Regulation of 53BP1 Protein Stability by RNF8 and RNF168 Is Important for Efficient DNA Double-Strand Break Repair

53BP1 regulates DNA double-strand break (DSB) repair. In functional assays for specific DSB repair pathways, we found that 53BP1 was important in the conservative non-homologous end-joining (C-NHEJ) pathway, and this activity was dependent upon RNF8 and RNF168. We observed that 53BP1 protein was diffusely abundant in nuclei, and upon ionizing radiation, 53BP1 was everywhere degraded except at DNA damage sites. Depletion of RNF8 or RNF168 blocked the degradation of the diffusely localized nuclear 53BP1, and ionizing radiation induced foci (IRIF) did not form. Furthermore, when 53BP1 degradation was inhibited, a subset of 53BP1 was bound to DNA damage sites but bulk, unbound 53BP1 remained in the nucleoplasm, and localization of its downstream effector RIF1 at DSBs was abolished. Our data suggest a novel mechanism for responding to DSB that upon ionizing radiation, 53BP1 was divided into two populations, ensuring functional DSB repair: damage site-bound 53BP1 whose binding signal is known to be generated by RNF8 and RNF168; and unbound bulk 53BP1 whose ensuing degradation is regulated by RNF8 and RNF168.     

Ring Finger Nuclear Factor RNF168 Is Important for Defects in Homologous Recombination Caused by Loss of the Breast Cancer Susceptibility Factor BRCA1

The RING finger nuclear factor RNF168 is required for recruitment of several DNA damage response factors to double strand breaks (DSBs), including 53BP1 and BRCA1. Because 53BP1 and BRCA1 function antagonistically during the DSB repair pathway homologous recombination (HR), the influence of RNF168 on HR has been unclear. We report that RNF168 depletion causes an elevated frequency of two distinct HR pathways (homology-directed repair and single strand annealing), suppresses defects in HR caused by BRCA1 silencing, but does not suppress HR defects caused by disruption of CtIP, RAD50, BRCA2, or RAD51. Furthermore, RNF168-depleted cells can form ionizing radiation-induced foci of the recombinase RAD51 without forming BRCA1 ionizing radiation-induced foci, indicating that this loss of BRCA1 recruitment to DSBs does not reflect a loss of function during HR. Additionally, we find that RNF168 and 53BP1 have a similar influence on HR. We suggest that RNF168 is important for HR defects caused by BRCA1 loss.     

RNF8- and RNF168-dependent degradation of KDM4A/JMJD2A triggers 53BP1 recruitment to DNA damage sites

In response to DNA damage, cells initiate complex signalling cascades leading to growth arrest and DNA repair. The recruitment of 53BP1 to damaged sites requires the activation of the ubiquitination cascade controlled by the E3 ubiquitin ligases RNF8 and RNF168, and methylation of histone H4 on lysine 20. However, molecular events that regulate the accessibility of methylated histones, to allow the recruitment of 53BP1 to DNA breaks, are unclear. Here, we show that like 53BP1, the JMJD2A (also known as KDM4A) tandem tudor domain binds dimethylated histone H4K20; however, JMJD2A is degraded by the proteasome following the DNA damage in an RNF8-dependent manner. We demonstrate that JMJD2A is ubiquitinated by RNF8 and RNF168. Moreover, ectopic expression of JMJD2A abrogates 53BP1 recruitment to DNA damage sites, indicating a role in antagonizing 53BP1 for methylated histone marks. The combined knockdown of JMJD2A and JMJD2B significantly rescued the ability of RNF8- and RNF168-deficient cells to form 53BP1 foci. We propose that the RNF8-dependent degradation of JMJD2A regulates DNA repair by controlling the recruitment of 53BP1 at DNA damage sites.     

