Sites Of DNA DNA Double-strand Breaks Research Articles

Knockdown of KDM5B Leads to DNA Damage and Cell Cycle Arrest in Granulosa Cells via MTF1

KDM5B is essential for early embryo development, which is under the control of maternal factors in oocytes. Granulosa cells (GCs) play a critical role during oocyte mature. However, the role of KDM5B in GCs remains to be elucidated. In the current study, we found that KDM5B expressed highly in the ovaries and located in goat GCs. Using an RNA sequence, we identified 1353 differentially expressed genes in the KDM5B knockdown GCs, which were mainly enriched in cell cycle, cell division, DNA replication and the cellular oxidative phosphorylation regulation pathway. Moreover, we reported a decrease in the percentage of proliferated cells but an increase in the percentage of apoptotic cells in the KDM5B knockdown GCs. In addition, in the KDM5B knockdown GCs, the percentage of GCs blocked at the S phase was increased compared to the NC group, suggesting a critical role of KDM5B in the cell cycle. Moreover, in the KDM5B knockdown GCs, the reactive oxygen species level, the mitochondrial depolarization ratio, and the expression of intracellular phosphorylated histone H2AX (γH2AX) increased, suggesting that knockdown of KDM5B leads to DNA damage, primarily in the form of DNA double-strand breaks (DSBs). Interestingly, we found a down-regulation of MTF1 in the KDM5B knockdown GCs, and the level of cell proliferation, as well as the cell cycle block in the S phase, was improved. In contrast, in the group with both KDM5B knockdown and MTF1 overexpression, the level of ROS, the expression of γH2AX and the number of DNA DSB sites decreased. Taken together, our results suggest that KDM5B inhibits DNA damage and promotes the cell cycle in GCs, which might occur through the up-regulation of MTF1.

And-1 is required for homologous recombination repair by regulating DNA end resection.

Homologous recombination (HR) is a major mechanism to repair DNA double-strand breaks (DSBs). Although tumor suppressor CtIP is critical for DSB end resection, a key initial event of HR repair, the mechanism regulating the recruitment of CtIP to DSB sites remains largely unknown. Here, we show that acidic nucleoplasmic DNA‐binding protein 1 (And‐1) forms complexes with CtIP as well as other repair proteins, and is essential for HR repair by regulating DSB end resection. Furthermore, And-1 is recruited to DNA DSB sites in a manner dependent on MDC1, BRCA1 and ATM, down-regulation of And-1 impairs end resection by reducing the recruitment of CtIP to damage sites, and considerably reduces Chk1 activation and other damage response during HR repair. These findings collectively demonstrate a hitherto unknown role of MDC1→And-1→CtIP axis that regulates CtIP-mediated DNA end resection and cellular response to DSBs.

Inhibition of Ku70 acetylation by INHAT subunit SET/TAF-Iβ regulates Ku70-mediated DNA damage response.

DNA double-strand breaks (DSBs) can cause either cell death or genomic instability. The Ku heterodimer Ku70/80 is required for the NHEJ (non-homologous end-joining) DNA DSB repair pathway. The INHAT (inhibitor of histone acetyltransferases) complex subunit, SET/TAF-Iβ, can inhibit p300- and PCAF-mediated acetylation of both histone and p53, thereby repressing general transcription and that of p53 target genes. Here, we show that SET/TAF-Iβ interacts with Ku70/80, and that this interaction inhibits CBP- and PCAF-mediated Ku70 acetylation in an INHAT domain-dependent manner. Notably, DNA damage by UV disrupted the interaction between SET/TAF-Iβ and Ku70. Furthermore, we demonstrate that overexpressed SET/TAF-Iβ inhibits recruitment of Ku70/80 to DNA damage sites. We propose that dysregulation of SET/TAF-Iβ expression prevents repair of damaged DNA and also contributes to cellular proliferation. All together, our findings indicate that SET/TAF-Iβ interacts with Ku70/80 in the nucleus and inhibits Ku70 acetylation. Upon DNA damage, SET/TAF-Iβ dissociates from the Ku complex and releases Ku70/Ku80, which are then recruited to DNA DSB sites via the NHEJ DNA repair pathway.

