TOPBP1 takes RADical command in recombinational DNA repair

TOPBP1 is a key player in DNA replication and DNA damage signaling. In this issue, Moudry et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201507042) uncover a crucial role for TOPBP1 in DNA repair by revealing its requirement for RAD51 loading during repair of double strand breaks by homologous recombination.Proper replication and maintenance of the eukaryotic genome requires the involvement of the scaffolding protein TOPBP1. Over the last 20 years, studies in yeast, frog, and mammals have revealed conserved roles for TOPBP1 in initiation of DNA replication and activation of DNA damage signaling. TOPBP1 has been shown to assemble ternary protein complexes necessary to jump-start DNA replication or to initiate DNA damage signaling events by recognizing distinct phosphoproteins via its multiple BRCA1 C terminus (BRCT) domains (Fig. 1 D; Wardlaw et al., 2014). In this issue, Moudry et al. add to the list of crucial TOPBP1 roles in genome biology and reveal that TOPBP1 is also required for proper repair of double strand breaks (DSBs) by homologous recombination (HR).Open in a separate windowFigure 1.Involvement of TOPBP1 in HR-mediated repair. (A) The work by Moudry et al. (2016) supports a model in which TOPBP1 directs the action of PLK1 kinase toward RAD51, mediating a phosphorylation event that licenses a second phosphorylation event mediated by the casein kinase 2 (CK2) kinase. These phosphorylation events are believed to facilitate the loading of RAD51, which replaces RPA, at damage sites and favor HR. (B) TOPBP1 as a hub for coordinating the action of multiple kinases toward genome maintenance. (C) Speculative model for the mutually exclusive engagement of TOPBP1 in distinct cellular processes as a strategy for coordinating genome replication and DNA damage responses (see text for details). (D) Current understanding of how TOPBP1 and its yeast orthologue Dpb11 mediate the formation of ternary complexes for replication initiation, DNA damage signaling, and recombinational repair. In mammals, the indicated proteins have been shown to interact with TOPBP1, but the roles of most of those interactions remain unclear. For simplicity, some TOPBP1 interactions and their cofactors are not depicted.Moudry et al. (2016) report that depletion of TOPBP1 makes cells highly sensitive to the poly (ADP-ribose) polymerase inhibitor olaparib, a drug known to sensitize cells with an already dysfunctional HR machinery. In particular, olaparib hypersensitizes cells that carry mutations in the bona fide HR factors and tumor suppressors BRCA1 or BRCA2. In this work, the authors first identified TOPBP1 as a hit in a high-content RNAi screen for proteins whose depletion resulted in higher toxicity after olaparib treatment in osteosarcoma cells, which suggests that loss or inactivation of TOPBP1 predicts the response of cancer cells to this drug. Moudry et al. (2016) observed that RNAi-mediated knockdown of TOPBP1 in cancer cells treated with olaparib increased the level of DNA damage and induced DNA DSB markers. The researchers subsequently examined whether olaparib sensitivity reflected defective HR in TOPBP1-depleted cells by measuring HR activity through several parameters and confirmed that TOPBP1-depleted cells showed reduced HR activity.The HR process encompasses several phases, including end resection and chromatin loading of RPA and RAD51, which can be visualized by formation of microscopically detectable foci. Moudry et al. (2016) searched for which step of HR was compromised in cells depleted for TOPBP1 and found that DNA end resection, i.e., the processing of the 5′ recessed end that exposes a 3′ overhang used for homology search, seemed not to be affected, as evaluated by the amounts of single stranded DNA detected by BrdU incorporation under nondenaturing conditions. Interestingly, they found that the next key stage in HR, in which the RAD51 recombinase protein is loaded at these 3′ overhangs (Fig. 1 A), was greatly impaired, based on the assessment of the formation of RAD51 foci by microscopy and of the biochemical analysis of RAD51 accumulation on chromatin. Although