Bloom's syndrome protein is required for correct relocalization of RAD50/MRE11/NBS1 complex after replication fork arrest

Bloom's syndrome (BS) is a rare genetic disorder characterized by a broad range of symptoms and, most importantly, a predisposition to many types of cancers. Cells derived from patients with BS exhibit an elevated rate of somatic recombination and hypermutability, supporting a role for bleomycin (BLM) in the maintenance of genomic integrity. BLM is thought to participate in several DNA transactions, the failure of which could give raise to genomic instability, and to interact with many proteins involved in replication, recombination, and repair. In this study, we show that BLM function is specifically required to properly relocalize the RAD50/MRE11/NBS1 (RMN) complex at sites of replication arrest, but is not essential in the activation of BRCA1 either after stalled replication forks or gamma-rays. We also provide evidence that BLM is phosphorylated after replication arrest in an Ataxia and RAD3-related protein (ATR)-dependent manner and that phosphorylation is not required for subnuclear relocalization. Therefore, in ATR dominant negative mutant cells, the assembly of the RMN complex in nuclear foci after replication blockage is almost completely abolished. Together, these results suggest a relationship between BLM, ATR, and the RMN complex in the response to replication arrest, proposing a role for BLM protein and RMN complex in the resolution of stalled replication forks.     

ATR and ATM-Dependent Movement of BLM Helicase during Replication Stress Ensures Optimal ATM Activation and 53BP1 Focus Formation

The BLM helicase, a deficiency in which markedly increases cancer incidence in humans, isrequired for optimal repair during DNA replication. We show that BLM rapidly moves fromPML nuclear bodies to damaged replication forks, returning to PML bodies several hours later,owing to activities of the DNA damage response kinases ATR and ATM, respectively.Immunofluorescence and cellular fractionation demonstrate that BLM partitions to different subcellularcompartments after replication stress. Unexpectedly, fibroblasts lacking BLM weredeficient in phospho-ATM (S-1981) and 53-binding protein-1 (53BP1), and these proteins failedto form foci following replication stress. Expression of a dominant p53 mutant or helicasedeficientBLM restored replication stress-induced 53BP1 foci, but only mutant p53 restoredoptimal ATM activation. Thus, optimal repair of damaged replication fork lesions likelyrequires both ATR and ATM, BLM recruits 53BP1 to these lesions independent of its helicaseactivity, and optimal activation of ATM requires both p53 and BLM helicase activities.

Supplemental material for this paper can be found at the following link:

http://www.landesbioscience.com/journals/cc/davalosCC3-12-sup.pdf     

BLM helicase-dependent transport of p53 to sites of stalled DNA replication forks modulates homologous recombination

Diverse functions, including DNA replication, recombination and repair, occur during S phase of the eukaryotic cell cycle. It has been proposed that p53 and BLM help regulate these functions. We show that p53 and BLM accumulated after hydroxyurea (HU) treatment, and physically associated and co-localized with each other and with RAD51 at sites of stalled DNA replication forks. HU-induced relocalization of BLM to RAD51 foci was p53 independent. However, BLM was required for efficient localization of either wild-type or mutated (Ser15Ala) p53 to these foci and for physical association of p53 with RAD51. Loss of BLM and p53 function synergistically enhanced homologous recombination frequency, indicating that they mediated the process by complementary pathways. Loss of p53 further enhanced the rate of spontaneous sister chromatid exchange (SCE) in Bloom syndrome (BS) cells, but not in their BLM-corrected counterpart, indicating that involvement of p53 in regulating spontaneous SCE is BLM dependent. These results indicate that p53 and BLM functionally interact during resolution of stalled DNA replication forks and provide insight into the mechanism of genomic fidelity maintenance by these nuclear proteins.     