Internal Tandem Duplication Mutation Of FLT3 (FLT3/ITD) Induces Increased Homologous Recombination DNA Repair Activity, Drug Resistance and Sister Chromatid Exchanges In Acute Myeloid Leukaemia (AML)

The majority of patients with adult acute myeloid leukaemia (AML) that present with an apparently normal karyotype (NK-AML) are grouped together in the “intermediate” risk category and constitute 40-45% of all adult AML patients. Mutations in the FMS-like tyrosine kinase 3 (FLT3) receptor whether it is internal tandem duplication (ITD) of its juxtamembrane domain or point mutations in its kinase domain are one of the most common mutations in NK-AML. The presence of FLT3-ITD mutation in NK-AML results in a more aggressive disease, resistance to therapy and poor survival. Acquired copy neutral loss of heterozygosity (CN-LOH) also referred to as uniparental disomy (UPD) is a common phenomenon of myeloid malignancies where an oncogenic allele is duplicated on the other chromosome. The use of single nucleotide phenotype analysis (SNP-A) karyotyping detects CN-LOH in 20% and 40% of newly diagnosed and relapsed AML respectively. CN-LOH at 6p or at the FLT3 locus 13q associated with a FLT3-ITD mutation in NK-AML results in an even more aggressive disease compared to NK-AML + FLT3-ITD without CN-LOH. It is has been proposed that CN-LOH is the result of a homologous recombination (HR) DNA repair event. However, the underlying mechanisms that confer CN-LOH have yet to be determined.To elucidate the mechanisms that produce CN-LOH in NK-AML we determined whether oncogenes such as FLT3-ITD have the propensity to generate CN-LOH through production of excessive DNA double strand breaks (DSB) and up-regulation of HR DNA repair. Primary NK-AML (n=17) were initially characterised by SNP-A and DNA sequencing. 12/17 primary NK-AML had FLT3/ITD mutations with a clone size ≥ 25%, but were wild type (WT) for RAS. From this cohort 6/12 possessed CN-LOH with 3/12 having CN-LOH at 13q. 5/17 primary NK-AML were WT for FLT3 and RAS, but had nucleophosmin (NPM) and DNA methyltransferase 1 (DNMT1) mutations. From this cohort, 1/5 primary NK-AML + WT FLT3 had CN-LOH.We showed that primary AML + FLT3/ITD cohort, the MOLM 13 cell line that possesses a FLT3/ITD mutation and WT FLT3 KG-1 cells transfected with a FLT3-ITD expression vector demonstrate increased ROS compared to the WT FLT3 primary NK-AML cohort, the WT FLT3 cell line, U937 and KG-1 transfected with empty vector. Immunofluorescence studies showed that primary AML with FLT3/ITD, MOLM 13 and FLT3-ITD transfected KG-1 cells have increased mobilization of phospho-γH2AX (a DNA DSB intermediate) and RAD51 (a HR factor) to sites of DNA DSB in response to mitomycin C (MMC) induced DNA damage compared to non-mutated FLT3 primary AML cells, cell lines and KG-1 transfected with empty vector (phospho-γH2AX, 29% vs. 21%, p<0.005 and RAD51, 25% VS 19%, p<0.01). Moreover, we show that HR is augmented in FLT3-ITD transfected cells and MOLM-13 with and without exogenous DNA damage using DR-GFP gene reporter assays. Increased HR activity has been associated with increased resistance to genotoxic agents. Using soft agar clonogenic assays, MOLM 13 and FLT3-ITD transfected KG-1 showed considerably increased resistance to MMC and the radiomimetic agent, bleomycin compared to U937 cells and KG-1 + empty vector. Finally, to evaluate FLT3-ITD induced genomic instability we measured sister chromatid exchanges (SCE) that result from HR mediated chromosome crossing over. FLT3/ITD primary AML, MOLM-13 and FLT3-ITD transfected KG-1 cells show a significant increase in SCE compared to WT FLT3 primary AML cells, U937 cells and KG-1 transfected with empty vector, (10 vs. 6 SCE per metaphase, p<0.001). Importantly, primary AML with CN-LOH at 13q + FLT3-ITD mutation gives significantly higher HR activity and SCE than non-13q CN-LOH primary AML + FLT3-ITD mutation (p<0.05). Moreover, treatment of FLT3/ITD primary AML, MOLM-13 and FLT3-ITD transfected KG-1 cells with the FLT3 inhibitor, CEP701 and the anti-oxidant agent, N-acetyl cysteine restored ROS levels, HR activity, sensitivity to MMC and SCE to levels observed for WT FLT3. These findings not only provide a strong proof of principle that FLT3-ITD derived DNA damage is the trigger for CN-LOH in NK-AML, but further our understanding of leukaemic progression and relapse. Disclosures:No relevant conflicts of interest to declare.