the mechanism by which TOPBP1 promotes the loading of RAD51 remains unclear, the authors propose an interesting model in which TOPBP1 plays a scaffolding role to direct Polo-like Kinase 1 (PLK1), which phosphorylates RAD51 and facilitates its loading to DNA damage sites (Fig. 1 A; Yata et al., 2012). Consistent with this model, they show that TOPBP1 physically interacts with PLK1 and that depletion of TOPBP1 impairs PLK1-dependent RAD51 phosphorylation. Although more work is needed to prove that the TOPBP1–PLK1 interaction is required for this phosphorylation event, the results are exciting as they suggest another important functional link between TOPBP1 and a kinase. During DNA damage signaling, TOPBP1 plays an established role in activating the ATR kinase (Kumagai et al., 2006) and is believed to direct ATR’s action toward specific substrates. This latter function is best understood in yeast, in which TOPBP1/Dpb11 forms a ternary complex to direct ATR/Mec1 action to phosphorylate the downstream kinase Rad53. Interestingly, recent data from fission yeast also suggest that TOPBP1 interacts with yet another kinase, CDK, and directs its kinase action (Qu et al., 2013). The emerging scenario is that TOPBP1 may function as a scaffolding hub for controlling the action of distinct kinases to ensure genome integrity (Fig. 1 B).Although the work of Moudry et al. (2016) is the first to show a clear role for TOPBP1 in RAD51 loading, studies in budding yeast have proposed links between the TOPBP1 orthologue Dpb11 and HR-mediated repair. It was shown that the temperature-sensitive dpb11-1 mutant displays a sensitivity to DNA damage that is not further increased by deletion of RAD51, suggesting that Dpb11 functions in HR repair (Ogiwara et al., 2006). In addition, other groups showed that TOPBP1/Dpb11 is required for DSB-induced mating-type switching and also reached the conclusion that TOPBP1/Dpb11 is required for HR-mediated repair of a DSB (Germann et al., 2011; Hicks et al., 2011). These studies provided compelling evidence that the role for TOPBP1/Dpb11 in DSB repair is independent of its roles in replication initiation and DNA damage signaling. In humans, there also is evidence pointing to potential roles for TOPBP1 in DNA repair, as depletion of TOPBP1 was found to increase sensitivity to ionizing radiation and lead to defective DSB repair by HR (Morishima et al., 2007).The new set of results provided by Moudry et al. (2016) clearly place TOPBP1 at the center stage of HR-mediated repair in what seems to be yet another key and evolutionarily conserved role for TOPBP1, in addition to replication initiation and DNA damage signaling. An intriguing and unanswered question relates to defining the evolutionary benefit conferred by maintaining these crucial roles in the same protein. It is tempting to speculate that having a single protein module in command of key licensing events helps ensure the ordered and mutually exclusive execution of distinct cellular processes (Fig. 1 C). This is a particularly attractive and well-suited idea for the established role of DNA damage signaling in inhibiting origin firing during DNA replication. Sequestration of TOPBP1 into a complex involved in DNA damage signaling would help ensure that replication initiation is inhibited. Consistent with this hypothesis, it is established in yeast that the same BRCT domains involved in replication initiation are also required for DNA damage signaling. In addition, it was recently shown that competition between DNA damage signaling proteins and DNA repair factors for binding to the BRCT domains of TOPBP1/Dpb11 is a mechanism to remove TOPBP1/Dpb11 from a pro-DNA damage signaling complex, resulting in dampening of DNA damage signaling (Ohouo et al., 2013; Cussiol et al., 2015). It will be exciting to further explore this competition-based regulatory mechanism in human cells, as well as in the coordination of DNA damage signaling with DNA repair. In this direction, it is crucial that the precise molecular mechanism by which TOPBP1 promotes HR repair is elucidated, including defining which TOPBP1 BRCT domains are required and which factors they are binding to favor