Ectopic expression of RNF168 and 53BP1 increases mutagenic but not physiological non-homologous end joining

DNA double strand breaks (DSBs) formed during S phase are preferentially repaired by homologous recombination (HR), whereas G₁ DSBs, such as those occurring during immunoglobulin class switch recombination (CSR), are repaired by non-homologous end joining (NHEJ). The DNA damage response proteins 53BP1 and BRCA1 regulate the balance between NHEJ and HR. 53BP1 promotes CSR in part by mediating synapsis of distal DNA ends, and in addition, inhibits 5’ end resection. BRCA1 antagonizes 53BP1 dependent DNA end-blocking activity during S phase, which would otherwise promote mutagenic NHEJ and genome instability. Recently, it was shown that supra-physiological levels of the E3 ubiquitin ligase RNF168 results in the hyper-accumulation of 53BP1/BRCA1 which accelerates DSB repair. Here, we ask whether increased expression of RNF168 or 53BP1 impacts physiological versus mutagenic NHEJ. We find that the anti-resection activities of 53BP1 are rate-limiting for mutagenic NHEJ but not for physiological CSR. As heterogeneity in the expression of RNF168 and 53BP1 is found in human tumors, our results suggest that deregulation of the RNF168/53BP1 pathway could alter the chemosensitivity of BRCA1 deficient tumors.     

ZMYM2 restricts 53BP1 at DNA double-strand breaks to favor BRCA1 loading and homologous recombination

An inability to repair DNA double-strand breaks (DSBs) threatens genome integrity and can contribute to human diseases, including cancer. Mammalian cells repair DSBs mainly through homologous recombination (HR) and nonhomologous end-joining (NHEJ). The choice between these pathways is regulated by the interplay between 53BP1 and BRCA1, whereby BRCA1 excludes 53BP1 to promote HR and 53BP1 limits BRCA1 to facilitate NHEJ. Here, we identify the zinc-finger proteins (ZnF), ZMYM2 and ZMYM3, as antagonizers of 53BP1 recruitment that facilitate HR protein recruitment and function at DNA breaks. Mechanistically, we show that ZMYM2 recruitment to DSBs and suppression of break-associated 53BP1 requires the SUMO E3 ligase PIAS4, as well as SUMO binding by ZMYM2. Cells deficient for ZMYM2/3 display genome instability, PARP inhibitor and ionizing radiation sensitivity and reduced HR repair. Importantly, depletion of 53BP1 in ZMYM2/3-deficient cells rescues BRCA1 recruitment to and HR repair of DSBs, suggesting that ZMYM2 and ZMYM3 primarily function to restrict 53BP1 engagement at breaks to favor BRCA1 loading that functions to channel breaks to HR repair. Identification of DNA repair functions for these poorly characterized ZnF proteins may shed light on their unknown contributions to human diseases, where they have been reported to be highly dysregulated, including in several cancers.     

The interplay between BRCA1 and 53BP1 influences death,aging, senescence and cancer

In proliferating cells DNA double strand breaks (DSBs) are a common occurrence during DNA replication. DSB repair using homologous recombination is essential for the error-free repair of such breaks and proliferating cells require some level of HR activity for their viability. The BRCA1 tumour suppressor has an important role in this process and is believed to channel the DSBs into the HR pathway. The related 53BP1 gene is known to positively regulate repair of DSBs outside of S phase, but via the NHEJ pathway. Two new studies suggest a new role for 53BP1 as an inhibitor of HR [1], [2]. These genetic studies establish that 53BP1, but not other components of the NHEJ machinery, can inhibit the early resection step of HR. In cells defective for BRCA1, which is required for efficient HR, the balance between promoting and inhibiting HR is thrown towards inhibition. Simultaneous loss of 53BP1 can rescue the HR defect of BRCA1-defective cells and restore cellular viability. Here, I provide an overview of these studies and discuss their implications for tumourigenesis.     