Human RNF169 is a negative regulator of the ubiquitin-dependent response to DNA double-strand breaks

Nonproteolytic ubiquitylation of chromatin surrounding deoxyribonucleic acid double-strand breaks (DSBs), mediated by the RNF8/RNF168 ubiquitin ligases, plays a key role in recruiting repair factors, including 53BP1 and BRCA1, to reestablish genome integrity. In this paper, we show that human RNF169, an uncharacterized E3 ubiquitin ligase paralogous to RNF168, accumulated in DSB repair foci through recognition of RNF168-catalyzed ubiquitylation products by its motif interacting with ubiquitin domain. Unexpectedly, RNF169 was dispensable for chromatin ubiquitylation and ubiquitin-dependent accumulation of repair factors at DSB sites. Instead, RNF169 functionally competed with 53BP1 and RAP80-BRCA1 for association with RNF168-modified chromatin independent of its catalytic activity, limiting the magnitude of their recruitment to DSB sites. By delaying accumulation of 53BP1 and RAP80 at damaged chromatin, RNF169 stimulated homologous recombination and restrained nonhomologous end joining, affecting cell survival after DSB infliction. Our results show that RNF169 functions in a noncanonical fashion to harness RNF168-mediated protein recruitment to DSB-containing chromatin, thereby contributing to regulation of DSB repair pathway utilization.     

Give me a break,but not in mitosis

DNA double-strand breaks (DSBs) are extremely cytotoxic with a single unrepaired DSB being sufficient to induce cell death. A complex signalling cascade, termed the DNA damage response (DDR), is in place to deal with such DNA lesions and maintain genome stability. Recent work by us and others has found that the signalling cascade activated by DSBs in mitosis is truncated, displaying apical, but not downstream, components of the DDR. The E3 Ubiquitin ligases RNF8, RNF168 and BRCA1, along with the DDR mediator 53BP1, are not recruited to DSB sites in mitosis, and activation of downstream checkpoint kinases is also impaired. Here, we show that RNF8 and RNF168 are recruited to DNA damage foci in late mitosis, presumably to prime sites for 53BP1 recruitment in early G1. Interestingly, we show that, although RNF8, RNF168 and 53BP1 are excluded from DSB sites during most of mitosis, they associate with mitotic structures such as the kinetochore, suggesting roles for these DDR factors during mitotic cell division. We discuss these and other recent findings and suggest how these novel data collectively contribute to our understanding of mitosis and how cells deal with DNA damage during this crucial cell cycle stage.     

Give me a break,but not in mitosis: The mitotic DNA damage response marks DNA double strand breaks with early signaling events

DNA double-strand breaks (DSBs) are extremely cytotoxic lesions with a single unrepaired DSB being sufficient to induce cell death. A complex signaling cascade, termed the DNA damage response (DDR), is in place to deal with such DNA lesions and maintain genome stability. Recent work by us and others has found that the signaling cascade activated by DSBs in mitosis is truncated, displaying apical, but not downstream, components of the DDR. The E3 Ubiquitin ligases RNF8, RNF168 and BRCA1, along with the DDR mediator 53BP1, are not recruited to DSB sites in mitosis, and activation of downstream checkpoint kinases is also impaired. Here, we show that RNF8 and RNF168 are recruited to DNA damage foci in late mitosis, presumably to prime sites for 53BP1 recruitment in early G₁. Interestingly, we show that, although RNF8, RNF168 and 53BP1 are excluded from DSB sites during most of mitosis, they associate with mitotic structures such as the kinetochore, suggesting roles for these DDR factors during mitotic cell division. We discuss these and other recent findings and suggest how these novel data collectively contribute to our understanding of mitosis and how cells deal with DNA damage during this crucial cell cycle stage.Key words: mitosis, DNA damage response, DNA double-strand breaks, signaling cascade, chromatin     

DYRK1A regulates the recruitment of 53BP1 to the sites of DNA damage in part through interaction with RNF169

