Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P16104 (H2AX)
 3,930 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:An oligomerized 53BP1 tudor domain suffices for recognition of DNA double-strand breaks. 1906 41

During meiosis in male mammals, X and Y chromosomes undergo the process of meiotic sex chromosome inactivation (MSCI). A crucial role in MSCI has recently been reported for BRCA1, ATR kinase, and phosphorylated histone H2AX, but the exact mechanism remains to be determined. Small ubiquitin-like modifier (SUMO) proteins have recently been shown to localize to the sex body in mouse meiotic spermatocytes, but the role they play during MSCI is unknown. In this study, in order to better understand the molecular events of MSCI, we followed dynamic changes in gammaH2AX and SUMO localization patterns during MSCI. Using confocal laser scanning microscopy (CLSM) as an analytical tool for visualizing numerous spermatocytes from the same development stage and for consecutively following the meiotic progression, we were able to demonstrate a very early appearance of SUMO-1, which preceded gammaH2AX accumulation on the sex chromosomes during their meiotic inactivation. In contrast to SUMO-1, SUMO-2/3 was undetectable in zygotene spermatocytes, suggesting a possible specific role for SUMO-1 in the initiation of MSCI.
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PMID:Sumoylation precedes accumulation of phosphorylated H2AX on sex chromosomes during their meiotic inactivation. 1915 30

DNA double-strand breaks (DSBs) not only interrupt the genetic information, but also disrupt the chromatin structure, and both impairments require repair mechanisms to ensure genome integrity. We showed previously that RNF8-mediated chromatin ubiquitylation protects genome integrity by promoting the accumulation of repair factors at DSBs. Here, we provide evidence that, while RNF8 is necessary to trigger the DSB-associated ubiquitylations, it is not sufficient to sustain conjugated ubiquitin in this compartment. We identified RNF168 as a novel chromatin-associated ubiquitin ligase with an ability to bind ubiquitin. We show that RNF168 interacts with ubiquitylated H2A, assembles at DSBs in an RNF8-dependent manner, and, by targeting H2A and H2AX, amplifies local concentration of lysine 63-linked ubiquitin conjugates to the threshold required for retention of 53BP1 and BRCA1. Thus, RNF168 defines a new pathway involving sequential ubiquitylations on damaged chromosomes and uncovers a functional cooperation between E3 ligases in genome maintenance.
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PMID:RNF168 binds and amplifies ubiquitin conjugates on damaged chromosomes to allow accumulation of repair proteins. 1920 79

The modification of eukaryotic proteins by covalent attachment of ubiquitin is a versatile signaling event with a wide range of possible consequences. Canonical poly-ubiquitination by Lys-48 linked chains usually destines a protein for degradation by the proteasome. By contrast, attachment of a single ubiquitin or ubiquitin chains linked through Lys-63 or Lys-6 serves a non-proteolytic role. Over the last years, evidence has accumulated that several nuclear proteins become ubiquitinated in response to DNA damage. Typically, these proteins carry mono-ubiquitin or non-classical ubiquitin chains and are localized close to the site of DNA damage. Of particular interest are PCNA and the variant histone H2AX, two key proteins whose ubiquitination serves to recruit factors needed by the cell to cope with the damage. A prerequisite for docking effector proteins to the site of the lesion is the detection of a specific ubiquitin modification, a process that can be mediated by a range of dedicated ubiquitin-binding domains (UBDs). As the same types of ubiquitin modification are involved in entirely different processes, the recognition of the ubiquitin mark has to go along with the recognition of the modified protein. Thus, ubiquitin-binding domains gain their specificity through combination with other recognition domains and motifs. This review discusses ubiquitin-binding domains relevant to the DNA damage response, including their binding mode, their specificity, and their interdependence with other factors. For several repair pathways, current knowledge of the events downstream of the ubiquitin mark is sketchy. A closer look at orphan UBD proteins might lead to the identification of missing pieces in the DNA response puzzle.
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PMID:Ubiquitin-binding domains and their role in the DNA damage response. 1921 13