An RNF168 fragment defective for focal accumulation at DNA damage is proficient for inhibition of homologous recombination in BRCA1 deficient cells

The E3 ubiquitin ligase RNF168 is a DNA damage response (DDR) factor that promotes monoubiquitination of H2A/H2AX at K13/15, facilitates recruitment of other DDR factors (e.g. 53BP1) to DNA damage, and inhibits homologous recombination (HR) in cells deficient in the tumor suppressor BRCA1. We have examined the domains of RNF168 important for these DDR events, including chromosomal HR that is induced by several nucleases (I-SceI, CAS9-WT and CAS9-D10A), since the inducing nuclease affects the relative frequency of distinct repair outcomes. We found that an N-terminal fragment of RNF168 (1-220/N221*) efficiently inhibits HR induced by each of these nucleases in BRCA1 depleted cells, and promotes recruitment of 53BP1 to DNA damage and H2AX monoubiquitination at K13/15. Each of these DDR events requires a charged residue in RNF168 (R57). Notably, RNF168-N221* fails to self-accumulate into ionizing radiation induced foci (IRIF). Furthermore, expression of RNF168 WT and N221* can significantly bypass the role of another E3 ubiquitin ligase, RNF8, for inhibition of HR in BRCA1 depleted cells, and for promotion of 53BP1 IRIF. We suggest that the ability for RNF168 to promote H2A/H2AX monoubiquitination and 53BP1 IRIF, but not RNF168 self-accumulation into IRIF, is important for inhibition of HR in BRCA1 deficient cells.     

NFBD1/MDC1, 53BP1 and BRCA1 have both redundant and unique roles in the ATM pathway

NFBD1/MDC1, 53BP1, and BRCA1 are DNA damage checkpoint proteins with twin BRCT domains. In order to determine if they have redundant roles in responses to ionizing radiation, we used siRNA and shRNA to deplete NFBD1, 53BP1, and BRCA1 in single, double, and triple combinations. These analyses were performed in early passage human foreskin fibroblasts so that checkpoint responses could be assessed in a normal genetic background. We report that NFBD1, 53BP1, and BRCA1 have both unique and redundant functions in radiation-induced phosphorylation and localization events in the ATM-Chk2 pathway. 53BP1, but not NFBD1 and BRCA1, mediates ionizing radiation-induced ATM S1981 autophosphorylation. In contrast, all three mediators collaborate to promote IR-induced Chk2 T68 phosphorylation. NFBD1 and 53BP1, but not BRCA1, work together to mediate pATMS1981, pChk2T68, and NBS1 ionizing radiation induced foci (IRIF). However, the relative importance of NFBD1 and 53BP1 in IRIF formation differ. We also determined the interdependence among mediators in IRIF recruitment. We extend previous findings in cancer cells and mouse cells that NFBD1 is upstream of 53BP1 and BRCA1 to primary human cells. Furthermore, NFBD1 promotes BRCA1 IRIF through both 53BP1-dependent and 53BP1-independent mechanisms.     

