Timely and proper cellular response to DNA damage is essential for maintenance of genome stability and integrity. B-cell lymphoma/leukemia 10 (BCL10) facilitates ubiquitination of NEMO in the cytosol, activating NFκB signaling. Translocation and/or point mutations of BCL10 associate with mucosa-associated lymphoid tissue lymphomas and other malignancies. However, the mechanisms by which the resulting aberrant expression of BCL10 leads to cellular oncogenesis are poorly understood. In this report, we found that BCL10 in the nucleus is enriched at the DNA damage sites in an ATM- and RNF8-dependent manner. ATM-dependent phosphorylation of BCL10 promotes its interaction with and presentation of UBC13 to RNF8, and RNF8-mediated ubiquitination of BCL10 enhances binding of BCL10 and UBC13 to RNF168. This allows mono-ubiquitination on H2AX by RNF168 and further poly-ubiquitination by the RNF8/RNF168-containing complex. Depletion of BCL10 compromised homology recombination-mediated DNA double-strand break (DSB) repair because of insufficient recruitment of BRCA1, RAD51, and the ubiquitinated DNA damage response factors. Taken together, our results demonstrate a novel function of BCL10 in delivering UBC13 to RNF8/RNF168 to regulate ubiquitination-mediated DSB signaling and repair.
BCL10; DNA damage; RNF168; RNF8; ubiquitination
Proper DNA damage response is essential for the maintenance of genome integrity. The E3 ligase RNF168 deficiency fully prevents both the initial recruitment and retention of 53BP1 at sites of DNA damage. In response to DNA damage, RNF168-dependent recruitment of the lysine-specific demethylase LSD1 to the site of DNA damage promotes local H3K4me2 demethylation and ubiquitination of H2A/H2AX, facilitating 53BP1 recruitment to sites of DNA damage. Alternatively, RNF168-mediated K63-linked ubiquitylation of 53BP1 is required for the initial recruitment of 53BP1 to sites of DNA damage and for its function in repair. We demonstrated here that phosphorylation and dephosphorylation of LSD1 at S131 and S137 was mediated by casein kinase 2 (CK2) and wild-type p53-induced phosphatase 1 (WIP1), respectively. LSD1, RNF168 and 53BP1 interacted with each other directly. CK2-mediated phosphorylation of LSD1 exhibited no impact on its interaction with 53BP1, but promoted its interaction with RNF168 and RNF168-dependent 53BP1 ubiquitination and subsequent recruitment to the DNA damage sites. Furthermore, overexpression of phosphorylation-defective mutants failed to restore LSD1 depletion-induced cellular sensitivity to DNA damage. Taken together, our results suggest that LSD1 phosphorylation modulated by CK2/WIP1 regulates RNF168-dependent 53BP1 recruitment directly in response to DNA damage and cellular sensitivity to DNA damaging agents.
Spinocerebellar ataxia type 10 (SCA10) is an autosomal dominant neurologic disorder caused by ATTCT expansion in the ATXN10 gene. Previous investigations have identified that depletion of Ataxin-10, the gene product, leads to cellular apoptosis and cytokinesis failure. Herein we identify the mitotic kinase Aurora B as an Ataxin-10 interacting partner. Aurora B interacts with and phosphorylates Ataxin-10 at S12, as evidenced by in vitro kinase and mass spectrometry analysis. Both endogenous and S12-phosphorylated Ataxin-10 localizes to the midbody during cytokinesis, and cytokinetic defects induced by inhibition of ATXN10 expression is not rescued by the S12A mutant. Inhibition of Aurora B or expression of the S12A mutant renders reduced interaction between Ataxin-10 and polo-like kinase 1 (Plk1), a kinase previously identified to regulate Ataxin-10 in cytokinesis. Taken together, we propose a model that Aurora B phosphorylates Ataxin-10 at S12 to promote the interaction between Ataxin-10 and Plk1 in cytokinesis. These findings identify an Aurora B-dependent mechanism that implicates Ataxin-10 in cytokinesis.