BLM is an early responder to DNA double-strand breaks

Bloom syndrome (BS) is an autosomal recessive disorder characterized by a marked predisposition to cancer and elevated genomic instability. The defective protein in BS, BLM, is a member of the RecQ helicase family and is believed to function in various DNA transactions, including in replication, repair, and recombination. Here, we show that both endogenous and overexpressed human BLM accumulates at sites of laser light-induced DNA double-strand breaks within 10s and colocalizes with gammaH2AX and ATM. Like its RecQ helicase family member, WRN, the defective protein in Werner syndrome, dissection of the BLM protein revealed that its HRDC domain is sufficient for its recruitment to the damaged sites. In addition, we confirmed that the C-terminal region spanning amino acids 1250-1292 within the HRDC domain is necessary for BLM recruitment. To identify additional proteins required for the recruitment of BLM, we examined the recruitment of BLM in various mutants generated from chicken DT40 cells and found that the early accumulation of BLM was not dependent on the presence of ATM, RAD17, DNA-PKcs, NBS1, XRCC3, RAD52, RAD54, or WRN. Thus, HRDC domain in DNA helicases is a common early responder to DNA double-strand breaks, enabling BLM and WRN to be involved in DNA repair.     

53BP1 ablation rescues genomic instability in mice expressing ‘RING‐less’ BRCA1

BRCA1 mutations strongly predispose affected individuals to breast and ovarian cancer, but the mechanism by which BRCA1 acts as a tumor suppressor is not fully understood. Homozygous deletion of exon 2 of the mouse Brca1 gene normally causes embryonic lethality, but we show that exon 2‐deleted alleles of Brca1 are expressed as a mutant isoform that lacks the N‐terminal RING domain. This “RING‐less” BRCA1 protein is stable and efficiently recruited to the sites of DNA damage. Surprisingly, robust RAD51 foci form in cells expressing RING‐less BRCA1 in response to DNA damage, but the cells nonetheless display the substantial genomic instability. Genomic instability can be rescued by the deletion of Trp53bp1, which encodes the DNA damage response factor 53BP1, and mice expressing RING‐less BRCA1 do not show an increased susceptibility to tumors in the absence of 53BP1. Genomic instability in cells expressing RING‐less BRCA1 correlates with the loss of BARD1 and a defect in restart of replication forks after hydroxyurea treatment, suggesting a role of BRCA1–BARD1 in genomic integrity that is independent of RAD51 loading.     

Functional interaction between BLM helicase and 53BP1 in a Chk1-mediated pathway during S-phase arrest

Bloom's syndrome is a rare autosomal recessive genetic disorder characterized by chromosomal aberrations, genetic instability, and cancer predisposition, all of which may be the result of abnormal signal transduction during DNA damage recognition. Here, we show that BLM is an intermediate responder to stalled DNA replication forks. BLM colocalized and physically interacted with the DNA damage response proteins 53BP1 and H2AX. Although BLM facilitated physical interaction between p53 and 53BP1, 53BP1 was required for efficient accumulation of both BLM and p53 at the sites of stalled replication. The accumulation of BLM/53BP1 foci and the physical interaction between them was independent of gamma-H2AX. The active Chk1 kinase was essential for both the accurate focal colocalization of 53BP1 with BLM and the consequent stabilization of BLM. Once the ATR/Chk1- and 53BP1-mediated signal from replicational stress is received, BLM functions in multiple downstream repair processes, thereby fulfilling its role as a caretaker tumor suppressor.     