Human Dual-specificity tyrosine (Y) Regulated Kinase 1A (DYRK1A) is encoded by a dosage dependent gene whereby either trisomy or haploinsufficiency result in developmental abnormalities. However, the function and regulation of this important protein kinase are not fully understood. Here, we report proteomic analysis of DYRK1A in human cells that revealed a novel role of DYRK1A in DNA double-strand breaks (DSBs) repair, mediated in part by its interaction with the ubiquitin-binding protein RNF169 that accumulates at the DSB sites and promotes homologous recombination repair (HRR) by displacing 53BP1, a key mediator of non-homologous end joining (NHEJ). We found that overexpression of active, but not the kinase inactive DYRK1A in U-2 OS cells inhibits accumulation of 53BP1 at the DSB sites in the RNF169-dependent manner. DYRK1A phosphorylates RNF169 at two sites that influence its ability to displace 53BP1 from the DSBs. Although DYRK1A is not required for the recruitment of RNF169 to the DSB sites and 53BP1 displacement, inhibition of DYRK1A or mutation of the DYRK1A phosphorylation sites in RNF169 decreases its ability to block accumulation of 53BP1 at the DSB sites. Interestingly, CRISPR-Cas9 knockout of DYRK1A in human and mouse cells also diminished the 53BP1 DSB recruitment in a manner that did not require RNF169, suggesting that dosage of DYRK1A can influence the DNA repair processes through both RNF169-dependent and independent mechanisms. Human U-2 OS cells devoid of DYRK1A display an increased HRR efficiency and resistance to DNA damage, therefore our findings implicate DYRK1A in the DNA repair processes.     

Discordance between phosphorylation and recruitment of 53BP1 in response to DNA double-strand breaks

During the DNA damage response (DDR), chromatin modifications contribute to localization of 53BP1 to sites of DNA double-strand breaks (DSBs). 53BP1 is phosphorylated during the DDR, but it is unclear whether phosphorylation is directly coupled to chromatin binding. In this study, we used human diploid fibroblasts and HCT116 tumor cells to study 53BP1 phosphorylation at Serine-25 and Serine-1778 during endogenous and exogenous DSBs (DNA replication and whole-cell or sub-nuclear microbeam irradiation, respectively). In non-stressed conditions, endogenous DSBs in S-phase cells led to accumulation of 53BP1 and γH2AX into discrete nuclear foci. Only the frank collapse of DNA replication forks following hydroxyurea treatment initiated 53BP1^Ser25 and 53BP1^Ser1778 phosphorylation. In response to exogenous DSBs, 53BP1^Ser25 and 53BP1^Ser1778 phosphoforms localized to sites of initial DSBs in a cell cycle-independent manner. 53BP1 phosphoforms also localized to late residual foci and associated with PML-NBs during IR-induced senescence. Using isogenic cell lines and small-molecule inhibitors, we observed that DDR-induced 53BP1 phosphorylation was dependent on ATM and DNA-PKcs kinase activity but independent of MRE11 sensing or RNF168 chromatin remodeling. However, loss of RNF168 blocked recruitment of phosphorylated 53BP1 to sites of DNA damage. Our results uncouple 53BP1 phosphorylation from DSB localization and support parallel pathways for 53BP1 biology during the DDR. As relative 53BP1 expression may be a biomarker of DNA repair capacity in solid tumors, the tracking of 53BP1 phosphoforms in situ may give unique information regarding different cancer phenotypes or response to cancer treatment.     

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.     

RNF8/RNF168信号通路在DNA损伤修复中的作用

细胞内DNA会受部分外界因素(如紫外辐射,电离辐射和化学毒素)和内部因素(如复制错误)的影响而发生损伤,包括DNA双链断裂、DNA错配和DNA交链等。DNA损伤发生后,损伤部位会被一些蛋白识别,进而招募一系列蛋白至损伤部位,形成一个修复系统。DNA双链断裂是最严重的一种DNA损伤,错误修复往往导致疾病的发生。DNA双链断裂(double strand break, DSB)后,细胞启动RNF8/RNF168信号通路进行修复。RNF8和RNF168是这条通路的枢纽蛋白;53BP和BRCA1是关键的效应蛋白,决定着DSB修复的方式;组蛋白泛素化、磷酸化和甲基化等翻译后修饰是这条通路顺利进行的基本条件;染色质重塑、泛素化酶/去泛素化酶平衡和蛋白稳定性是这条通路的主要调节方式。本综述对RNF8/RNF168信号通路进行了梳理总结,希望其能对相关研究者起到参考作用。     