DNA repair pathway choice—a PTIP of the hat to 53BP1

In the June issue of Cell, Nussenzweig and colleagues identify PTIP/PAXIP as a 53BP1 effector protein in the regulatory network that controls DSB repair pathway choice.Cell (2013) 153 6, 1266–1280 doi: 10.1016/j.cell.2013.05.023DNA double-stranded breaks (DSBs) are highly cytotoxic lesions that can induce genome rearrangements if not accurately repaired. DSBs can be repaired either through homologous recombination (HR) or non-homologous end-joining (NHEJ). HR is the preferred repair pathway during the S and G2 cell cycle phases because a sister chromatid provides a perfect template for ‘error-free'' repair. During G1, when HR is suppressed to prevent recombination with homologues, repair is achieved primarily by NHEJ. Molecularly, DSB repair pathway choice is largely regulated at the level of 5′ to 3′ DNA end resection, that is, the formation of the 3′ end single-stranded DNA overhangs that are used to initiate HR. End resection inhibits NHEJ and promotes HR.In the June issue of Cell, Nussenzweig and colleagues identified the protein PTIP (also known as PAXIP) as a new component of the regulatory network that controls DSB repair pathway choice [1]. This work has important implications for our understanding of the mechanisms by which genomic integrity is underpinned, and is especially germane to those interested in the genesis of breast and ovarian cancer caused by a defective BRCA1 protein, which is crucial for DSB repair by HR.53BP1 (also known as TP53BP1) is a key determinant of DSB repair pathway choice [2]. In response to DSBs, 53BP1 binds to chromatin at damaged sites, where it promotes NHEJ by blocking end resection. 53BP1 has a crucial role during class switch recombination (CSR) in B cells and the fusion of dysfunctional telomeres. An even more striking phenotype was observed in mice in which loss of 53BP1 reversed most of the phenotypes associated with BRCA1 deficiency, including cell and embryonic lethality as well as tumorigenesis [2]. These findings suggest that 53BP1 and BRCA1 battle each other to influence DSB repair pathway choice.Molecularly, 53BP1 is responsible for the defective HR seen in BRCA1-deficient cells. Furthermore, in those cells, 53BP1 promotes the formation of characteristic radial chromosomes that are caused by toxic NHEJ events, presumably during S phase. Understanding exactly how 53BP1 carries out its many functions has been a major challenge to the field as 53BP1 does not harbour any enzymatic activity. However, it has been shown that 53BP1 must accumulate on chromatin to be functional. In addition, a mutant 53BP1 allele in which all 28 ataxia telangiectasia-mutated (ATM) phosphorylation sites were changed to alanine (53BP1^28A) failed to rescue 53BP1 deficiency, suggesting that 53BP1 acts through phosphorylation-dependent protein interactions to promote NHEJ [2].RIF1 was identified as the first effector of 53BP1 in DSB repair [3,4,5,6,7]. RIF1 accumulates at DSB sites by binding to phosphorylated 53BP1 but, intriguingly, the loss of RIF1 has a milder effect than the loss of 53BP1 with respect to the fusion of dysfunctional telomeres [3], and RIF1 deficiency does not fully restore HR in BRCA1-deficient cells [7]. As the 53BP1^28A mutant is nearly as defective as the complete loss of 53BP1 for these activities, these observations indicate that additional 53BP1 effector proteins contribute to some of the 53BP1 functions.Nussenzweig and colleagues provide compelling evidence that the BRCT domain-containing protein PTIP is the missing 53BP1 effector protein [1]. The authors identified a separation-of-function mutation in 53BP1 that disrupted the first eight amino-terminal ATM sites (53BP1^8A). The 53BP1^8A mutant behaved the same as the wild-type protein with respect to CSR—a physiological process dependent on NHEJ—but failed to promote genome instability (radial chromosome formation) in BRCA1-deficient cells after treatment with a PARP inhibitor. Since RIF1-deficient cells have impaired CSR and RIF1 can localize to break sites in cells expressing the 53BP1^8A mutant, this suggests that a protein other than RIF1 binds to the N-terminal region of 53BP1 to inhibit HR.The newly identified 53BP1 effector protein PTIP is a multifunctional DNA repair factor that interacts with phosphorylated Ser 25 of 53BP1 through its tandem BRCT domains [8]—a site that was mutated in the 53BP1^8A allele. PTIP is also part of the MLL3/MLL4 histone H3 Lys 4 methyltransferase complexes but this function seems to be unrelated to its role as a 53BP1 co-factor.Nussenzweig and co-workers found that PTIP-deficient cells are sensitive to ionizing radiation but tolerant of DNA damaging agents that are toxic to HR-deficient cells, which suggests a role for PTIP in NHEJ. In agreement with this, the fusion frequency of uncapped telomeres was reduced in PTIP-deficient cells. Interestingly, as in the case of the 53BP1^8A allele, PTIP-deficient B cells were proficient in switching their immunoglobulin locus, although this switching event is impaired in RIF1^−/− B cells. This suggests that PTIP might participate selectively in pathological NHEJ.Nussenzweig and colleagues next generated a conditional BRCA1^−/− PTIP^−/− mouse