Reversible topoisomerase I (Top1)-DNA cleavage complexes are the key DNA lesion induced by anticancer camptothecins (CPTs) (e.g. topotecan and irinotecan) as well as structurally perturbed DNAs (e.g. oxidatively damaged, UV-irradiated, or alkylated DNA). It has been proposed that Top1 cleavage complexes arrest advancing replication forks, triggering the formation of DNA double strand breaks (DSBs) because of replication fork runoff at the Top1 cleavage complex sites on the leading strand. In this study, we show that the formation of replication-dependent DSBs requires the ubiquitin-proteasome pathway in CPT-treated cells. First, the proteasome inhibitor MG-132 specifically inhibited CPT-induced but not ionizing radiation- or hydroxyurea-induced DSBs as revealed by both the neutral comet assay and measurements of the specific DNA damage signals (e.g. gamma-H2AX, phosphorylated ataxia telangiectasia mutated (Ser-1981), and phosphorylated Chk2 (Ser-33/35)) that are characteristic for DSBs. Knocking down the 20 S proteasome maturation protein also supported the requirement of the proteasome activity for CPT-induced DSBs. Second, CPT-induced DSB signals were shown to require ubiquitin, ubiquitin-activating enzyme (E1), a CUL-3-based ubiquitin ligase (E3), and the formation of Lys-48-linked polyubiquitin chains on Top1. Third, immunocytochemical studies revealed that the CPT-induced formation of gamma-H2AX foci occurred at the replication forks and was attenuated by co-treatment with the proteasome inhibitor MG-132. In the aggregate, these results support a replication fork collision model in which Top1 cleavage complexes at the arrested replication forks are degraded by proteasome prior to replication fork runoff on the leading strand to generate DSBs.
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PMID:Proteasome-dependent processing of topoisomerase I-DNA adducts into DNA double strand breaks at arrested replication forks. 1966 69

Rearranged Ig V regions undergo activation-induced cytidine deaminase (AID)-initiated diversification in sequence to produce either nontemplated or templated mutations, in the related pathways of somatic hypermutation and gene conversion. In chicken DT40 B cells, gene conversion normally predominates, producing mutations templated by adjacent pseudo-V regions, but impairment of gene conversion switches mutagenesis to a nontemplated pathway. We recently showed that the activator, E2A, functions in cis to promote diversification, and that G(1) phase of cell cycle is the critical window for E2A action. By single-cell imaging of stable AID-yellow fluorescent protein transfectants, we now demonstrate that AID-yellow fluorescent protein can stably localize to the nucleus in G(1) phase, but undergoes ubiquitin-dependent proteolysis later in cell cycle. By imaging of DT40 polymerized lactose operator-lambda(R) cells, in which polymerized lactose operator tags the rearranged lambda(R) gene, we show that both the repair polymerase Poleta and the multifunctional factor MRE11/RAD50/NBS1 localize to lambda(R), and that lambda(R)/Poleta colocalizations occur predominately in G(1) phase, when they reflect repair of AID-initiated damage. We find no evidence of induction of gamma-H2AX, the phosphorylated variant histone that is a marker of double-strand breaks, and Ig gene conversion may therefore proceed by a pathway involving templated repair at DNA nicks rather than double-strand breaks. These results lead to a model in which Ig gene conversion initiates and is completed or nearly completed in G(1) phase. AID deaminates ssDNA, and restriction of mutagenesis to G(1) phase would contribute to protecting the genome from off-target attack by AID when DNA replication occurs in S phase.
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PMID:Temporal regulation of Ig gene diversification revealed by single-cell imaging. 1974 85

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 gamma-H2AX foci. Because the increased gamma-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 gamma-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.
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PMID:SUMO modification regulates BLM and RAD51 interaction at damaged replication forks. 1995 65

