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)

The mouse meiotic mutant Mei1 was isolated in a screen for infertile mice descended from chemically mutagenized embryonic stem cells. Homozygotes of both sexes are sterile due to meiotic arrest caused by defects in chromosome synapsis. Notably, RAD51 protein does not load onto Mei1 mutant meiotic chromosomes, suggesting that there is a defect in either recombinational repair or the production of double-strand breaks (DSBs) that require such repair. Here, we show that treatment of mutant males with cisplatin restores RAD51 loading, suggesting that mutant spermatocytes have intact recombinational repair mechanisms. Levels of histone H2AX phosphorylation (gammaH2AX) at leptonema are significantly reduced compared with wild-type controls but comparable to that seen in animals deficient for SPO11, the molecule required for catalyzing DSB formation during meiosis. These observations provide evidence that genetically programmed DSB induction is defective in Mei1 leptotene spermatocytes. We also report the positional cloning of Mei1, which encodes a product without significant homology to any known protein. Expressed almost exclusively in gonads, Mei1 has no apparent homologs in yeast, worms, or flies. However, Mei1 orthologs are present in the genomes of mammals, chickens, and zebrafish. Thus, Mei1 is required for vertebrate meiosis. To our knowledge, Mei1 is the first meiosis-specific mutation identified by forward genetic approaches in mammals.
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PMID:Positional cloning and characterization of Mei1, a vertebrate-specific gene required for normal meiotic chromosome synapsis in mice. 1467 27

Bloom's syndrome (BS) is a human genetic disorder associated with cancer predisposition. The BS gene product, BLM, is a member of the RecQ helicase family, which is required for the maintenance of genome stability in all organisms. In budding and fission yeasts, loss of RecQ helicase function confers sensitivity to inhibitors of DNA replication, such as hydroxyurea (HU), by failure to execute normal cell cycle progression following recovery from such an S-phase arrest. We have examined the role of the human BLM protein in recovery from S-phase arrest mediated by HU and have probed whether the stress-activated ATR kinase, which functions in checkpoint signaling during S-phase arrest, plays a role in the regulation of BLM function. We show that, consistent with a role for BLM in protection of human cells against the toxicity associated with arrest of DNA replication, BS cells are hypersensitive to HU. BLM physically associates with ATR (ataxia telangiectasia and rad3(+) related) protein and is phosphorylated on two residues in the N-terminal domain, Thr-99 and Thr-122, by this kinase. Moreover, BS cells ectopically expressing a BLM protein containing phosphorylation-resistant T99A/T122A substitutions fail to adequately recover from an HU-induced replication blockade, and the cells subsequently arrest at a caffeine-sensitive G(2)/M checkpoint. These abnormalities are not associated with a failure of the BLM-T99A/T122A protein to localize to replication foci or to colocalize either with ATR itself or with other proteins that are required for response to DNA damage, such as phosphorylated histone H2AX and RAD51. Our data indicate that RecQ helicases play a conserved role in recovery from perturbations in DNA replication and are consistent with a model in which RecQ helicases act to restore productive DNA replication following S-phase arrest and hence prevent subsequent genomic instability.
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PMID:Phosphorylation of the Bloom's syndrome helicase and its role in recovery from S-phase arrest. 1472 72

Poly(ADP-ribose) polymerase (PARP1) facilitates DNA repair by binding to DNA breaks and attracting DNA repair proteins to the site of damage. Nevertheless, PARP1-/- mice are viable, fertile and do not develop early onset tumours. Here, we show that PARP inhibitors trigger gamma-H2AX and RAD51 foci formation. We propose that, in the absence of PARP1, spontaneous single-strand breaks collapse replication forks and trigger homologous recombination for repair. Furthermore, we show that BRCA2-deficient cells, as a result of their deficiency in homologous recombination, are acutely sensitive to PARP inhibitors, presumably because resultant collapsed replication forks are no longer repaired. Thus, PARP1 activity is essential in homologous recombination-deficient BRCA2 mutant cells. We exploit this requirement in order to kill BRCA2-deficient tumours by PARP inhibition alone. Treatment with PARP inhibitors is likely to be highly tumour specific, because only the tumours (which are BRCA2-/-) in BRCA2+/- patients are defective in homologous recombination. The use of an inhibitor of a DNA repair enzyme alone to selectively kill a tumour, in the absence of an exogenous DNA-damaging agent, represents a new concept in cancer treatment.
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PMID:Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase. 1582 66