to investigate the contribution of PTIP to the genome instability of BRCA1-deficient B cells. Loss of PTIP restored normal growth kinetics and genome stability to BRCA1-deficient cells treated with a PARP inhibitor. In addition, RAD51 IR-induced focus formation was restored in BRCA1^−/− PTIP^−/− cells. As the primary defect of BRCA1-deficient cells with respect to HR seems to be at the level of resection, the accumulation of the single-stranded DNA-binding protein RPA into IR-induced foci was then analysed. The finding that PTIP-deficient cells have an increased number of RPA foci per cell supports a role for PTIP in blocking resection. Together, this suggests that PTIP opposes DNA end resection and mutagenic DSB repair in BRCA1-deficient cells.These results were surprising as they revealed that the 53BP1 activities relating to physiological NHEJ (during CSR) and mutagenic NHEJ (after PARP inhibition) can be separated, and that they are carried out by two distinct proteins that ‘read'' ATM-dependent 53BP1 phosphorylation. The relationship between 53BP1, RIF1 and PTIP is probably complex, as suggested by the possible competition between RIF1 and PTIP, and the observation that both proteins contribute in an additive manner to the fusion of dysfunctional telomeres, downstream from 53BP1.According to these findings, multiple phosphorylation events in 53BP1 seem to integrate ATM activity to control distinct aspects of DSB repair pathway choice (Fig 1). Establishing exactly how an increase of ATM activity at break sites is translated into the coordination of 53BP1 phosphorylation, with RIF1 and PTIP binding, will be an important milestone towards understanding 53BP1 function. Indeed, multi-site phosphorylation and its recognition by binding proteins can be used to develop switch-like responses that might be important for organizing the chromatin at DSB sites.Open in a separate windowFigure 153BP1 phospho-dependent interactions involved in DSB repair. PTIP and RIF1 interact with chromatin-bound and ATM-phosphorylated 53BP1 at DSB sites. PTIP binds directly to 53BP1 phosphorylated on Ser 14;25 (within the first eight Ser/Thr-Q sites). RIF1 binds to phosphorylated 53BP1 either directly or through an intermediate factor (X). The carboxy-terminal seven Ser/Thr-Q sites (9–15 Ser/Thr-Q sites) are involved in the interaction of RIF1–53BP1, although the amino-terminal eight Ser/Thr-Q sites might stabilize the binding. It is unknown whether PTIP and RIF1 can associate simultaneously with 53BP1 (left side of the figure), or if the binding is exclusive, due to either differential phosphorylation of the Ser/Thr-Q sites or steric hindrance (right side of the figure). 53BP1, PTIP and RIF1 block DNA end-resection and promote NHEJ repair. Although both PTIP and RIF1 contribute to dysfunctional telomere fusions, they also have distinct functions downstream from 53BP1. While RIF1 is essential for CSR and has a milder effect on toxic NHEJ events, PTIP is dispensable for CSR and has a more prominent role in toxic NHEJ events that lead to genome instability in BRCA1-deficient cells. ATM, ataxia telangiectasia-mutated; CSR, class switch recombination; DSB, double-stranded break; NHEJ, non-homologous end-joining.The identification of PTIP as a new 53BP1 effector also deepens the mystery of DSB repair pathway choice regulation by 53BP1. Future studies are needed to elucidate how 53BP1 and its effector proteins block resection. Are PTIP and RIF1 blocking specific nucleases? Do they act in a temporally distinct fashion or are they distributed in distinct subdomains of the chromatin flanking DSB sites? What is the function of PTIP in relation to the cell cycle? Testing whether RIF1 binds directly to 53BP1, and if so to which phosphorylated site, might answer some of the above questions. The identification of a RIF1 mutation that selectively disrupts 53BP1 binding would enable surgical manipulation of the 53BP1–RIF1–PTIP circuit at DSB sites.Another unresolved issue is whether 53BP1 acts solely by recruiting RIF1 and PTIP, or whether 53BP1 has a more active role in blocking resection. We have shown that 53BP1 localizes to the chromatin flanking the DSBs by binding to methylated and ubiquitinated nucleosomes, in a wheel clamp-like manner [9]. This suggests that 53BP1 might modify the nucleosomal array structure in a way that makes it refractory to the resection machinery. Recognizing how nucleosomes modified by 53BP1 cooperate with RIF1 and PTIP might provide clues to the role of these two proteins in end protection.It is important to note that in human cells, PTIP might not be recruited to DSB sites in a 53BP1- and ATM-dependent manner [8]. Furthermore, in the avian B-cell line DT40, PTIP promotes HR instead of inhibiting it [10]. It will be important to revisit these studies to tease out whether these differences are due to context-, experiment- or species-specific effects.The identification of PTIP as a candidate genetic modifier of BRCA1-deficient tumours is an important finding. As noted by the authors, disabling the PTIP–53BP1 interaction pharmacologically might selectively restore HR in BRCA1-deficient cells, which might be useful in certain contexts, for example as a chemopreventive strategy.     