Molecular Radiotherapy Using Cleavable Radioimmunoconjugates That Target EGFR and γH2AX

Many anticancer therapies, including ionizing radiation (IR), cause cytotoxicity through generation of DNA double-strand breaks (DSB). Delivery of therapeutic radionuclides to DNA DSB sites can amplify this DNA damage, for additional therapeutic gain. Herein, we report on two radiopharmaceuticals, radiolabeled with the Auger electron emitter (111)In, with dual specificity for both the intranuclear, DNA damage repair signaling protein γH2AX and the EGF receptor (EGFR). The EGFR ligand EGF was conjugated to a fluorophore- or (111)In-labeled anti-γH2AX antibody, linked via a nuclear localization sequence (NLS) to ensure nuclear translocation. EGF conjugation was achieved either through a noncleavable PEG linker (PEO6) or a cleavable disulfide bond. Both conjugates selectively bound EGFR on fixed cells and γH2AX in cell extracts. Both compounds enter EGFR-expressing cells in an EGF/EGFR-dependent manner. However, only the cleavable compound was seen to associate with γH2AX foci in the nuclei of irradiated cells. Intracellular retention of the cleavable compound was prolonged in γH2AX-expressing cells. Clonogenic survival was significantly reduced when cells were exposed to IR (to induce γH2AX) plus (111)In-labeled cleavable compound compared to either alone and compared to nonspecific controls. In vivo, uptake of (111)In-labeled cleavable compound in MDA-MB-468 xenografts in athymic mice was 2.57 ± 0.47 percent injected dose/g (%ID/g) but increased significantly to 6.30 ± 1.47%ID/g in xenografts where γH2AX was induced by IR (P < 0.01). This uptake was dependent on EGF/EGFR and anti-γH2AX/γH2AX interactions. We conclude that tumor-specific delivery of radiolabeled antibodies directed against intranuclear epitopes is possible using cleavable antibody-peptide conjugates.

Abstract 639: Repair of DNA double strand breaks by 20-1000 bp “patches” derived from distant genomic regions.

Abstract It has been suggested that errors in the repair of DNA double strand breaks (DSB) can result in gross chromosomal rearrangements (GCR). To study DNA DSB repair in vivo, we generated a vector with the EF1a promoter driving expression of the herpes simplex thymidine kinase (HsTK); interspersed between the EF1a promoter and Hstk was the recognition site for the meganuclease I-SceI. The EF1aTk vector was introduced into U937 cells and a clone (designated F5) with a single copy of the vector was isolated. Transfection of F5 cells with an I-SceI expression vector, followed by ganciclovir (GCV) selection, identified clones that lost expression of HsTK. We recovered no GCR, as most of the GCV-resistant clones had deletions of the EF1a promoter or the HsTK. However ∼10% of the clones had insertions (47-756 bp) of DNA sequences derived from distant regions of the genome; all inserted fragments were derived from gene and/or retrotransposon repeat elements such as LINE or SINE sequences. Therefore, we hypothesized that the inserted sequences used to “patch” the DNA DSB could be based on reverse transcription of an RNA template. Since sequences derived from human RNA and human genomic DNA are identical (with the exception of RNA splice events, poly-A tails, and RNA-edited nucleotides), we co-transfected the F5 cells with murine RNA and an I-SceI expression vector to determine if patches at the DNA DSB sites could be derived from RNA. We identified 121 independent sequences which represented insertions of 20-609 bp at the I-SceI cleavage site. Of these 121 insertions, 6 were vector or E. coli capture events and 7 were too short to identify. The chromosomal origin of 5 insertions could not be assigned as these insertions consisted of 5 or 6 bp tandem repeat sequences; 3 had a (CCCTAA)n repeat, identical to a mammalian telomere repeat. Of the remaining 103 insertions, 94 were derived from a single genomic loci, and 9 samples contained sequences from more than 2 distinct genomic regions. The sequences were derived from 22 of the 24 human chromosomes. 65% of the sequences were found to contain sequences from a transcribed gene region, and 52% contained repeat sequences such as LINE, SINE, or LTR. The involvement of retrotransposon sequences, which are known to be reverse transcribed and integrated into the genome, supports the hypothesis that at least some of the insertions may be templated from RNA. Although most insertions were derived from human sequences; 9 insertions matched murine sequences, and were derived from the co-transfected mouse RNA. All of these were derived from gene and/or retrotransposon sequence. Taken together, these findings demonstrate that I-SceI-induced DNA DSB can be repaired by “patches” derived from distant regions of the genome, and suggest that transcribed mRNA and retrotransposons can play an important role in the repair of DNA DSB and preservation of genomic integrity. Citation Format: Masahiro Onozawa, Tamas Varga, Zhenhua Zhang, Yoo Jung Kim, Peter Aplan. Repair of DNA double strand breaks by 20-1000 bp “patches” derived from distant genomic regions. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 639. doi:10.1158/1538-7445.AM2013-639

Repair of DNA Double Strand Breaks by RNA/DNA Patches in U937 Cells.