RAD51 loading or other pro-HR functions. Through truncation mutation analyses, Moudry et al. (2016) show that the specific BRCT domains 7/8 of TOPBP1 are essential for TOPBP1’s role in promoting HR. However, it remains unclear how this is accomplished mechanistically.To make the scenario even more complicated, TOPBP1 is known to physically interact with an extensive network of repair factors, including, but not limited to, BRCA1, 53BP1, MRN, FANCJ, and BLM (Greenberg et al., 2006; Wardlaw et al., 2014). This points to an extremely complex system by which TOPBP1 could be coordinating the action of a range of repair factors and repair pathways (Fig. 1 D). It would not be surprising if TOPBP1 was found to be key for the regulation of other steps in HR-mediated repair as well as other repair pathways in response to varied types of genotoxic insults, including DNA replication stress. In yeast, the interaction between TOPBP1/Dpb11 and the repair scaffold Slx4 provides an additional example of the rich range of possibilities by which TOPBP1/Dpb11 functions in DNA repair. In addition to sequestering TOPBP1/Dpb11 and dampening DNA damage signaling (Ohouo et al., 2013; Cussiol et al., 2015), the Slx4–TOPBP1/Dpb11 interaction was recently found to control DNA end resection (Dibitetto et al., 2015) and was proposed to affect the late step of resolution of repair intermediates (Gritenaite et al., 2014). The TOPBP1–SLX4 interaction is conserved in humans; however, it remains unclear how this interaction impacts DNA repair in higher eukaryotes. Moreover, whereas in yeast it is possible to clearly define a pro-DNA damage signaling complex and a pro-recombinational repair complex (Fig. 1 D), in mammals the scenario is more complex and it is currently unclear what the precise contributions of different TOPBP1 interactions are in DNA damage signaling and/or recombinational repair. Finally, because the ATR kinase is expected to regulate several DNA repair factors, it is likely that the ATR-activating function of TOPBP1 plays important roles in some aspect of DNA repair. A major experimental avenue to explore this possibility and improve our understanding of the other roles for TOPBP1 in DNA repair will be the generation of separation-of-function mutants that do not interfere with DNA replication or DNA damage signaling.Following the findings reported by Moudry et al. (2016), it is interesting to speculate on the implications of understanding TOPBP1’s role in HR-mediated repair for cancer research and treatment. Little is known about the role of TOPBP1 in carcinogenesis. It was found that TOPBP1 expression and subcellular localization are altered in a subset of breast cancer samples (Going et al., 2007; Liu et al., 2009; Forma et al., 2012) and Moudry et al. (2016) also report altered TOPBP1 protein expression in ovarian cancers, although at modest frequencies. Nonetheless, as we learn more about TOPBP1 mechanisms of action in HR, it is possible that it may become an important target for manipulating the HR response by using small molecules such as Calcein AM, which targets BRCT domains 7/8 of TOPBP1 (Chowdhury et al., 2014) and was shown by Moudry et al. (2016) to impair HR. Concerning the finding that TOPBP1 plays a pro-HR function very much like BRCA1 and BRCA2, whose genes are most frequently mutated in ovarian and breast cancers, it is intriguing that although TOPBP1 mutations have been found in cancers (Rebbeck et al., 2009; Forma et al., 2013), they are relatively infrequent and are likely not driver mutations. If TOPBP1 plays a key role in RAD51 loading, which is the step severely perturbed in BRCA1- or BRCA2-mutated cancer cells, it is not clear how more cancer-driving mutations have not been identified in TOPBP1. One possibility is that TOPBP1 mutations affecting TOPBP1’s pro-HR function also affect DNA replication and DNA damage signaling and impair the replicative capacity of cancer cells. Disentangling potential antagonistic roles for TOPBP1 in both suppressing and supporting tumorigenesis could lead to exciting new directions to study this complex multifunctional protein and to potentially develop new therapeutic strategies.     