BRCA1-Ku80 Protein Interaction Enhances End-joining Fidelity of Chromosomal Double-strand Breaks in the G1 Phase of the Cell Cycle

Quality control of DNA double-strand break (DSB) repair is vital in preventing mutagenesis. Non-homologous end-joining (NHEJ), a repair process predominant in the G₁ phase of the cell cycle, rejoins DSBs either accurately or with errors, but the mechanisms controlling its fidelity are poorly understood. Here we show that BRCA1, a tumor suppressor, enhances the fidelity of NHEJ-mediated DSB repair and prevents mutagenic deletional end-joining through interaction with canonical NHEJ machinery during G₁. BRCA1 binds and stabilizes Ku80 at DSBs through its N-terminal region, promotes precise DSB rejoining, and increases cellular resistance to radiation-induced DNA damage in a G₁ phase-specific manner. These results suggest that BRCA1, as a central player in genome integrity maintenance, ensures high fidelity repair of DSBs by not only promoting homologous recombination repair in G₂/M phase but also facilitating fidelity of Ku80-dependent NHEJ repair, thus preventing deletional end-joining of chromosomal DSBs during G₁.     

BRCA1 functions independently of homologous recombination in DNA interstrand crosslink repair

Brca1 is required for DNA repair by homologous recombination (HR) and normal embryonic development. Here we report that deletion of the DNA damage response factor 53BP1 overcomes embryonic lethality in Brca1-nullizygous mice and rescues HR deficiency, as measured by hypersensitivity to polyADP-ribose polymerase (PARP) inhibition. However, Brca1,53BP1 double-deficient cells are hypersensitive to DNA interstrand crosslinks (ICLs), indicating that BRCA1 has an additional role in DNA crosslink repair that is distinct from HR. Disruption of the nonhomologous end-joining (NHEJ) factor, Ku, promotes DNA repair in Brca1-deficient cells; however deletion of either Ku or 53BP1 exacerbates genomic instability in cells lacking FANCD2, a mediator of the Fanconi anemia pathway for ICL repair. BRCA1 therefore has two separate roles in ICL repair that can be modulated by manipulating NHEJ, whereas FANCD2 provides a key activity that cannot be bypassed by ablation of 53BP1 or Ku.     

SHLD2/FAM35A co‐operates with REV7 to coordinate DNA double‐strand break repair pathway choice