CLASPIN is an essential mediator in the DNA replication checkpoint, responsible for ATR (ataxia telangiectasia and Rad3-related protein)-dependent activation of CHK1 (checkpoint kinase 1). Here we found a dynamic signaling pathway that regulates CLASPIN turn over. Under unperturbed conditions, the E3 ubiquitin ligase HERC2 regulates the stability of the deubiquitinating enzyme USP20 by promoting ubiquitination-mediated proteasomal degradation. Under replication stress, ATR-mediated phosphorylation of USP20 results in the disassociation of HERC2 from USP20. USP20 in turn deubiquitinates K48-linked-polyubiquitinated CLASPIN, stabilizing CLASPIN and ultimately promoting CHK1 phosphorylation and CHK1-directed checkpoint activation. Inhibition of USP20 expression promotes chromosome instability and xenograft tumor growth. Taken together, our findings demonstrated a novel function of HERC2/USP20 in coordinating CHK1 activation by modulating CLASPIN stability, which ultimately promotes genome stability and suppresses tumor growth.
XRCC4-like factor (XLF) is involved in non-homologous end joining-mediated repair of DNA double-strand breaks (DSBs). Mutations in the WRN gene results in the development of Werner syndrome (WS), a rare autosomal recessive disorder characterized by premature ageing and genome instability. In the present study, it was identified that XLF protein levels were lower in WRN-deficient fibroblasts, compared with normal fibroblasts. Depletion of WRN in HeLa cells led to a decrease of XLF mRNA and its promoter activity. Chromatin immunoprecipitation assays demonstrated that WRN was associated with the XLF promoter. Depletion of XLF in normal human fibroblasts increased the percentage of β-galactosidase (β-gal) staining-positive cells, indicating acceleration in cellular senescence. Taken together, the results suggest that XLF is a transcriptional target of WRN and may be involved in the regulation of cellular senescence.
Werner syndrome; XLF; transcriptional regulation; cellular senescence
DNA endonuclease CtIP is involved in both DNA double-strand break (DSB) repair and transcriptional repression/activation. The cyclin-dependent kinase inhibitor P21, which is induced at transcription level in response to a variety of stresses, controls G1/S transition. In this report, we found that CtIP bound to the P21 promoter, and this binding was enhanced in response to DNA damage. Concomitantly, ectopic expression of CtIP increased P21 promoter activity, and this increment was enhanced upon camptothecin treatment. Conversely, DNA damage failed to induce P21 gene expression in CtIP-deficient cells. Taken together, our data demonstrate that CtIP is required for DNA damage-induced P21 induction.
DNA endonuclease CtIP; DNA damage; P21
DNA double-strand break (DSB) is the most severe form of DNA damage, which is repaired mainly through high-fidelity homologous recombination (HR) or error-prone non-homologous end joining (NHEJ). Defects in the DNA damage response lead to genomic instability and ultimately predispose organs to cancer. Nicotinamide phosphoribosyltransferase (Nampt), which is involved in nicotinamide adenine dinucleotide metabolism, is overexpressed in a variety of tumors. In this report, we found that Nampt physically associated with CtIP and DNA-PKcs/Ku80, which are key factors in HR and NHEJ, respectively. Depletion of Nampt by small interfering RNA (siRNA) led to defective NHEJ-mediated DSB repair and enhanced HR-mediated repair. Furthermore, the inhibition of Nampt expression promoted proliferation of cancer cells and normal human fibroblasts and decreased β-galactosidase staining, indicating a delay in the onset of cellular senescence in normal human fibroblasts. Taken together, our results suggest that Nampt is a suppressor of HR-mediated DSB repair and an enhancer of NHEJ-mediated DSB repair, contributing to the acceleration of cellular senescence.
Nampt; DNA-PKcs/Ku80; CtIP; DNA repair; cellular senescence
DNA double-strand breaks (DSBs) are among the most lethal lesions associated with genome stability, which, when destabilized, predisposes organs to cancers. DSBs are primarily fixed either with little fidelity by non-homologous end joining (NHEJ) repair or with high fidelity by homology-directed repair (HDR). The phosphorylated form of H2AX on serine 139 (γ-H2AX) is a marker of DSBs. In this study, we explored if the protein phosphatase PP6 is involved in DSB repair by depletion of its expression in human cancer cell lines, and determined PP6 expression in human breast cancer tissues by immunohistochemistry staining. We found that bacterially produced PP6c (the catalytic subunit of PP6)-containing heterotrimeric combinations exhibit phosphatase activity against γ-H2AX in the in vitro phosphatase assays. Depletion of PP6c or PP6R2 led to persistent high levels of γ-H2AX after DNA damage and a defective HDR. Chromatin immunoprecipitation assays demonstrated that PP6c was recruited to the region adjacent to the DSB sites. Expression of PP6c, PP6R2 and PP6R3 in human breast tumors was significantly lower than those in benign breast diseases. Taken together, our results suggest that γ-H2AX is a physiological substrate of PP6 and PP6 is required for HDR and its expression may harbor a protective role during the development of breast cancer.