Homologous recombination is an important mechanism in DNA replication to ensure faithful DNA synthesis and genomic stability. In this study, we investigated the role of XRCC2, a member of the RAD51 paralog family, in cellular recovery from replication arrest via homologous recombination. The protein expression of XRCC2, as well as its binding partner RAD51D, is dramatically increased in S- and G2-phases, suggesting that these proteins function during and after DNA synthesis. XRCC2 mutant irs1 cells exhibit hypersensitivity to hydroxyurea (HU) and are defective in the induction of RAD51 foci after HU treatment. In addition, the HU-induced chromatin association of RAD51 is deficient in irs1 mutant. Interestingly, irs1 cells are only slightly sensitive to thymidine and able to form intact RAD51 foci in S-phase cells arrested with thymidine. Irs1 cells showed increased level of chromatin-bound RAD51 as well as the wild type cells after thymidine treatment. Both HU and thymidine induce gamma-H2AX foci in arrested S-phase nuclei. These results suggest that XRCC2 is involved in repair of HU-induced damage, but not thymidine-induced damage, at the stalled replication forks. Our data suggest that there are at least two sub-pathways in homologous recombination, XRCC2-dependent and -independent, for repair of stalled replication forks and assembly of RAD51 foci following replication arrest in S-phase.
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PMID:Differential roles of XRCC2 in homologous recombinational repair of stalled replication forks. 1586 95

The Mei1(m1Jcs) allele contains a point mutation in a novel gene required for normal meiosis in male and female mice. We previously hypothesized that Mei1 is likely required for the formation of genetically programmed double-strand breaks (DSBs), the initiating event of meiotic recombination because in mutant spermatocytes (1) RAD51 foci are greatly reduced at zygonema; (2) RAD51 foci can be restored by cisplatin-induced DNA damage; and (3) phosphorylated H2AX is greatly reduced at leptonema. If this hypothesis is correct, Mei1 would act upstream of genes required for repair of DSBs by homologous recombination. To test this, we examined meiosis in Mei(m1Jcs)/Mei1(m1Jcs) (Mei1(-/-)) and Dmc1(tm1Jcs)/Dmc1(tm1Jcs) (Dmc1(-/-)) mice and mice homozygous at both loci (Dmc1(-/-) Mei1(-/-)), exploiting the fact that oogenesis is much more severely affected by the absence of DMC1 than by the absence of MEI1. The phenotypes of both male and female double mutants were identical to that of Mei1(-/-) animals. Therefore, Mei1 can be positioned upstream of Dmc1 in the genetic pathway that operates during mammalian meiosis. Furthermore, this epistatic interaction provides additional evidence in support of the hypothesis that Mei1 is required for the initiating events of meiotic recombination.
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PMID:Mei1 is epistatic to Dmc1 during mouse meiosis. 1592 51

The relationship between meiotic recombination events and different patterns of pairing and synapsis has been analysed in prophase I spermatocytes of the grasshopper Stethophyma grossum, which exhibit very unusual meiotic characteristics, namely (1) the three shortest bivalents achieve full synapsis and do not show chiasma localisation; (2) the remaining eight bivalents show restricted synapsis and proximal chiasma localisation, and (3) the X chromosome remains unsynapsed. We have studied by means of immunofluorescence the localisation of the phosphorylated histone H2AX (gamma-H2AX), which marks the sites of double-strand breaks; the SMC3 cohesin subunit, which is thought to have a close relationship to the development of the axial element (a synaptonemal complex component); and the recombinase RAD51. We observed a marked nuclear polarization of both the maturation of SMC3 cohesin axis and the ulterior appearance of gamma-H2AX and RAD51 foci, these being exclusively restricted to those chromosomal regions that first form cohesin axis stretches. This polarised distribution of recombination events is maintained throughout prophase I over those autosomal regions that are undergoing, or about to undergo, synapsis. We propose that the restricted distribution of recombination events along the chromosomal axes in the spermatocytes is responsible for the incomplete presynaptic homologous alignment and, hence, for the partial synaptonemal complex formation displayed by most bivalents.
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PMID:DNA double-strand breaks and homology search: inferences from a species with incomplete pairing and synapsis. 1597 53

