damaged DNA. We wondered if Rad51 recombinase (Rad51), a factor that escorts replication forks, aids replication across UV lesions. We found that depletion of Rad51 impairs S-phase progression and increases cell death after UV irradiation. Interestingly, Rad51 and the TLS polymerase polη modulate the elongation of nascent DNA in different ways, suggesting that DNA elongation after UV irradiation does not exclusively rely on TLS events. In particular, Rad51 protects the DNA synthesized immediately before UV irradiation from degradation and avoids excessive elongation of nascent DNA after UV irradiation. In Rad51-depleted samples, the degradation of DNA was limited to the first minutes after UV irradiation and required the exonuclease activity of the double strand break repair nuclease (Mre11). The persistent dysregulation of nascent DNA elongation after Rad51 knockdown required Mre11, but not its exonuclease activity, and PrimPol, a DNA polymerase with primase activity. By showing a crucial contribution of Rad51 to the synthesis of nascent DNA, our results reveal an unanticipated complexity in the regulation of DNA elongation across UV-damaged templates.The DNA-binding protein Rad51 is a central component of homologous recombination repair (HRR). HRR repairs double-strand breaks (DSBs) in an error-free way and processes one-ended DSBs to reactivate collapsed replication forks (1). During HRR, DSBs are processed by the 3′-to-5′ exonuclease activity of the double strand break repair nuclease (Mre11) to generate protruding 3′ ssDNA at DSBs. The ssDNA is then coated with Rad51, a factor that catalyzes homology search and strand invasion. The loading and stabilization of Rad51/ssDNA complexes are supported by multiple mediators, such as the tumor suppressor BRCA2 (breast cancer 2) (1). Moreover, Rad51 promotes XPF1- and Exo1-mediated DSB formation after gemcitabine-induced irreversible ribonucleotide reductase inhibition, thus promoting cell death (2). The signals that redirect Rad51 into a DSB formation pathway rather than DSB repair are not yet known.The functions of Rad51 are not limited to the processing/generation of DSBs. Over the past few years, it has become evident that Rad51 escorts ongoing replication forks regardless of the presence of DSBs (3–5). Specifically, Rad51 protects persistently stalled replication forks from Mre11-mediated nucleolytic degradation and facilitates replication fork restart when the replication-halting agent hydroxyurea (HU) or aphidicolin (APH) is removed (6–19). Such novel functions of Rad51 require many HRR factors, including BCRA2, FANCD2 (Fanconi Anemia Complementation group protein D2), CtIP, BRCA1, and the WRN helicase, but are independent of HRR effectors, such as Rad54 (6, 7). Rad51-dependent fork-restart and fork-protection are distinct mechanisms, because proteins like the BLM helicase promote the former, but not the latter, process after HU treatment (6, 20). Mre11-mediated nucleolytic degradation of nascent DNA in BCRA2- and FANCD2-depleted, HU-treated cells was suggested to take place at the unprotected ends of reversed forks, which may mimic DSBs (6–8). Conversely, two recent reports suggest that Rad51 prevents pathological Mre11-dependent nucleolytic degradation of nonreversed stalled forks and promotes controlled DNA2-dependent exonucleolytic processing of reversed forks (15, 21).DSB-independent Rad51 functions were revealed by the use of agents that cause a significant degree (more than 40%) of replication fork stalling, such as HU, camptothecin (CPT), and mitomycin C (MMC) (15). Much less is known about the participation of Rad51 in the replication across DNA lesions that do not persistently halt replication forks and only cause a moderate reduction in the replication speed (e.g., UV-induced DNA lesions) (22). Multiple mechanisms aid DNA replication after UV irradiation. First, strongly distorting UV lesions are effectively removed by nucleotide excision repair (NER). Second, mildly distorting lesions, which are less efficiently removed by NER, can be used as replication templates in translesion DNA synthesis (TLS) events (23). TLS avoids fork stalling by loading specialized DNA polymerases that use damaged DNA as replication templates (24).It is currently unclear whether non-TLS events aid DNA replication across UV-damaged DNA in mammalian systems. Importantly, Rad51 is recruited to DNA after UV irradiation (4, 21, 25). HRR factors contribute to the repair of infrequent DSBs generated by high UV doses (80 J/m²) in NER-deficient backgrounds (26). Interestingly, however, cancer cells depleted from Rad51 were sensitive to much lower UV doses (1–5 J/m²) (26), thus suggesting DSB-independent functions of Rad51 in the cellular response to UV light. More recently, Rad51 was shown to maintain continuous DNA replication after treatment with methyl-methane sulfonate (MMS), a DNA-damaging agent that induces bulky lesions similar to the lesions caused by UV radiation (4). Whether Rad51 prevents nucleolytic degradation of nascent DNA in response to UV irradiation has not yet been explored. Remarkably, fork reversal takes place frequently after UV irradiation, similar to the case with HU, MMC, and CPT (21).We therefore set out to investigate the contribution of Rad51 to DNA replication across UV lesions. Using the DNA stretching technique (27), we uncovered two DSB-independent roles of Rad51 in the replication of UV-damaged DNA. First, Rad51 protected the nascent DNA from rapid and time-limited Mre11-dependent exonucleolytic degradation. Second, Rad51 prevented excessive DNA elongation after UV irradiation. Such dysregulated elongation of DNA was orchestrated by Mre11, but not by its exonuclease activity, and a DNA polymerase with primase activity, PrimPol (primase polymerase). Our results therefore suggest that Rad51 depletion increases repriming after UV irradiation. Intriguingly, both Rad51-mediated functions affected the accumulation of DNA damage response (DDR) markers at later time points, but only the excessive fork elongation in Rad51-depleted cells was associated with cell death. Finally, we demonstrate that the TLS DNA polymerase polη and Rad51 are both required to achieve optimal elongation of nascent UV-damaged DNA.