Functional interaction of p53 and BLM DNA helicase in apoptosis

The Bloom syndrome (BS) protein, BLM, is a member of the RecQ DNA helicase family that also includes the Werner syndrome protein, WRN. Inherited mutations in these proteins are associated with cancer predisposition of these patients. We recently discovered that cells from Werner syndrome patients displayed a deficiency in p53-mediated apoptosis and WRN binds to p53. Here, we report that analogous to WRN, BLM also binds to p53 in vivo and in vitro, and the C-terminal domain of p53 is responsible for the interaction. p53-mediated apoptosis is defective in BS fibroblasts and can be rescued by expression of the normal BLM gene. Moreover, lymphoblastoid cell lines (LCLs) derived from BS donors are resistant to both gamma-radiation and doxorubicin-induced cell killing, and sensitivity can be restored by the stable expression of normal BLM. In contrast, BS cells have a normal Fas-mediated apoptosis, and in response to DNA damage normal accumulation of p53, normal induction of p53 responsive genes, and normal G(1)-S and G(2)-M cell cycle arrest. BLM localizes to nuclear foci referred to as PML nuclear bodies (NBs). Cells from Li-Fraumeni syndrome patients carrying p53 germline mutations and LCLs lacking a functional p53 have a decreased accumulation of BLM in NBs, whereas isogenic lines with functional p53 exhibit normal accumulation. Certain BLM mutants (C1055S or Delta133-237) that have a reduced ability to localize to the NBs when expressed in normal cells can impair the localization of wild type BLM to NBs and block p53-mediated apoptosis, suggesting a dominant-negative effect. Taken together, our results indicate both a novel mechanism of p53 function by which p53 mediates nuclear trafficking of BLM to NBs and the cooperation of p53 and BLM to induce apoptosis.     

Regulation and localization of the Bloom syndrome protein in response to DNA damage

Bloom syndrome (BS) is an autosomal recessive disorder characterized by a high incidence of cancer and genomic instability. BLM, the protein defective in BS, is a RecQ-like helicase, presumed to function in DNA replication, recombination, or repair. BLM localizes to promyelocytic leukemia protein (PML) nuclear bodies and is expressed during late S and G2. We show, in normal human cells, that the recombination/repair proteins hRAD51 and replication protein (RP)-A assembled with BLM into a fraction of PML bodies during late S/G2. Biochemical experiments suggested that BLM resides in a nuclear matrix-bound complex in which association with hRAD51 may be direct. DNA-damaging agents that cause double strand breaks and a G2 delay induced BLM by a p53- and ataxia-telangiectasia mutated independent mechanism. This induction depended on the G2 delay, because it failed to occur when G2 was prevented or bypassed. It coincided with the appearance of foci containing BLM, PML, hRAD51 and RP-A, which resembled ionizing radiation-induced foci. After radiation, foci containing BLM and PML formed at sites of single-stranded DNA and presumptive repair in normal cells, but not in cells with defective PML. Our findings suggest that BLM is part of a dynamic nuclear matrix-based complex that requires PML and functions during G2 in undamaged cells and recombinational repair after DNA damage.     

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.     

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

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

hSSB1 associates with and promotes stability of the BLM helicase

Background

Maintenance of genome stability is critical in human cells. Mutations in or loss of genome stability pathways can lead to a number of pathologies including cancer. hSSB1 is a critical DNA repair protein functioning in the repair and signalling of stalled DNA replication forks, double strand DNA breaks and oxidised DNA lesions. The BLM helicase is central to the repair of both collapsed DNA replication forks and double strand DNA breaks by homologous recombination.

Results

In this study, we demonstrate that hSSB1 and BLM helicase form a complex in cells and the interaction is altered in response to ionising radiation (IR). BLM and hSSB1 also co-localised at nuclear foci following IR-induced double strand breaks and stalled replication forks. We show that hSSB1 depleted cells contain less BLM protein and that this deficiency is due to proteasome mediated degradation of BLM. Consequently, there is a defect in recruitment of BLM to chromatin in response to ionising radiation-induced DSBs and to hydroxyurea-induced stalled and collapsed replication forks.

Conclusions

Our data highlights that BLM helicase and hSSB1 function in a dynamic complex in cells and that this complex is likely required for BLM protein stability and function.