Abstract 2375It has been suggested that errors in the repair of DNA double strand breaks (DSB) can result in gross chromosomal rearrangements (GCR), including chromosomal amplifications, deletions, inversions, and translocations. To study repair of DNA DSB in vivo, we generated a vector containing the EF1a promoter driving expression of the herpes simplex thymidine kinase (HsTK); interspersed between the EF1a promoter and Hstk cDNA was the recognition site for the rare-cutting meganuclease I-SceI. This vector was electroporated into U937 cells, and a clone containing a single copy, of the EF1aTK vector, designated F5, was isolated. Transfection of the F5 cells with an I-SceI expression vector, followed by ganciclovir selection, identified clones that had lost expression of HsTK. We recovered no GCR with this approach; most of the GCV-resistant clones had partial or complete deletions of the EF1a promoter, the HsTK cDNA, or both. However we noted that ∼5–10% of repair elements involved insertions of DNA sequences derived from distant regions of the genome; the length of the inserted fragment varied from 47 to 756 bp. Surprisingly, all of the inserted fragments were derived from gene and/or retrotransposon repeat elements such as LINE (Long Interspersed Nuclear Element) or SINE (Short Interspersed Nuclear Element) sequences. Therefore, we hypothesized that the inserted sequences used to “patch” the DNA DSB could be based on reverse transcription of an RNA template. Since sequences derived from human RNA and human genomic DNA are identical (with the exception of RNA splice events, poly-A tails, and RNA-edited nucleotides), we co-transfected the F5 cells with murine RNA and an I-SceI expression vector to test the hypothesis that the patches at the DNA DSB sites could be derived from RNA. After hygromycin selection for successfully transfected cells, genomic DNA was isolated, and amplified with primers that flanked the I-SceI cleavage site. DNA fragments of 500–1000bp (larger than the size of the uncleaved EF!aTK PCR product, which was 400 bp) containing insertions at the I-SceI site were isolated, subcloned, and sequenced. We identified 51 independent sequences which had insertions of 23–266 bp at I-SceI cleavage site. Of these 51 insertions, 4 were vector capture events (derived form the expression vector), and 4 were too short to identify unambiguously. Of the remaining 43 insertions, 39 were derived from a single genomic loci, and 4 samples contained identifiable sequences from 2 distinct genomic regions. The sequences were derived from 16 of the 24 human chromosomes, with no clear preference for any specific chromosome. 62% of the sequences were found to contain sequences from a transcribed gene region, and 64% contained repeat sequences such as LINE, SINE, or LTR. The involvement of retrotransposon sequences, which are known to be reverse transcribed and integrated into the genome, supports the hypothesis that at least some of the insertions may be templated from RNA. Furthermore, 45 of the 47 inserted sequences were derived from endogenous human sequences; however 2 insertions matched to mouse sequences, and were derived from the co-transfected mouse RNA. One sample matched an intronic region of murine Vwa3b, which we confirmed was highly expressed in the mouse cells used to harvest the RNA used for the co-transfection. A second sample was not a gene sequence but contained a LINE element.Taken together, these findings demonstrate that I-SceI-induced DNA DSB can be repaired by “patches” derived from distant regions of the genome. This is the first systematic study focused on captured sequences at DNA DSB sites in the mammalian genome, and suggests that transcribed mRNA and retrotransposons can play an important role in the repair of DNA DSB and preservation of genomic integrity. Finally, the observation that I-SceI-induced DNA DSBs are often repaired by insertions suggests that these insertions could be mistaken for chromosome translocations, if only one “side” of the DNA DSB is sequenced. Disclosures:No relevant conflicts of interest to declare.