Solving the RIDDLE of 53BP1 recruitment to sites of damage

The cellular response to DNA double strand breaks is a complex, integrated network of pathways, coordinated by the PI-3-kinase-like family of kinases, which includes ATM, ATR and DNA-PK, that function to preserve the integrity of the genome. Mutations in genes that control these pathways are associated with increased genomic instability, neurodegeneration, immunodeficiency, premature aging and tumour predisposition. Indeed a significant proportion of our understanding regarding the mechanisms controlling DNA double strand break (DSB) repair has come from the study of cells derived from patients with inherited mutations in these genes. The discovery of the E3 ubiquitin ligase, RNF8, as a regulator of DNA DSB repair has brought to light a critical role for the ubiquitin system in regulating the cellular DSBs. Recently, identification of mutations in a second E3 ubiquitin ligase, RNF168, as the underlying genetic cause of the DNA repair deficiency disorder, RIDDLE syndrome, has provided the first link between ubiquitin-dependent DSB repair and immune system development in man. The finding that RNF168 functions downstream of RNF8 to orchestrate the recruitment of repair proteins, such as BRCA1 and 53BP1, to sites of DNA damage suggests that these two E3 ligases define a ubiquitylation cascade that regulates the spatial relocalisation of DSB repair proteins.     

RNF4 regulates DNA double-strand break repair in a cell cycle-dependent manner

Both RNF4 and KAP1 play critical roles in the response to DNA double-strand breaks (DSBs), but the functional interplay of RNF4 and KAP1 in regulating DNA damage response remains unclear. We have previously demonstrated the recruitment and degradation of KAP1 by RNF4 require the phosphorylation of Ser824 (pS824) and SUMOylation of KAP1. In this report, we show the retention of DSB-induced pS824-KAP1 foci and RNF4 abundance are inversely correlated as cell cycle progresses. Following irradiation, pS824-KAP1 foci predominantly appear in the cyclin A (-) cells, whereas RNF4 level is suppressed in the G0-/G1-phases and then accumulates during S-/G2-phases. Notably, 53BP1 foci, but not BRCA1 foci, co-exist with pS824-KAP1 foci. Depletion of KAP1 yields opposite effect on the dynamics of 53BP1 and BRCA1 loading, favoring homologous recombination repair. In addition, we identify p97 is present in the RNF4-KAP1 interacting complex and the inhibition of p97 renders MCF7 breast cancer cells relatively more sensitive to DNA damage. Collectively, these findings suggest that combined effect of dynamic recruitment of RNF4 to KAP1 regulates the relative occupancy of 53BP1 and BRCA1 at DSB sites to direct DSB repair in a cell cycle-dependent manner.     

The proteasomal de‐ubiquitinating enzyme POH1 promotes the double‐strand DNA break response

The regulation of Ubiquitin (Ub) conjugates generated by the complex network of proteins that promote the mammalian DNA double‐strand break (DSB) response is not fully understood. We show here that the Ub protease POH1/rpn11/PSMD14 resident in the 19S proteasome regulatory particle is required for processing poly‐Ub formed in the DSB response. Proteasome activity is required to restrict tudor domain‐dependent 53BP1 accumulation at sites of DNA damage. This occurs both through antagonism of RNF8/RNF168‐mediated lysine 63‐linked poly‐Ub and through the promotion of JMJD2A retention on chromatin. Consistent with this role POH1 acts in opposition to RNF8/RNF168 to modulate end‐joining DNA repair. Additionally, POH1 acts independently of 53BP1 in homologous recombination repair to promote RAD51 loading. Accordingly, POH1‐deficient cells are sensitive to DNA damaging agents. These data demonstrate that proteasomal POH1 is a key de‐ubiquitinating enzyme that regulates ubiquitin conjugates generated in response to damage and that several aspects of the DSB response are regulated by the proteasome.     