     

Physical and functional interaction between the Bloom's syndrome gene product and the largest subunit of chromatin assembly factor 1

Bloom's syndrome (BS) is a genomic instability disorder characterized by cancer susceptibility. The protein defective in BS, BLM, belongs to the RecQ family of DNA helicases. In this study, we found that BLM interacts with hp150, the largest subunit of chromatin assembly factor 1 (CAF-1), in vitro and in vivo. Colocalization of a proportion of the cellular complement of these two proteins is found at specific nuclear foci coinciding with sites of DNA synthesis in the S phase. This colocalization increases in the presence of agents that damage DNA or inhibit DNA replication. In support of a functional interaction between BLM and CAF-1, we show that BLM inhibits CAF-1-mediated chromatin assembly during DNA repair in vitro. Although CAF-1 activity is not altered in BLM-deficient cells, the absence of BLM does impair the ability of CAF-1 to be mobilized within the nucleus in response to hydroxyurea treatment. Our results provide the first link between BLM and chromatin assembly coupled to DNA repair and suggest that BLM and CAF-1 function in a coordinated way to promote survival in response to DNA damage and/or replication blockade.     

The yeast Sgs1 helicase is differentially required for genomic and ribosomal DNA replication

The members of the RecQ family of DNA helicases play conserved roles in the preservation of genome integrity. RecQ helicases are implicated in Bloom and Werner syndromes, which are associated with genomic instability and predisposition to cancers. The human BLM and WRN helicases are required for normal S phase progression. In contrast, Saccharomyces cerevisiae cells deleted for SGS1 grow with wild-type kinetics. To investigate the role of Sgs1p in DNA replication, we have monitored S phase progression in sgs1Delta cells. Unexpectedly, we find that these cells progress faster through S phase than their wild-type counterparts. Using bromodeoxyuridine incorporation and DNA combing, we show that replication forks are moving more rapidly in the absence of the Sgs1 helicase. However, completion of DNA replication is strongly retarded at the rDNA array of sgs1Delta cells, presumably because of their inability to prevent recombination at stalled forks, which are very abundant at this locus. These data suggest that Sgs1p is not required for processive DNA synthesis but prevents genomic instability by coordinating replication and recombination events during S phase.     

Endogenous gamma-H2AX-ATM-Chk2 checkpoint activation in Bloom's syndrome helicase deficient cells is related to DNA replication arrested forks

The Bloom syndrome helicase (BLM) is critical for genomic stability. A defect in BLM activity results in the cancer-predisposing Bloom syndrome (BS). Here, we report that BLM-deficient cell lines and primary fibroblasts display an endogenously activated DNA double-strand break checkpoint response with prominent levels of phosphorylated histone H2AX (gamma-H2AX), Chk2 (p(T68)Chk2), and ATM (p(S1981)ATM) colocalizing in nuclear foci. Interestingly, the mitotic fraction of gamma-H2AX foci did not seem to be higher in BLM-deficient cells, indicating that these lesions form transiently during interphase. Pulse labeling with iododeoxyuridine and immunofluorescence microscopy showed the colocalization of gamma-H2AX, ATM, and Chk2 together with replication foci. Those foci costained for Rad51, indicating homologous recombination at these replication sites. We therefore analyzed replication in BS cells using a single molecule approach on combed DNA fibers. In addition to a higher frequency of replication fork barriers, BS cells displayed a reduced average fork velocity and global reduction of interorigin distances indicative of an elevated frequency of origin firing. Because BS is one of the most penetrant cancer-predisposing hereditary diseases, it is likely that the lack of BLM engages the cells in a situation similar to precancerous tissues with replication stress. To our knowledge, this is the first report of high ATM-Chk2 kinase activation and its linkage to replication defects in a BS model.     