protein phosphatase; PP6; γ-H2AX; DNA double-strand break; homology-directed repair
KRAB domain–associated protein 1 (KAP1, also known as TIF1β) is a universal transcriptional corepressor that is susceptible to phosphorylation at Ser824 by ataxia-telangiectasia mutated (ATM) and to modification by small ubiquitin-like modifying (SUMO) proteins. Here, we found that whereas protein phosphatase 1α (PP1α) directly interacted with KAP1 under unstressed conditions, PP1β interacted with KAP1 under conditions of genotoxic stress. Changes in the abundance of PP1α or PP1β led to a differential inverse-coregulation of the phosphorylation and SUMOylation states of KAP1 under basal conditions and in response to DNA double-strand breaks (DSBs). Chromatin immunoprecipitation and re-immunoprecipitation experiments revealed that PP1α and PP1β were recruited to KAP1 with different kinetics before and after the induction of DNA DSBs, which provided a mechanistic basis for the switch in the dephosphorylation and SUMOylation states of KAP1. PP1β-stimulated SUMOylation of KAP1 occurred by mechanisms that were dependent and independent of the phosphorylation status of Ser824. We posit a mechanism whereby the combined actions of PP1α and PP1β dynamically cause dephosphorylation of KAP1 Ser824 and assure the SUMOylation of KAP1 to counter the effect of ATM, thereby regulating the transcription of KAP1 target genes in unstressed and stressed cells.
MDC1 is a key mediator of the DNA-damage response in mammals with several phosphorylation-dependent protein interaction domains. The function of its N-terminal forkhead-associated (FHA) domain remains elusive. Here, we show with structural, biochemical and cellular data that the FHA domain mediates phosphorylation-dependent dimerization of MDC1 in response to DNA damage. Crystal structures of the FHA domain reveal a face-to-face dimer with pseudo-dyad symmetry. We found that the FHA domain recognizes phosphothreonine 4 (pT4) at the N-terminus of MDC1 and determined its crystal structure in complex with a pT4 peptide. Biochemical analysis further revealed that in the dimer, the FHA domain binds in trans to pT4 from the other subunit, which greatly stabilizes the otherwise unstable dimer. We show that T4 is phosphorylated primarily by ATM upon DNA damage. MDC1 mutants with the FHA domain deleted or impaired in its ability to dimerize formed fewer foci at DNA-damage sites, but the localization defect was largely rescued by an artificial dimerization module, suggesting that dimerization is the primary function of the MDC1 FHA domain. Our results suggest a novel mechanism for the regulation of MDC1 function through T4 phosphorylation and FHA-mediated dimerization.
The histone H2A variant H2AX is rapidly phosphorylated in response to DNA double-stranded breaks to produce γ-H2AX. γ-H2AX stabilizes cell cycle checkpoint proteins and DNA repair factors at the break site. We previously found that the protein phosphatase PP2A is required to resolve γ-H2AX foci and complete DNA repair after exogenous DNA damage. Here we describe a three-protein PP4 phosphatase complex in mammalian cells, containing PP4C, PP4R2 and PP4R3β, that specifically dephosphorylates ATR-mediated γ-H2AX generated during DNA replication. PP4 efficiently dephosphorylates γ-H2AX within mononucleosomes in vitro. The effect of PP4 on γ-H2AX is independent of ATR and checkpoint kinase activity. When the PP4 complex is silenced, repair of DNA replication mediated breaks is inefficient, and cells are hypersensitive to DNA replication inhibitors, but not radiomimetic drugs. Therefore γ-H2AX elimination at DNA damage foci is required for DNA damage repair, but accomplishing this task involves distinct phosphatases with potentially overlapping roles.
The catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) plays a major role in the repair of DNA double-strand breaks (DSBs) by nonhomologous end joining (NHEJ). We have previously shown that DNA-PKcs is autophosphorylated in response to ionizing radiation (IR) and that dephosphorylation by a protein phosphatase 2A (PP2A)-like protein phosphatase (PP2A, PP4, or PP6) regulates the protein kinase activity of DNA-PKcs. Here we report that DNA-PKcs interacts with the catalytic subunits of PP6 (PP6c) and PP2A (PP2Ac), as well as with the PP6 regulatory subunits PP6R1, PP6R2, and PP6R3. Consistent with a role in the DNA damage response, silencing of PP6c by small interfering RNA (siRNA) induced sensitivity to IR and delayed release from the G2/M checkpoint. Furthermore, siRNA silencing of either PP6c or PP6R1 led to sustained phosphorylation of histone H2AX on serine 139 (γ-H2AX) after IR. In contrast, silencing of PP6c did not affect the autophosphorylation of DNA-PKcs on serine 2056 or that of the ataxia-telangiectasia mutated (ATM) protein on serine 1981. We propose that a novel function of DNA-PKcs is to recruit PP6 to sites of DNA damage and that PP6 contributes to the dephosphorylation of γ-H2AX, the dissolution of IR-induced foci, and release from the G2/M checkpoint in vivo.
Human Rap1-interacting protein 1 (RIF1) contributes to the ataxia telangiectasia, mutated-mediated DNA damage response against the dexterous effect of DNA lesions and plays a critical role in the S-phase checkpoint. However, the molecular mechanisms by which human RIF1 conquers DNA aberrations remain largely unknown. We here showed that inhibition of RIF1 expression by small interfering RNA led to defective homologous recombination-mediated DNA double-strand break repair and sensitized cancer cells to camptothecin or staurosporine treatment. RIF1 underwent caspase-dependent cleavage upon apoptosis. We further found that RIF1 was highly expressed in human breast tumors, and its expression status was positively correlated with differentiation degrees of invasive ductal carcinoma of the breast. Our results suggest that RIF1 encodes an anti-apoptotic factor required for DNA repair and is a potential target for cancer treatment.
The combination of paclitaxel and doxorubicin is among the most successful chemotherapy regimens in cancer treatment. CDK5RAP2, when mutated, causes primary microcephaly. We show here that inhibition of CDK5RAP2 expression causes chromosome mis-segregation, fails to maintain the spindle checkpoint, and is associated with reduced expression of the spindle checkpoint proteins BUBR1 and MAD2 and an increase in chromatin-associated CDC20. CDK5RAP2 resides on the BUBR1 and MAD2 promoters and regulates their transcription. Furthermore, CDK5RAP2-knockdown cells have increased resistance to paclitaxel and doxorubicin, and this resistance is partially rescued upon restoration of CDK5RAP2 expression. Cancer cells cultured in the presence of paclitaxel or doxorubicin exhibit dramatically decreased CDK5RAP2 levels. These results suggest that CDK5RAP2 is required for spindle checkpoint function and is a common target in paclitaxel and doxorubicin resistance.
CDK5RAP2; primary microcephaly; spindle checkpoint; BUBR1; MAD2; drug resistance; paclitaxel; doxorubicin
The tumor suppressor gene CHK2 encodes a versatile effector serine/threonine kinase involved in responses to DNA damage. Chk2 has an amino-terminal SQ/TQ cluster domain (SCD), followed by a forkhead-associated (FHA) domain and a carboxyl-terminal kinase catalytic domain. Mutations in the SCD or FHA domain impair Chk2 checkpoint function. We show here that autophosphorylation of Chk2 produced in a cell-free system requires trans phosphorylation by a wortmannin-sensitive kinase, probably ATM or ATR. Both SQ/TQ sites and non-SQ/TQ sites within the Chk2 SCD can be phosphorylated by active Chk2. Amino acid substitutions in the SCD and the FHA domain impair auto- and trans-kinase activities of Chk2. Chk2 forms oligomers that minimally require the FHA domain of one Chk2 molecule and the SCD within another Chk2 molecule. Chk2 oligomerization in vivo increases after DNA damage, and when damage is induced by gamma irradiation, this increase requires ATM. Chk2 oligomerization is phosphorylation dependent and can occur in the absence of other eukaryotic proteins. Chk2 can cross-phosphorylate another Chk2 molecule in an oligomeric complex. Induced oligomerization of a Chk2 chimera in vivo concomitant with limited DNA damage augments Chk2 kinase activity. These results suggest that Chk2 oligomerization regulates Chk2 activation, signal amplification, and transduction in DNA damage checkpoint pathways.