DNA double‐strand breaks (DSBs) can be repaired by two major pathways: non‐homologous end‐joining (NHEJ) and homologous recombination (HR). DNA repair pathway choice is governed by the opposing activities of 53BP1, in complex with its effectors RIF1 and REV7, and BRCA1. However, it remains unknown how the 53BP1/RIF1/REV7 complex stimulates NHEJ and restricts HR to the S/G2 phases of the cell cycle. Using a mass spectrometry (MS)‐based approach, we identify 11 high‐confidence REV7 interactors and elucidate the role of SHLD2 (previously annotated as FAM35A and RINN2) as an effector of REV7 in the NHEJ pathway. FAM35A depletion impairs NHEJ‐mediated DNA repair and compromises antibody diversification by class switch recombination (CSR) in B cells. FAM35A accumulates at DSBs in a 53BP1‐, RIF1‐, and REV7‐dependent manner and antagonizes HR by limiting DNA end resection. In fact, FAM35A is part of a larger complex composed of REV7 and SHLD1 (previously annotated as C20orf196 and RINN3), which promotes NHEJ and limits HR. Together, these results establish SHLD2 as a novel effector of REV7 in controlling the decision‐making process during DSB repair.     

HP1 promotes tumor suppressor BRCA1 functions during the DNA damage response

The DNA damage response (DDR) involves both the control of DNA damage repair and signaling to cell cycle checkpoints. Therefore, unraveling the underlying mechanisms of the DDR is important for understanding tumor suppression and cellular resistance to clastogenic cancer therapeutics. Because the DDR is likely to be influenced by chromatin regulation at the sites of DNA damage, we investigated the role of heterochromatin protein 1 (HP1) during the DDR process. We monitored double-strand breaks (DSBs) using the γH2AX foci marker and found that depleting cells of HP1 caused genotoxic stress, a delay in the repair of DSBs and elevated levels of apoptosis after irradiation. Furthermore, we found that these defects in repair were associated with impaired BRCA1 function. Depleting HP1 reduced recruitment of BRCA1 to DSBs and caused defects in two BRCA1-mediated DDR events: (i) the homologous recombination repair pathway and (ii) the arrest of cell cycle at the G₂/M checkpoint. In contrast, depleting HP1 from cells did not affect the non-homologous end-joining (NHEJ) pathway: instead it elevated the recruitment of the 53BP1 NHEJ factor to DSBs. Notably, all three subtypes of HP1 seemed to be almost equally important for these DDR functions. We suggest that the dynamic interaction of HP1 with chromatin and other DDR factors could determine DNA repair choice and cell fate after DNA damage. We also suggest that compromising HP1 expression could promote tumorigenesis by impairing the function of the BRCA1 tumor suppressor.     

RIF1: A novel regulatory factor for DNA replication and DNA damage response signaling

DNA double strand breaks (DSBs) are highly toxic to the cells and accumulation of DSBs results in several detrimental effects in various cellular processes which can lead to neurological, immunological and developmental disorders. Failure of the repair of DSBs spurs mutagenesis and is a driver of tumorigenesis, thus underscoring the importance of the accurate repair of DSBs. Two major canonical DSB repair pathways are the non-homologous end joining (NHEJ) and homologous recombination (HR) pathways. 53BP1 and BRCA1 are the key mediator proteins which coordinate with other components of the DNA repair machinery in the NHEJ and HR pathways respectively, and their exclusive recruitment to DNA breaks/ends potentially decides the choice of repair by either NHEJ or HR. Recently, Rap1 interacting factor 1 has been identified as an important component of the DNA repair pathway which acts downstream of the ATM/53BP1 to inhibit the 5′–3′ end resection of broken DNA ends, in-turn facilitating NHEJ repair and inhibiting homology directed repair. Rif1 is conserved from yeast to humans but its function has evolved from telomere length regulation in yeast to the maintenance of genome integrity in mammalian cells. Recently its role in the maintenance of genomic integrity has been expanded to include the regulation of chromatin structure, replication timing and intra-S phase checkpoint. We present a summary of these important findings highlighting the various aspects of Rif1 functions and discuss the key implications for genomic integrity.     