Polymerase eta and p53 jointly regulate cell survival,apoptosis and Mre11 recombination during S phase checkpoint arrest after UV irradiation

Xeroderma pigmentosum variant (XPV) cells lack the damage-specific polymerase eta and undergo a protracted arrest at the S phase checkpoint(s) following UV damage. The S phase checkpoints encompass several qualitatively different processes, and stimulate downstream events that are dependent on the functional state of p53. Primary fibroblasts with wild-type p53 arrest in S, and require a functional polymerase eta (pol eta) to carry out bypass replication, but do not recruit recombination factors for recovery. XPV cells with non-functional p53, as a result of transformation by SV40 or HPV16 (E6/E7), recruit the hMre11/hRad50/Nbs1 complex to arrested replication forks, coincident with PCNA, whereas normal transformed cells preferentially use the pol eta bypass replication pathway. The formation of hMre11 foci implies that arrested replication forks rapidly undergo a collapse involving double strand breakage and rejoining. Apoptosis occurs after UV only in cells transformed by SV40, and not in normal or XPV fibroblasts or HPV16 (E6/E7) transformed cells. Conversely, ultimate cell survival in XPV cells was much less in HPV16 (E6/E7) transformed cells than in SV40 transformed cells, indicating that apoptosis was not a reliable predictor of cell survival. Inhibition of p53 transactivation by pifithrin-alpha or inhibition of protein synthesis by cycloheximide did not induce hMre11 foci or apoptosis in UV damaged fibroblasts. Inhibition of kinase activity with wortmannin did not increase killing by UV, unlike the large increase seen with caffeine. Since HPV16 (E6/E7) transformed XPV cells were highly UV sensitive and not further sensitized by caffeine, it appears likely that caffeine sensitization proceeds through a p53 pathway. The S phase checkpoints are therefore, a complex set of different checkpoints that are coordinated by p53 with the capacity to differentially modulate cell survival, apoptosis, bypass replication and hMre11 recombination.     

Selective cleavage of BLM, the bloom syndrome protein, during apoptotic cell death

Bloom syndrome (BS) is an autosomal recessive disorder characterized by a high incidence of cancer and genomic instability. BLM, the protein defective in BS, is a RECQ-like helicase that is presumed to function in mammalian DNA replication, recombination, or repair. We show here that BLM, but not the related RECQ-like helicase WRN, is rapidly cleaved in cells undergoing apoptosis. BLM was cleaved to 47- and 110-kDa major fragments, with kinetics similar to the apoptotic cleavage of poly(A)DP-ribose polymerase. BLM cleavage was prevented by a caspase 3 inhibitor and did not occur in caspase 3-deficient cells. Moreover, recombinant BLM was cleaved to 47- and 110-kDa fragments by caspase 3, but not caspase 6, in vitro. The caspase 3 recognition sequence (412)TEVD(415) was verified by mutating aspartate 415 to glycine and showing that this mutation rendered BLM resistant to caspase 3 cleavage. Cleavage did not abolish the BLM helicase activity but abolished BLM nuclear foci and the association of BLM with condensed DNA and the insoluble matrix. The results suggest that BLM, but not WRN, is an early selected target during the execution of apoptosis.     

Rif1 provides a new DNA‐binding interface for the Bloom syndrome complex to maintain normal replication

BLM, the helicase defective in Bloom syndrome, is part of a multiprotein complex that protects genome stability. Here, we show that Rif1 is a novel component of the BLM complex and works with BLM to promote recovery of stalled replication forks. First, Rif1 physically interacts with the BLM complex through a conserved C‐terminal domain, and the stability of Rif1 depends on the presence of the BLM complex. Second, Rif1 and BLM are recruited with similar kinetics to stalled replication forks, and the Rif1 recruitment is delayed in BLM‐deficient cells. Third, genetic analyses in vertebrate DT40 cells suggest that BLM and Rif1 work in a common pathway to resist replication stress and promote recovery of stalled forks. Importantly, vertebrate Rif1 contains a DNA‐binding domain that resembles the αCTD domain of bacterial RNA polymerase α; and this domain preferentially binds fork and Holliday junction (HJ) DNA in vitro and is required for Rif1 to resist replication stress in vivo. Our data suggest that Rif1 provides a new DNA‐binding interface for the BLM complex to restart stalled replication forks.     