The ubiquitin- and SUMO-dependent signaling response to DNA double-strand breaks

DNA double-strand breaks (DSBs) represent the most destructive type of chromosomal lesion and trigger rapid chromatin restructuring accompanied by accumulation of proteins in the vicinity of the DSB. Non-proteolytic ubiquitylation of chromatin surrounding DSBs, mediated by the RNF8/RNF168 ubiquitin ligase cascade, has emerged as a key mechanism for restoration of genome integrity by licensing the DSB-modified chromatin to concentrate genome caretaker proteins such as 53BP1 and BRCA1 near the lesions. In parallel, SUMOylation of upstream DSB regulators is also required for execution of this ubiquitin-dependent chromatin response, but its molecular basis is currently unclear. Here, we discuss recent insights into how ubiquitin- and SUMO-dependent signaling processes cooperate to orchestrate protein interactions with sites of DNA damage to facilitate DSB repair.     

A small ubiquitin binding domain inhibits ubiquitin-dependent protein recruitment to DNA repair foci

The rapid ubiquitination of chromatin surrounding DNA double-stranded breaks (DSB) drives the formation of large structures called ionizing radiation-induced foci (IRIF), comprising many DNA damage response (DDR) proteins. This process is regulated by RNF8 and RNF168 ubiquitin ligases and is thought to be necessary for DNA repair and activation of signaling pathways involved in regulating cell cycle checkpoints. Here we demonstrate that it is possible to interfere with ubiquitin-dependent recruitment of DDR factors by expressing proteins containing ubiquitin binding domains (UBDs) that bind to lysine 63-linked polyubiquitin chains. Expression of the E3 ubiquitin ligase RAD18 prevented chromatin spreading of 53BP1 at DSBs, and this phenomenon was dependent upon the integrity of the RAD18 UBD. An isolated RAD18 UBD interfered with 53BP1 chromatin spreading, as well as other important DDR mediators, including RAP80 and the BRCA1 tumor suppressor protein, consistent with the model that the RAD18 UBD is blocking access of proteins to ubiquitinated chromatin. Using the RAD18 UBD as a tool to impede localization of 53BP1 and BRCA1 to repair foci, we found that DDR signaling, DNA DSB repair, and radiosensitivity were unaffected. We did find that activated ATM (S1981P) and phosphorylated SMC1 (a specific target of ATM) were not detectable in DNA repair foci, in addition to upregulated homologous recombination repair, revealing 2 DDR responses that are dependent upon chromatin spreading of certain DDR factors at DSBs. These data demonstrate that select UBDs containing targeting motifs may be useful probes in determining the biological significance of protein–ubiquitin interactions.     

USP3 counteracts RNF168 via deubiquitinating H2A and γH2AX at lysine 13 and 15

Histone ubiquitination plays a vital role in DNA damage response (DDR), which is important for maintaining genomic integrity in eukaryotic cells. In DDR, ubiquitination of histone H2A and γH2AX by the concerted action of ubiquitin (Ub) ligases, RNF168 and RNF8, generates a cascade of ubiquitination signaling. However, little is known about deubiquitinating enzymes (DUBs) that may catalyze the removal of Ub from these histones. This study demonstrated that USP3, an apparent DUB for mono-ubiquitinated H2A, is indeed the enzyme for deubiquitinating Ub conjugates of γH2AX and H2A from lysine sites, where the ubiquitination is initiated by RNF168. Here, we showed that ectopic expression of USP3 led to the deubiquitination of both H2A and γH2AX in response to UV-induced DNA damage. Moreover, ectopic USP3 expression abrogated FK2 antibody-reactive Ub-conjugate foci, which co-localize with damage-induced γH2AX foci. In addition, USP3 overexpression impaired the accumulation of downstream repair factors BRCA1 and 53BP1 at the damage sites in response to both UV and γ-irradiation. We further identified that the USP3 removes Ub at lysine 13 and 15 of H2A and γH2AX, as well as lysine 118 and 119 of H2AX in response to DNA damage. Taken together, the results suggested that USP3 is a negative regulator of ubiquitination signaling, counteracting RNF168- and RNF8-mediated ubiquitination.     