Ubiquitin-activating enzyme UBA1 is required for cellular response to DNA damage

The cellular DNA damage response (DDR) machinery that maintains genomic integrity and prevents severe pathologies, including cancer, is orchestrated by signaling through protein modifications. Protein ubiquitylation regulates repair of DNA double-strand breaks (DSBs), toxic lesions caused by various metabolic as well as environmental insults such as ionizing radiation (IR). Whereas several components of the DSB-evoked ubiquitylation cascade have been identified, including RNF168 and BRCA1 ubiquitin ligases, whose genetic defects predispose to a syndrome mimicking ataxia-telangiectasia and cancer, respectively, the identity of the apical E1 enzyme involved in DDR has not been established. Here, we identify ubiquitin-activating enzyme UBA1 as the E1 enzyme required for responses to IR and replication stress in human cells. We show that siRNA-mediated knockdown of UBA1, but not of another UBA family member UBA6, impaired formation of both ubiquitin conjugates at the sites of DNA damage and IR-induced foci (IRIF) by the downstream components of the DSB response pathway, 53BP1 and BRCA1. Furthermore, chemical inhibition of UBA1 prevented IRIF formation and severely impaired DSB repair and formation of 53BP1 bodies in G₁, a marker of response to replication stress. In contrast, the upstream steps of DSB response, such as phosphorylation of histone H2AX and recruitment of MDC1, remained unaffected by UBA1 depletion. Overall, our data establish UBA1 as the apical enzyme critical for ubiquitylation-dependent signaling of both DSBs and replication stress in human cells, with implications for maintenance of genomic integrity, disease pathogenesis and cancer treatment.     

H4K20me2 distinguishes pre-replicative from post-replicative chromatin to appropriately direct DNA repair pathway choice by 53BP1-RIF1-MAD2L2

The main pathways for the repair of DNA double strand breaks (DSBs) are non-homologous end-joining (NHEJ) and homologous recombination directed repair (HDR). These operate mutually exclusive and are activated by 53BP1 and BRCA1, respectively. As HDR can only succeed in the presence of an intact copy of replicated DNA, cells employ several mechanisms to inactivate HDR in the G1 phase of cell cycle. As cells enter S-phase, these inhibitory mechanisms are released and HDR becomes active. However, during DNA replication, NHEJ and HDR pathways are both functional and non-replicated and replicated DNA regions co-exist, with the risk of aberrant HDR activity at DSBs in non-replicated DNA. It has become clear that DNA repair pathway choice depends on inhibition of DNA end-resection by 53BP1 and its downstream factors RIF1 and MAD2L2. However, it is unknown how MAD2L2 accumulates at DSBs to participate in DNA repair pathway control and how the NHEJ and HDR repair pathways are appropriately activated at DSBs with respect to the replication status of the DNA, such that NHEJ acts at DSBs in pre-replicative DNA and HDR acts on DSBs in post-replicative DNA. Here we show that MAD2L2 is recruited to DSBs in H4K20 dimethylated chromatin by forming a protein complex with 53BP1 and RIF1 and that MAD2L2, similar to 53BP1 and RIF1, suppresses DSB accumulation of BRCA1. Furthermore, we show that the replication status of the DNA locally ensures the engagement of the correct DNA repair pathway, through epigenetics. In non-replicated DNA, saturating levels of the 53BP1 binding site, di-methylated lysine 20 of histone 4 (H4K20me2), lead to robust 53BP1-RIF1-MAD2L2 recruitment at DSBs, with consequent exclusion of BRCA1. Conversely, replication-associated 2-fold dilution of H4K20me2 promotes the release of the 53BP1-RIF1-MAD2L2 complex and favours the access of BRCA1. Thus, the differential H4K20 methylation status between pre-replicative and post-replicative DNA represents an intrinsic mechanism that locally ensures appropriate recruitment of the 53BP1-RIF1-MAD2L2 complex at DNA DSBs, to engage the correct DNA repair pathway.     

Structural Maintenance of Chromosomes Flexible Hinge Domain Containing 1 (SMCHD1) Promotes Non-homologous End Joining and Inhibits Homologous Recombination Repair upon DNA Damage

Structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1) has been shown to be involved in gene silencing and DNA damage. However, the exact mechanisms of how SMCHD1 participates in DNA damage remains largely unknown. Here we present evidence that SMCHD1 recruitment to DNA damage foci is regulated by 53BP1. Knocking out SMCHD1 led to aberrant γH2AX foci accumulation and compromised cell survival upon DNA damage, demonstrating the critical role of SMCHD1 in DNA damage repair. Following DNA damage induction, SMCHD1 depletion resulted in reduced 53BP1 foci and increased BRCA1 foci, as well as less efficient non-homologous end joining (NHEJ) and elevated levels of homologous recombination (HR). Taken together, these results suggest an important function of SMCHD1 in promoting NHEJ and repressing HR repair in response to DNA damage.     