Fanconi anemia and Bloom's syndrome crosstalk through FANCJ-BLM helicase interaction

Fanconi anemia (FA) and Bloom's syndrome (BS) are rare hereditary chromosomal instability disorders. FA displays bone marrow failure, acute myeloid leukemia, and head and neck cancers, whereas BS is characterized by growth retardation, immunodeficiency, and a wide spectrum of cancers. The BLM gene mutated in BS encodes a DNA helicase that functions in a protein complex to suppress sister-chromatid exchange. Of the 15 FA genetic complementation groups implicated in interstrand crosslink repair, FANCJ encodes a DNA helicase involved in recombinational repair and replication stress response. Based on evidence that BLM and FANCJ interact we suggest that crosstalk between BLM and FA pathways is more complex than previously thought. We propose testable models for how FANCJ and BLM coordinate to help cells deal with stalled replication forks or double-strand breaks (DSB). Understanding how BLM and FANCJ cooperate will help to elucidate an important pathway for maintaining genomic stability.     

RING finger and WD repeat domain 3 (RFWD3) associates with replication protein A (RPA) and facilitates RPA-mediated DNA damage response

DNA damage response is crucial for maintaining genomic integrity and preventing cancer by coordinating the activation of checkpoints and the repair of damaged DNA. Central to DNA damage response are the two checkpoint kinases ATM and ATR that phosphorylate a wide range of substrates. RING finger and WD repeat domain 3 (RFWD3) was initially identified as a substrate of ATM/ATR from a proteomic screen. Subsequent studies showed that RFWD3 is an E3 ubiquitin ligase that ubiquitinates p53 in vitro and positively regulates p53 levels in response to DNA damage. We report here that RFWD3 associates with replication protein A (RPA), a single-stranded DNA-binding protein that plays essential roles in DNA replication, recombination, and repair. Binding of RPA to single-stranded DNA (ssDNA), which is generated by DNA damage and repair, is essential for the recruitment of DNA repair factors to damaged sites and the activation of checkpoint signaling. We show that RFWD3 is physically associated with RPA and rapidly localizes to sites of DNA damage in a RPA-dependent manner. In vitro experiments suggest that the C terminus of RFWD3, which encompass the coiled-coil domain and the WD40 domain, is necessary for binding to RPA. Furthermore, DNA damage-induced phosphorylation of RPA and RFWD3 is dependent upon each other. Consequently, loss of RFWD3 results in the persistent foci of DNA damage marker γH2AX and the repair protein Rad51 in damaged cells. These findings suggest that RFWD3 is recruited to sites of DNA damage and facilitates RPA-mediated DNA damage signaling and repair.     

RAD18 promotes DNA double-strand break repair during G1 phase through chromatin retention of 53BP1

Recruitment of RAD18 to stalled replication forks facilitates monoubiquitination of PCNA during S-phase, promoting translesion synthesis at sites of UV irradiation-induced DNA damage. In this study, we show that RAD18 is also recruited to ionizing radiation (IR)-induced sites of DNA double-strand breaks (DSBs) forming foci which are co-localized with 53BP1, NBS1, phosphorylated ATM, BRCA1 and γ-H2AX. RAD18 associates with 53BP1 and is recruited to DSB sites in a 53BP1-dependent manner specifically during G1-phase, RAD18 monoubiquitinates KBD domain of 53BP1 at lysine 1268 in vitro. A monoubiquitination-resistant 53BP1 mutant harboring a substitution at lysine 1268 is not retained efficiently at the chromatin in the vicinity of DSBs. In Rad18-null cells, retention of 53BP1 foci, efficiency of DSB repair and post-irradiation viability are impaired compared with wild-type cells. Taken together, these results suggest that RAD18 promotes 53BP1-directed DSB repair by enhancing retention of 53BP1, possibly through an interaction between RAD18 and 53BP1 and the modification of 53BP1.