PARP1 and CHK1 coordinate PLK1 enzymatic activity during the DNA damage response to promote homologous recombination-mediated repair

Polo-like kinase 1 (PLK1) is a master kinase that regulates cell cycle progression. How its enzymatic activity is regulated in response to DNA damage is not fully understood. We show that PLK1 is enriched at double strand breaks (DSBs) within seconds of UV laser irradiation in a PARP-1-dependent manner and then disperses within 10 min in a PARG-dependent manner. Poly(ADP-)ribose (PAR) chains directly bind to PLK1 in vitro and inhibit its enzymatic activity. CHK1-mediated PLK1 phosphorylation at S137 prevents its binding to PAR and recruitment to DSBs but ensures PLK1 phosphorylation at T210 and its enzymatic activity toward RAD51 at S14. This subsequent phosphorylation event at S14 primes RAD51 for CHK1-mediated phosphorylation at T309, which is essential for full RAD51 activation. This CHK1–PLK1–RAD51 axis ultimately promotes homologous recombination (HR)-mediated repair and ensures chromosome stability and cellular radiosensitivity. These findings provide biological insight for combined cancer therapy using inhibitors of PARG and CHK1.     

TOPORS-mediated RAD51 SUMOylation facilitates homologous recombination repair

Homologous recombination (HR) is critical for error-free repair of DNA double-strand breaks. Chromatin loading of RAD51, a key protein that mediates the recombination, is a crucial step in the execution of the HR repair. Here, we present evidence that SUMOylation of RAD51 is crucial for the RAD51 recruitment to chromatin and HR repair. We found that topoisomerase 1-binding arginine/serine-rich protein (TOPORS) induces the SUMOylation of RAD51 at lysine residues 57 and 70 in response to DNA damaging agents. The SUMOylation was facilitated by an ATM-induced phosphorylation of TOPORS at threonine 515 upon DNA damage. Knockdown of TOPORS or expression of SUMOylation-deficient RAD51 mutants caused reduction in supporting normal RAD51 functions during the HR repair, suggesting the physiological importance of the modification. We found that the SUMOylation-deficient RAD51 reduces the association with its crucial binding partner BRCA2, explaining its deficiency in supporting the HR repair. These findings altogether demonstrate a crucial role for TOPORS-mediated RAD51 SUMOylation in promoting HR repair and genomic maintenance.     

The USP1-UAF1 complex interacts with RAD51AP1 to promote homologous recombination repair

USP1 deubiquitinating enzyme and its stoichiometric binding partner UAF1 play an essential role in promoting DNA homologous recombination (HR) repair in response to various types of DNA damaging agents. Deubiquitination of FANCD2 may be attributed to the key role of USP1-UAF1 complex in regulating HR repair, however whether USP1-UAF1 promotes HR repair independently of FANCD2 deubiquitination is not known. Here we show evidence that the USP1-UAF1 complex has a FANCD2-independent function in promoting HR repair. Proteomic search of UAF1-interacting proteins revealed that UAF1 associates with RAD51AP1, a RAD51-interacting protein implicated in HR repair. We show that UAF1 mediates the interaction between USP1 and RAD51AP1, and that depletion of USP1 or UAF1 led to a decreased stability of RAD51AP1. Protein interaction mapping analysis identified some key residues within RAD51AP1 required for interacting with the USP1-UAF1 complex. Cells expressing the UAF1 interaction-deficient mutant of RAD51AP1 show increased chromosomal aberrations in response to Mitomycin C treatment. Moreover, similar to the RAD51AP1 depleted cells, the cells expressing UAF1-interaction deficient RAD51AP1 display persistent RAD51 foci following DNA damage exposure, indicating that these factors regulate a later step during the HR repair. These data altogether suggest that the USP1-UAF1 complex promotes HR repair via multiple mechanisms: through FANCD2 deubiquitination, as well as by interacting with RAD51AP1.     

RAD51AP1 mediates RAD51 activity through nucleosome interaction

RAD51-associated protein 1 (RAD51AP1) is a key protein in the homologous recombination (HR) DNA repair pathway. Loss of RAD51AP1 leads to defective HR, genome instability, and telomere erosion. RAD51AP1 physically interacts with the RAD51 recombinase and promotes RAD51-mediated capture of donor DNA, synaptic complex assembly, and displacement-loop formation when tested with nucleosome-free DNA substrates. In cells, however, DNA is packaged into chromatin, posing an additional barrier to the complexities of the HR reaction. In this study, we show that RAD51AP1 binds to nucleosome core particles (NCPs), the minimum basic unit of chromatin in which approximately two superhelical turns of 147 bp double-stranded DNA are wrapped around one histone octamer with no free DNA ends remaining. We identified a C-terminal region in RAD51AP1, including its previously mapped DNA-binding domain, as critical for mediating the association between RAD51AP1 and both the NCP and the histone octamer. Using in vitro surrogate assays of HR activity, we show that RAD51AP1 is capable of promoting duplex DNA capture and initiating joint-molecule formation with the NCP and chromatinized template DNA, respectively. Together, our results suggest that RAD51AP1 directly assists in the RAD51-mediated search for donor DNA in chromatin. We present a model, in which RAD51AP1 anchors the DNA template through affinity for its nucleosomes to the RAD51-ssDNA nucleoprotein filament.     

SUMO Modification Regulates BLM and RAD51 Interaction at Damaged Replication Forks