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.     

Ring Finger Protein RNF169 Antagonizes the Ubiquitin-dependent Signaling Cascade at Sites of DNA Damage

Ubiquitin signals emanating from DNA double-strand breaks (DSBs) trigger the ordered assembly of DNA damage mediator and repair proteins. This highly orchestrated process is accomplished, in part, through the concerted action of the RNF8 and RNF168 E3 ligases, which have emerged as core signaling intermediates that promote DSB-associated ubiquitylation events. In this study, we report the identification of RNF169 as a negative regulator of the DNA damage signaling cascade. We found that RNF169 interacted with ubiquitin structures and relocalized to DSBs in an RNF8/RNF168-dependent manner. Moreover, ectopic expression of RNF169 attenuated ubiquitin signaling and compromised 53BP1 accumulation at DNA damage sites, suggesting that RNF169 antagonizes RNF168 functions at DSBs. Our study unveils RNF169 as a component in DNA damage signal transduction and adds to the complexity of regulatory ubiquitylation in genome stability maintenance.     

Polo-like kinase 1 inhibits DNA damage response during mitosis

In response to genotoxic stress, cells protect their genome integrity by activation of a conserved DNA damage response (DDR) pathway that coordinates DNA repair and progression through the cell cycle. Extensive modification of the chromatin flanking the DNA lesion by ATM kinase and RNF8/RNF168 ubiquitin ligases enables recruitment of various repair factors. Among them BRCA1 and 53BP1 are required for homologous recombination and non-homologous end joining, respectively. Whereas mechanisms of DDR are relatively well understood in interphase cells, comparatively less is known about organization of DDR during mitosis. Although ATM can be activated in mitotic cells, 53BP1 is not recruited to the chromatin until cells exit mitosis. Here we report mitotic phosphorylation of 53BP1 by Plk1 and Cdk1 that impairs the ability of 53BP1 to bind the ubiquitinated H2A and to properly localize to the sites of DNA damage. Phosphorylation of 53BP1 at S1618 occurs at kinetochores and in cytosol and is restricted to mitotic cells. Interaction between 53BP1 and Plk1 depends on the activity of Cdk1. We propose that activity of Cdk1 and Plk1 allows spatiotemporally controlled suppression of 53BP1 function during mitosis.     

Polo-like kinase 1 inhibits DNA damage response during mitosis

In response to genotoxic stress, cells protect their genome integrity by activation of a conserved DNA damage response (DDR) pathway that coordinates DNA repair and progression through the cell cycle. Extensive modification of the chromatin flanking the DNA lesion by ATM kinase and RNF8/RNF168 ubiquitin ligases enables recruitment of various repair factors. Among them BRCA1 and 53BP1 are required for homologous recombination and non-homologous end joining, respectively. Whereas mechanisms of DDR are relatively well understood in interphase cells, comparatively less is known about organization of DDR during mitosis. Although ATM can be activated in mitotic cells, 53BP1 is not recruited to the chromatin until cells exit mitosis. Here we report mitotic phosphorylation of 53BP1 by Plk1 and Cdk1 that impairs the ability of 53BP1 to bind the ubiquitinated H2A and to properly localize to the sites of DNA damage. Phosphorylation of 53BP1 at S1618 occurs at kinetochores and in cytosol and is restricted to mitotic cells. Interaction between 53BP1 and Plk1 depends on the activity of Cdk1. We propose that activity of Cdk1 and Plk1 allows spatiotemporally controlled suppression of 53BP1 function during mitosis.