NBN Phosphorylation regulates the accumulation of MRN and ATM at sites of DNA double-strand breaks

In response to ionizing radiation, the MRE11/RAD50/NBN (MRN) complex re-distributes to the sites of DNA double strand breaks (DSBs) where each of its individual components is phosphorylated by the serine-threonine kinase, ATM. ATM phosphorylation of NBN is required for activation of the S-phase checkpoint, but the mechanism whereby these phosphorylation events signal the checkpoint machinery remains unexplained. Here, we describe the use of direct protein transduction of the homing endonuclease, I-PpoI, into human cells to generate site-specific DSBs. Direct transduction of I-PpoI protein results in rapid accumulation and turnover of the endonuclease in live cells, facilitating comparisons across multiple cell lines. We demonstrate the utility of this system by introducing I-PpoI into isogenic cell lines carrying mutations at the ATM phosphorylation sites in NBN and assaying the effects of these mutations on the spatial distribution and temporal accumulation of NBN and ATM at DSBs by chromatin immunoprecipitation, as well as timing and extent of DSB repair. Although the spatial distribution of NBN and ATM recruited to the sites of DSBs was comparable between control cells and those expressing phosphorylation mutants of NBN, the timing of accumulation of NBN and ATM was altered. Serine to alanine mutations that blocked phosphorylation resulted in delayed recruitment of both NBN and ATM to DSBs. Serine to glutamic acid substitutions that mimicked the phosphorylation event resulted in both increased and prolonged accumulation of both NBN and ATM at DSBs. The repair of DSBs in cells lacking full-length NBN was significantly delayed compared to control cells while blocking phosphorylation of NBN resulted in a more modest delay in repair. These data indicate that following the induction of DSBs, phosphorylation of NBN regulates its accumulation, and that of ATM, at sites of DNA DSB as well as the timing of the repair of these sites.

Sp1 Facilitates DNA Double-Strand Break Repair through a Nontranscriptional Mechanism

Sp1 is a ubiquitously expressed transcription factor that is phosphorylated by ataxia telangiectasia mutated kinase (ATM) in response to ionizing radiation and H(2)O(2). Here, we show by indirect immunofluorescence that Sp1 phosphorylated on serine 101 (pSp1) localizes to ionizing radiation-induced foci with phosphorylated histone variant γH2Ax and members of the MRN (Mre11, Rad50, and Nbs1) complex. More precise analysis of occupancy of DNA double-strand breaks (DSBs) by chromatin immunoprecipitation (ChIP) shows that Sp1, like Nbs1, resides within 200 bp of DSBs. Using laser microirradiation of cells, we demonstrate that pSp1 is present at DNA DSBs by 7.5 min after induction of damage and remains at the break site for at least 8 h. Depletion of Sp1 inhibits repair of site-specific DNA breaks, and the N-terminal 182-amino-acid peptide, which contains targets of ATM kinase but lacks the zinc finger DNA binding domain, is phosphorylated, localizes to DSBs, and rescues the repair defect resulting from Sp1 depletion. Together, these data demonstrate that Sp1 is rapidly recruited to the region immediately adjacent to sites of DNA DSBs and is required for DSB repair, through a mechanism independent of its sequence-directed transcriptional effects.

Mutational showers during carcinogenesis

Mutational showers during carcinogenesis

Bi-directional routing of DNA mismatch repair protein human exonuclease 1 to replication foci and DNA double strand breaks

Human exonuclease 1 (hEXO1) is implicated in DNA metabolism, including replication, recombination and repair, substantiated by its interactions with PCNA, DNA helicases BLM and WRN, and several DNA mismatch repair (MMR) proteins. We investigated the sub-nuclear localization of hEXO1 during S-phase progression and in response to laser-induced DNA double strand breaks (DSBs). We show that hEXO1 and PCNA co-localize in replication foci. This apparent interaction is sustained throughout S-phase. We also demonstrate that hEXO1 is rapidly recruited to DNA DSBs. We have identified a PCNA interacting protein (PIP-box) region on hEXO1 located in its COOH-terminal ( 788QIKLNELW 795). This motif is essential for PCNA binding and co-localization during S-phase. Recruitment of hEXO1 to DNA DSB sites is dependent on the MMR protein hMLH1. We show that two distinct hMLH1 interaction regions of hEXO1 (residues 390–490 and 787–846) are required to direct the protein to the DNA damage site. Our results reveal that protein domains in hEXO1 in conjunction with specific protein interactions control bi-directional routing of hEXO1 between on-going DNA replication and repair processes in living cells.