DNA double-strand breaks (DSBs) are highly cytotoxic lesions that are generated by ionizing radiation and various DNA-damaging chemicals. Following DSB formation, cells activate the DNA-damage response (DDR) protein kinases ATM, ATR and DNA-PK (also known as PRKDC). These then trigger histone H2AX (also known as H2AFX) phosphorylation and the accumulation of proteins such as MDC1, 53BP1 (also known as TP53BP1), BRCA1, CtIP (also known as RBBP8), RNF8 and RNF168/RIDDLIN into ionizing radiation-induced foci (IRIF) that amplify DSB signalling and promote DSB repair. Attachment of small ubiquitin-related modifier (SUMO) to target proteins controls diverse cellular functions. Here, we show that SUMO1, SUMO2 and SUMO3 accumulate at DSB sites in mammalian cells, with SUMO1 and SUMO2/3 accrual requiring the E3 ligase enzymes PIAS4 and PIAS1. We also establish that PIAS1 and PIAS4 are recruited to damage sites via mechanisms requiring their SAP domains, and are needed for the productive association of 53BP1, BRCA1 and RNF168 with such regions. Furthermore, we show that PIAS1 and PIAS4 promote DSB repair and confer ionizing radiation resistance. Finally, we establish that PIAS1 and PIAS4 are required for effective ubiquitin-adduct formation mediated by RNF8, RNF168 and BRCA1 at sites of DNA damage. These findings thus identify PIAS1 and PIAS4 as components of the DDR and reveal how protein recruitment to DSB sites is controlled by coordinated SUMOylation and ubiquitylation.
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PMID:Mammalian SUMO E3-ligases PIAS1 and PIAS4 promote responses to DNA double-strand breaks. 2001 86

Gastrointestinal stromal tumors (GIST) are caused by activating mutations in the KIT or PDGFRA receptor tyrosine kinase genes. Although >85% of GIST patients treated with the small-molecule inhibitor imatinib mesylate (Gleevec) achieve disease stabilization, complete remissions are rare and a substantial proportion of patients develop resistance to imatinib over time. Upregulation of soluble, non-chromatin-bound histone H2AX has an important role in imatinib-induced apoptosis of GIST cells. Additionally, H2AX levels in untreated GIST are maintained at low levels by a pathway that involves KIT, phosphoinositide 3-kinase, and the ubiquitin-proteasome system. In this study, we asked whether bortezomib-mediated inhibition of the ubiquitin-proteasome machinery could lead to upregulation of histone H2AX and GIST cell death. We show that bortezomib rapidly triggers apoptosis in GIST cells through a combination of mechanisms involving H2AX upregulation and loss of KIT protein expression. Downregulation of KIT transcription was an underlying mechanism for bortezomib-mediated inhibition of KIT expression. In contrast, the nuclear factor-kappaB signaling pathway did not seem to play a major role in bortezomib-induced GIST cell death. Significantly, we found that bortezomib would induce apoptosis in two imatinib-resistant GIST cell lines as well as a short-term culture established from a primary imatinib-resistant GIST. Collectively, our results provide a rationale to test the efficacy of bortezomib in GIST patients with imatinib-sensitive or -resistant tumors.
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PMID:Proapoptotic activity of bortezomib in gastrointestinal stromal tumor cells. 2002 60

HIV-1 viral protein R (Vpr) induces cell cycle arrest at the G(2)/M phase by a mechanism involving the activation of the DNA damage sensor ATR. We and others recently showed that Vpr performs this function by subverting the activity of the DDB1-CUL4A (VPRBP) E3 ubiquitin ligase. Vpr could thus act as a connector between the E3 ligase and an unknown cellular factor whose ubiquitination would induce G(2) arrest. While attractive, this model is based solely on the indirect observation that some mutants of Vpr retain their interaction with the E3 ligase but fail to induce G(2) arrest. Using a tandem affinity purification approach, we observed that Vpr interacts with ubiquitinated cellular proteins and that this association requires the recruitment of an active E3 ligase given that the depletion of VPRBP by RNA interference or the overexpression of a dominant negative mutant of CUL4A decreased this association. Importantly, G(2)-arrest-defective mutants of Vpr in the C-terminal putative substrate-interacting domain displayed a decreased association with ubiquitinated proteins. We also found that the inhibition of proteasomal activity increased this association and that the ubiquitin chains were at least in part constituted of classical K48 linkages. Interestingly, the inhibition of K48 polyubiquitination specifically impaired the Vpr-induced phosphorylation of H2AX, an early target of ATR, but did not affect UV-induced H2AX phosphorylation. Overall, our results provide direct evidence that the association of Vpr with the DDB1-CUL4A (VPRBP) E3 ubiquitin ligase induces the K48-linked polyubiquitination of as-yet-unknown cellular proteins, resulting in their proteasomal degradation and ultimately leading to the activation of ATR and G(2) arrest.
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PMID:HIV-1 Vpr induces the K48-linked polyubiquitination and proteasomal degradation of target cellular proteins to activate ATR and promote G2 arrest. 2008 62


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