The gene mutated in Bloom''s syndrome, BLM, is important in the repair of damaged replication forks, and it has both pro- and anti-recombinogenic roles in homologous recombination (HR). At damaged forks, BLM interacts with RAD51 recombinase, the essential enzyme in HR that catalyzes homology-dependent strand invasion. We have previously shown that defects in BLM modification by the small ubiquitin-related modifier (SUMO) cause increased γ-H2AX foci. Because the increased γ-H2AX could result from defective repair of spontaneous DNA damage, we hypothesized that SUMO modification regulates BLM''s function in HR repair at damaged forks. To test this hypothesis, we treated cells that stably expressed a normal BLM (BLM+) or a SUMO-mutant BLM (SM-BLM) with hydroxyurea (HU) and examined the effects of stalled replication forks on RAD51 and its DNA repair functions. HU treatment generated excess γ-H2AX in SM-BLM compared to BLM+ cells, consistent with a defect in replication-fork repair. SM-BLM cells accumulated increased numbers of DNA breaks and were hypersensitive to DNA damage. Importantly, HU treatment failed to induce sister-chromatid exchanges in SM-BLM cells compared to BLM+ cells, indicating a specific defect in HR repair and suggesting that RAD51 function could be compromised. Consistent with this hypothesis, RAD51 localization to HU-induced repair foci was impaired in SM-BLM cells. These data suggested that RAD51 might interact noncovalently with SUMO. We found that in vitro RAD51 interacts noncovalently with SUMO and that it interacts more efficiently with SUMO-modified BLM compared to unmodified BLM. These data suggest that SUMOylation controls the switch between BLM''s pro- and anti-recombinogenic roles in HR. In the absence of BLM SUMOylation, BLM perturbs RAD51 localization at damaged replication forks and inhibits fork repair by HR. Conversely, BLM SUMOylation relieves its inhibitory effects on HR, and it promotes RAD51 function.     

PLK1 depletion alters homologous recombination and synaptonemal complex disassembly events during mammalian spermatogenesis

Homologous recombination (HR) is an essential meiotic process that contributes to the genetic variation of offspring and ensures accurate chromosome segregation. Recombination is facilitated by the formation and repair of programmed DNA double-strand breaks. These DNA breaks are repaired via recombination between maternal and paternal homologous chromosomes and a subset result in the formation of crossovers. HR and crossover formation is facilitated by synapsis of homologous chromosomes by a proteinaceous scaffold structure known as the synaptonemal complex (SC). Recent studies in yeast and worms have indicated that polo-like kinases (PLKs) regulate several events during meiosis, including DNA recombination and SC dynamics. Mammals express four active PLKs (PLK1–4), and our previous work assessing localization and kinase function in mouse spermatocytes suggested that PLK1 coordinates nuclear events during meiotic prophase. Therefore, we conditionally mutated Plk1 in early prophase spermatocytes and assessed stages of HR, crossover formation, and SC processes. Plk1 mutation resulted in increased RPA foci and reduced RAD51/DMC1 foci during zygonema, and an increase of both class I and class II crossover events. Furthermore, the disassembly of SC lateral elements was aberrant. Our results highlight the importance of PLK1 in regulating HR and SC disassembly during spermatogenesis.     

NEK8 regulates DNA damage-induced RAD51 foci formation and replication fork protection

Proteins essential for homologous recombination play a pivotal role in the repair of DNA double strand breaks, DNA inter-strand crosslinks and replication fork stability. Defects in homologous recombination also play a critical role in the development of cancer and the sensitivity of these cancers to chemotherapy. RAD51, an essential factor for homologous recombination and replication fork protection, accumulates and forms immunocytochemically detectable nuclear foci at sites of DNA damage. To identify kinases that may regulate RAD51 localization to sites of DNA damage, we performed a human kinome siRNA library screen, using DNA damage-induced RAD51 foci formation as readout. We found that NEK8, a NIMA family kinase member, is required for efficient DNA damage-induced RAD51 foci formation. Interestingly, knockout of Nek8 in murine embryonic fibroblasts led to cellular sensitivity to the replication inhibitor, hydroxyurea, and inhibition of the ATR kinase. Furthermore, NEK8 was required for proper replication fork protection following replication stall with hydroxyurea. Loading of RAD51 to chromatin was decreased in NEK8-depleted cells and Nek8-knockout cells. Single-molecule DNA fiber analyses revealed that nascent DNA tracts were degraded in the absence of NEK8 following treatment with hydroxyurea. Consistent with this, Nek8-knockout cells showed increased chromosome breaks following treatment with hydroxyurea. Thus, NEK8 plays a critical role in replication fork stability through its regulation of the DNA repair and replication fork protection protein RAD51.     