TopBP1 functions with 53BP1 in the G1 DNA damage checkpoint

TopBP1 is a checkpoint protein that colocalizes with ATR at sites of DNA replication stress. In this study, we show that TopBP1 also colocalizes with 53BP1 at sites of DNA double-strand breaks (DSBs), but only in the G1-phase of the cell cycle. Recruitment of TopBP1 to sites of DNA replication stress was dependent on BRCT domains 1-2 and 7-8, whereas recruitment to sites of DNA DSBs was dependent on BRCT domains 1-2 and 4-5. The BRCT domains 4-5 interacted with 53BP1 and recruitment of TopBP1 to sites of DNA DSBs in G1 was dependent on 53BP1. As TopBP1 contains a domain important for ATR activation, we examined whether it contributes to the G1 cell cycle checkpoint. By monitoring the entry of irradiated G1 cells into S-phase, we observed a checkpoint defect after siRNA-mediated depletion of TopBP1, 53BP1 or ATM. Thus, TopBP1 may mediate the checkpoint function of 53BP1 in G1.

An Oligomerized 53BP1 Tudor Domain Suffices for Recognition of DNA Double-Strand Breaks

53BP1, the vertebrate ortholog of the budding yeast Rad9 and fission yeast Crb2/Rhp9 checkpoint proteins, is recruited rapidly to sites of DNA double-strand breaks (DSBs). A tandem tudor domain in human 53BP1 that recognizes methylated residues in the histone core is necessary, but not sufficient, for efficient recruitment. By analysis of deletion mutants, we identify here additional elements in 53BP1 that facilitate recognition of DNA DSBs. The first element corresponds to an independently folding oligomerization domain. Replacement of this domain with heterologous tetramerization domains preserves the ability of 53BP1 to recognize DNA DSBs. A second element is only about 15 amino acids long and appears to be a C-terminal extension of the tudor domain, rather than an independently functioning domain. Recruitment of 53BP1 to sites of DNA DSBs is facilitated by histone H2AX phosphorylation and ubiquitination. However, none of the 53BP1 domains/elements important for recruitment are known to bind phosphopeptides or ubiquitin, suggesting that histone phosphorylation and ubiquitination regulate 53BP1 recruitment to sites of DNA DSBs indirectly.

Constitutive phosphorylation of MDC1 physically links the MRE11–RAD50–NBS1 complex to damaged chromatin

The MRE11–RAD50–Nijmegen breakage syndrome 1 (NBS1 [MRN]) complex accumulates at sites of DNA double-strand breaks (DSBs) in microscopically discernible nuclear foci. Focus formation by the MRN complex is dependent on MDC1, a large nuclear protein that directly interacts with phosphorylated H2AX. In this study, we identified a region in MDC1 that is essential for the focal accumulation of the MRN complex at sites of DNA damage. This region contains multiple conserved acidic sequence motifs that are constitutively phosphorylated in vivo. We show that these motifs are efficiently phosphorylated by caseine kinase 2 (CK2) in vitro and directly interact with the N-terminal forkhead-associated domain of NBS1 in a phosphorylation-dependent manner. Mutation of these conserved motifs in MDC1 or depletion of CK2 by small interfering RNA disrupts the interaction between MDC1 and NBS1 and abrogates accumulation of the MRN complex at sites of DNA DSBs in vivo. Thus, our data reveal the mechanism by which MDC1 physically couples the MRN complex to damaged chromatin.

Valosin-Containing Protein Phosphorylation at Ser784 in Response to DNA Damage

The response of eukaryotic cells to DNA damage includes the activation of phosphatidylinositol-3 kinase-related kinases (PIKK), such as ATM, ATR, and DNA-dependent protein kinase (DNA-PK). These three kinases have very similar substrate specificities in vitro, but in vivo, their substrates overlap only partially. Several in vivo substrates of ATM and ATR have been identified and almost all of them are involved in DNA damage-induced cell cycle arrest and/or apoptosis. In contrast, few in vivo substrates of DNA-PK have been identified. These include histone H2AX and DNA-PK itself. We identify here valosin-containing protein (VCP) as a novel substrate of DNA-PK and other PIKK family members. VCP is phosphorylated at Ser784 within its COOH terminus, a region previously shown to target VCP to specific intracellular compartments. Furthermore, VCP phosphorylated at Ser784 accumulated at sites of DNA double-strand breaks (DSBs). VCP is a protein chaperone that unfolds and translocates proteins. Its phosphorylation in response to DNA damage and its recruitment to sites of DNA DSBs could indicate a role of VCP in DNA repair.