53BP1, BRCA1, and the Choice between Recombination and End Joining at DNA Double-Strand Breaks

When DNA double-strand breaks occur, the cell cycle stage has a major influence on the choice of the repair pathway employed. Specifically, nonhomologous end joining is the predominant mechanism used in the G₁ phase of the cell cycle, while homologous recombination becomes fully activated in S phase. Studies over the past 2 decades have revealed that the aberrant joining of replication-associated breaks leads to catastrophic genome rearrangements, revealing an important role of DNA break repair pathway choice in the preservation of genome integrity. 53BP1, first identified as a DNA damage checkpoint protein, and BRCA1, a well-known breast cancer tumor suppressor, are at the center of this choice. Research on how these proteins function at the DNA break site has advanced rapidly in the recent past. Here, we review what is known regarding how the repair pathway choice is made, including the mechanisms that govern the recruitment of each critical factor, and how the cell transitions from end joining in G₁ to homologous recombination in S/G₂.     

Regulation of repair choice: Cdk1 suppresses recruitment of end joining factors at DNA breaks

Cell cycle plays a crucial role in regulating the pathway used to repair DNA double-strand breaks (DSBs). In Saccharomyces cerevisiae, homologous recombination is primarily limited to non-G₁ cells as the formation of recombinogenic single-stranded DNA requires CDK1-dependent 5′ to 3′ resection of DNA ends. However, the effect of cell cycle on non-homologous end joining (NHEJ) is not yet clearly defined. Using an assay to quantitatively measure the contributions of each repair pathway to repair product formation and cellular survival after DSB induction, we found that NHEJ is most efficient at G₁, and markedly repressed at G₂. Repression of NHEJ at G₂ is achieved by efficient end resection and by the reduced association of core NHEJ proteins with DNA breaks, both of which depend on the CDK1 activity. Importantly, repression of 5′ end resection by CDK1 inhibition at G₂ alone did not fully restore either physical association of Ku/Dnl4-Lif1 with DSBs or NHEJ proficiency to the level at G₁. Expression of excess Ku can partially offset the inhibition of end joining at G₂. The results suggest that regulation of Ku/Dnl4-Lif1 affinity for DNA ends may contribute to the cell cycle-dependent modulation of NHEJ efficiency.     

Opposing roles for 53BP1 during homologous recombination

Although DNA non-homologous end-joining repairs most DNA double-strand breaks (DSBs) in G2 phase, late repairing DSBs undergo resection and repair by homologous recombination (HR). Based on parallels to the situation in G1 cells, previous work has suggested that DSBs that undergo repair by HR predominantly localize to regions of heterochromatin (HC). By using H3K9me3 and H4K20me3 to identify HC regions, we substantiate and extend previous evidence, suggesting that HC-DSBs undergo repair by HR. Next, we examine roles for 53BP1 and BRCA1 in this process. Previous studies have shown that 53BP1 is pro-non-homologous end-joining and anti-HR. Surprisingly, we demonstrate that in G2 phase, 53BP1 is required for HR at HC-DSBs with its role being to promote phosphorylated KAP-1 foci formation. BRCA1, in contrast, is dispensable for pKAP-1 foci formation but relieves the barrier caused by 53BP1. As 53BP1 is retained at irradiation-induced foci during HR, we propose that BRCA1 promotes displacement but retention of 53BP1 to allow resection and any necessary HC modifications to complete HR. In contrast to this role for 53BP1 in HR in G2 phase, we show that it is dispensable for HR in S phase, where HC regions are likely relaxed during replication.