Beta human papillomavirus 8 E6 allows colocalization of non-homologous end joining and homologous recombination repair factors

Beta human papillomavirus (β-HPV) are hypothesized to make DNA damage more mutagenic and potentially more carcinogenic. Double strand breaks (DSBs) are the most deleterious DNA lesion. They are typically repaired by homologous recombination (HR) or non-homologous end joining (NHEJ). HR occurs after DNA replication while NHEJ can occur at any point in the cell cycle. HR and NHEJ are not thought to occur in the same cell at the same time. HR is restricted to cells in phases of the cell cycle where homologous templates are available, while NHEJ occurs primarily during G1. β-HPV type 8 protein E6 (8E6) attenuates both repair pathways. We use a series of immunofluorescence microscopy and flow cytometry experiments to better define the impact of this attenuation. We found that 8E6 causes colocalization of HR factors (RPA70 and RAD51) with an NHEJ factor (activated DNA-PKcs or pDNA-PKcs) at persistent DSBs. 8E6 also causes RAD51 foci to form during G1. The initiation of NHEJ and HR at the same lesion could lead to antagonistic DNA end processing. Further, HR cannot be readily completed in an error-free manner during G1. Both aberrant repair events would cause deletions. To determine if these mutations were occurring, we used next generation sequencing of the 200kb surrounding a CAS9-induced DSB. 8E6 caused a 21-fold increase in deletions. Chemical and genetic inhibition of p300 as well as an 8E6 mutant that is incapable of destabilizing p300 demonstrates that 8E6 is acting via p300 destabilization. More specific chemical inhibitors of DNA repair provided mechanistic insight by mimicking 8E6-induced dysregulation of DNA repair in a virus-free system. Specifically, inhibition of NHEJ causes RAD51 foci to form in G1 and colocalization of RAD51 with pDNA-PKcs.     

ATP-dependent nucleosome unwrapping catalyzed by human RAD51

Double-strand breaks (DSB) occur in chromatin following replication fork collapse and chemical or physical damage [Symington and Gautier (Double-strand break end resection and repair pathway choice. Annu. Rev. Genet. 2011;45:247–271.)] and may be repaired by homologous recombination (HR) and non-homologous end-joining. Nucleosomes are the fundamental units of chromatin and must be remodeled during DSB repair by HR [Andrews and Luger (Nucleosome structure(s) and stability: variations on a theme. Annu. Rev. Biophys. 2011;40:99–117.)]. Physical initiation of HR requires RAD51, which forms a nucleoprotein filament (NPF) that catalyzes homologous pairing and strand exchange (recombinase) between DNAs that ultimately bridges the DSB gap [San Filippo, Sung and Klein. (Mechanism of eukaryotic HR. Annu. Rev. Biochem. 2008;77:229–257.)]. RAD51 forms an NPF on single-stranded DNA and double-stranded DNA (dsDNA). Although the single-stranded DNA NPF is essential for recombinase initiation, the role of the dsDNA NPF is less clear. Here, we demonstrate that the human RAD51 (HsRAD51) dsDNA NPF disassembles nucleosomes by unwrapping the DNA from the core histones. HsRAD51 that has been constitutively or biochemically activated for recombinase functions displays significantly reduced nucleosome disassembly activity. These results suggest that HsRAD51 can perform ATP hydrolysis-dependent nucleosome disassembly in addition to its recombinase functions.     

Involvement of ATM in homologous recombination after end resection and RAD51 nucleofilament formation

Ataxia-telangiectasia mutated (ATM) is needed for the initiation of the double-strand break (DSB) repair by homologous recombination (HR). ATM triggers DSB end resection by stimulating the nucleolytic activity of CtIP and MRE11 to generate 3′-ssDNA overhangs, followed by RPA loading and RAD51 nucleofilament formation. Here we show for the first time that ATM is also needed for later steps in HR after RAD51 nucleofilament formation. Inhibition of ATM after completion of end resection did not affect RAD51 nucleofilament formation, but resulted in HR deficiency as evidenced by (i) an increase in the number of residual RAD51/γH2AX foci in both S and G2 cells, (ii) the decrease in HR efficiency as detected by HR repair substrate (pGC), (iii) a reduced SCE rate and (iv) the radiosensitization of cells by PARP inhibition. This newly described role for ATM was found to be dispensable in heterochromatin-associated DSB repair, as KAP1-depletion did not alleviate the HR-deficiency when ATM was inhibited after end resection. Moreover, we demonstrated that ATR can partly compensate for the deficiency in early, but not in later, steps of HR upon ATM inhibition. Taken together, we describe here for the first time that ATM is needed not only for the initiation but also for the completion of HR.     

GEMIN2 promotes accumulation of RAD51 at double-strand breaks in homologous recombination

RAD51 is a key factor in homologous recombination (HR) and plays an essential role in cellular proliferation by repairing DNA damage during replication. The assembly of RAD51 at DNA damage is strictly controlled by RAD51 mediators, including BRCA1 and BRCA2. We found that human RAD51 directly binds GEMIN2/SIP1, a protein involved in spliceosome biogenesis. Biochemical analyses indicated that GEMIN2 enhances the RAD51–DNA complex formation by inhibiting RAD51 dissociation from DNA, and thereby stimulates RAD51-mediated homologous pairing. GEMIN2 also enhanced the RAD51-mediated strand exchange, when RPA was pre-bound to ssDNA before the addition of RAD51. To analyze the function of GEMIN2, we depleted GEMIN2 in the chicken DT40 line and in human cells. The loss of GEMIN2 reduced HR efficiency and resulted in a significant decrease in the number of RAD51 subnuclear foci, as observed in cells deficient in BRCA1 and BRCA2. These observations and our biochemical analyses reveal that GEMIN2 regulates HR as a novel RAD51 mediator.     

Rad10 exhibits lesion-dependent genetic requirements for recruitment to DNA double-strand breaks in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, the Rad1–Rad10 protein complex participates in nucleotide excision repair (NER) and homologous recombination (HR). During HR, the Rad1–Rad10 endonuclease cleaves 3′ branches of DNA and aberrant 3′ DNA ends that are refractory to other 3′ processing enzymes. Here we show that yeast strains expressing fluorescently labeled Rad10 protein (Rad10-YFP) form foci in response to double-strand breaks (DSBs) induced by a site-specific restriction enzyme, I-SceI or by ionizing radiation (IR). Additionally, for endonuclease-induced DSBs, Rad10-YFP localization to DSB sites depends on both RAD51 and RAD52, but not MRE11 while IR-induced breaks do not require RAD51. Finally, Rad10-YFP colocalizes with Rad51-CFP and with Rad52-CFP at DSB sites, indicating a temporal overlap of Rad52, Rad51 and Rad10 functions at DSBs. These observations are consistent with a putative role of Rad10 protein in excising overhanging DNA ends after homology searching and refine the potential role(s) of the Rad1–Rad10 complex in DSB repair in yeast.     

RAD51 foci formation in response to DNA damage is modulated by TIP49

Chromatin modification plays an important role in modulating the access of homologous recombination proteins to the sites of DNA damage. TIP49 is highly conserved component of chromatin modification/remodeling complexes, but its involvement in homologous recombination repair in mammalian cells has not been examined in details. In the present communication we studied the role of TIP49 in recruitment of the key homologous recombination protein RAD51 to sites of DNA damage. RAD51 redistribution to chromatin and nuclear foci formation induced by double-strand breaks and interstrand crosslinks were followed under conditions of TIP49 depletion by RNA interference. TIP49 silencing reduced RAD51 recruitment to chromatin and nuclear foci formation to about 50% of that of the control. Silencing of TIP48, which is closely related to TIP49, induced a similar reduction in RAD51 foci formation. RAD51 foci reduction in TIP49-silenced cells was not a result of defective DNA damage checkpoint signaling as judged by the normal histone H2AX phosphorylation and cell cycle distribution. Treatment with the histone deacetylase inhibitor sodium butyrate restored RAD51 foci formation in the TIP49-depleted cells. The results suggest that as a constituent of chromatin modification complexes TIP49 may facilitate the access of the repair machinery to the sites of DNA damage.     

RPA mediates recombination repair during replication stress and is displaced from DNA by checkpoint signalling in human cells

The replication protein A (RPA) is involved in most, if not all, nuclear metabolism involving single-stranded DNA. Here, we show that RPA is involved in genome maintenance at stalled replication forks by the homologous recombination repair system in humans. Depletion of the RPA protein inhibited the formation of RAD51 nuclear foci after hydroxyurea-induced replication stalling leading to persistent unrepaired DNA double-strand breaks (DSBs). We demonstrate a direct role of RPA in homology directed recombination repair. We find that RPA is dispensable for checkpoint kinase 1 (Chk1) activation and that RPA directly binds RAD52 upon replication stress, suggesting a direct role in recombination repair. In addition we show that inhibition of Chk1 with UCN-01 decreases dissociation of RPA from the chromatin and inhibits association of RAD51 and RAD52 with DNA. Altogether, our data suggest a direct role of RPA in homologous recombination in assembly of the RAD51 and RAD52 proteins. Furthermore, our data suggest that replacement of RPA with the RAD51 and RAD52 proteins is affected by checkpoint signalling.     

BRIT1/MCPH1 Is Essential for Mitotic and Meiotic Recombination DNA Repair and Maintaining Genomic Stability in Mice

BRIT1 protein (also known as MCPH1) contains 3 BRCT domains which are conserved in BRCA1, BRCA2, and other important molecules involved in DNA damage signaling, DNA repair, and tumor suppression. BRIT1 mutations or aberrant expression are found in primary microcephaly patients as well as in cancer patients. Recent in vitro studies suggest that BRIT1/MCPH1 functions as a novel key regulator in the DNA damage response pathways. To investigate its physiological role and dissect the underlying mechanisms, we generated BRIT1 ^−/− mice and identified its essential roles in mitotic and meiotic recombination DNA repair and in maintaining genomic stability. Both BRIT1 ^−/− mice and mouse embryonic fibroblasts (MEFs) were hypersensitive to γ-irradiation. BRIT1 ^−/− MEFs and T lymphocytes exhibited severe chromatid breaks and reduced RAD51 foci formation after irradiation. Notably, BRIT1 ^−/− mice were infertile and meiotic homologous recombination was impaired. BRIT1-deficient spermatocytes exhibited a failure of chromosomal synapsis, and meiosis was arrested at late zygotene of prophase I accompanied by apoptosis. In mutant spermatocytes, DNA double-strand breaks (DSBs) were formed, but localization of RAD51 or BRCA2 to meiotic chromosomes was severely impaired. In addition, we found that BRIT1 could bind to RAD51/BRCA2 complexes and that, in the absence of BRIT1, recruitment of RAD51 and BRCA2 to chromatin was reduced while their protein levels were not altered, indicating that BRIT1 is involved in mediating recruitment of RAD51/BRCA2 to the damage site. Collectively, our BRIT1-null mouse model demonstrates that BRIT1 is essential for maintaining genomic stability in vivo to protect the hosts from both programmed and irradiation-induced DNA damages, and its depletion causes a failure in both mitotic and meiotic recombination DNA repair via impairing RAD51/BRCA2''s function and as a result leads to infertility and genomic instability in mice.     

SQSTM1/p62 mediates crosstalk between autophagy and the UPS in DNA repair

SQSTM1/p62 (sequestosome 1) selectively targets polyubiquitinated proteins for degradation via macroautophagy and the proteasome. Additionally, SQSTM1 shuttles between the cytoplasmic and nuclear compartments, although its role in the nucleus is relatively unknown. Here, we report that SQSTM1 dynamically associates with DNA damage foci (DDF) and regulates DNA repair. Upon induction of DNA damage SQSTM1 interacts with FLNA (filamin A), which has previously been shown to recruit DNA repair protein RAD51 (RAD51 recombinase) to double-strand breaks and facilitate homologous recombination (HR). SQSTM1 promotes proteasomal degradation of FLNA and RAD51 within the nucleus, resulting in reduced levels of nuclear RAD51 and slower DNA repair. SQSTM1 regulates the ratio between HR and nonhomologous end joining (NHEJ) by promoting the latter at the expense of the former. This SQSTM1-dependent mechanism mediates the effect of macroautophagy on DNA repair. Moreover, nuclear localization of SQSTM1 and its association with DDF increase with aging and are prevented by life-span-extending dietary restriction, suggesting that an imbalance in the mechanism identified here may contribute to aging and age-related diseases.     

RAD51 localization and activation following DNA damage

The efficient repair of double-strand breaks in DNA is critical for the maintenance of genome stability. In response to ionizing radiation and other DNA-damaging agents, the RAD51 protein, which is essential for homologous recombination, relocalizes within the nucleus to form distinct foci that can be visualized by microscopy and are thought to represent sites where repair reactions take place. The formation of RAD51 foci in response to DNA damage is dependent upon BRCA2 and a series of proteins known as the RAD51 paralogues (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3), indicating that the components present within foci assemble in a carefully orchestrated and ordered manner. By contrast, RAD51 foci that form spontaneously as cells undergo DNA replication at S phase occur without the need for BRCA2 or the RAD51 paralogues. It is known that BRCA2 interacts directly with RAD51 through a series of degenerative motifs known as the BRC repeats. These interactions modulate the ability of RAD51 to bind DNA. Taken together, these observations indicate that BRCA2 plays a critical role in controlling the actions of RAD51 at both the microscopic (focus formation) and molecular (DNA binding) level.