Observation and counting of gamma-H2AX foci in untreated cells as well as in cells exposed to cytotoxic agents is a widely used method for documenting the presence of double-strand breaks (DSBs) in the DNA and for analysis of their repair. Similar methods are employed to analyze formation of foci by a variety of proteins implicated in the cellular responses to DNA damage. Despite the wide application of the approach, the manual counting that is frequently used is prone to inaccuracies and investigator-related biases and artifacts. To alleviate this limitation, we developed and describe here personal computer-based algorithms, operating as utilities on available software, that allow an objective and quantitative analysis of foci from confocal images. The algorithms allow focus counting as well as size definition and correct for focus coincidence due to the overlap normally occurring with an increasing number of foci per nucleus. Furthermore, the software allows measurement of the integrated optical density (IOD) of each individual focus, which enables analysis of properties of foci as a function of time. Finally, the information generated by the above analysis algorithms can be employed to evaluate colocalization between foci formed by different proteins. A validation of the software is presented for radiation-induced gamma-H2AX foci in three widely used human cell lines and colocalization tested with RAD51 and gamma-H2AX foci. The computational methods presented extend to images generated by digital cameras.
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PMID:Computational Methods for analysis of foci: validation for radiation-induced gamma-H2AX foci in human cells. 1639 69

DNA repair by homologous recombination is essential for preserving genomic integrity. The RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3) play important roles in this process. In this study, we show that human RAD51 interacts with RAD51C-XRCC3 or RAD51B-C-D-XRCC2. In addition to being critical for RAD51 focus formation, RAD51C localizes to DNA damage sites. Inhibition of RAD51C results in a decrease in cellular proliferation consistent with a role in repairing double-strand breaks (DSBs) that occur naturally. To monitor a single DNA repair event, we developed immunofluorescence and chromatin immunoprecipitation (ChIP) methods on human cells where a unique DSB can be created in vivo. Using this system, we observed a single focus of RAD51C, RAD51 and 53BP1, which colocalized with gamma-H2AX. ChIPs revealed that endogenous human RAD51, RAD51C, RAD51D, XRCC2, XRCC3 and MRE11 proteins are recruited in the S-G2 phase of the cell cycle, while Ku80 is recruited during G1. We propose that RAD51C ensures a tight regulation of RAD51 assembly during DSB repair and plays a direct role in repairing DSBs in vivo.
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PMID:Interplay between human DNA repair proteins at a unique double-strand break in vivo. 1639 35

Spermatocytes normally sustain many meiotically induced double-strand DNA breaks (DSBs) early in meiotic prophase; in autosomal chromatin, these are repaired by initiation of meiotic homologous-recombination processes. Little is known about how spermatocytes respond to environmentally induced DNA damage after recombination-related DSBs have been repaired. The experiments described here tested the hypothesis that, even though actively completing meiotic recombination, pachytene spermatocytes cultured in the absence of testicular somatic cells initiate appropriate chromatin remodeling and cell-cycle responses to environmentally induced DNA damage. Two DNA-damaging agents were employed for in vitro treatment of pachytene spermatocytes: gamma-irradiation and etoposide, a topoisomerase II (TOP2) inhibitor that results in persistent unligated DSBs. Chromatin modifications associated with DSBs were monitored after exposure by labeling surface-spread chromatin with antibodies against RAD51 (which recognizes DSBs) and the phosphorylated variant of histone H2AFX (herein designated by its commonly used symbol, H2AX), gammaH2AX (which modifies chromatin associated with DSBs). Both gammaH2AX and RAD51 were rapidly recruited to irradiation- or etoposide-damaged chromatin. These chromatin modifications imply that spermatocytes recruit active DNA damage responses, even after recombination is substantially completed. Furthermore, irradiation-induced DNA damage inhibited okadaic acid-induced progression of spermatocytes from meiotic prophase to metaphase I (MI), implying efficacy of DNA damage checkpoint mechanisms. Apoptotic responses of spermatocytes with DNA damage differed, with an increase in frequency of early apoptotic spermatocytes after etoposide treatment, but not following irradiation. Taken together, these results demonstrate modification of pachytene spermatocyte chromatin and inhibition of meiotic progress after DNA damage by mechanisms that may ensure gametic genetic integrity.
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PMID:Spermatocyte responses in vitro to induced DNA damage. 1670 71

Fanconi anemia (FA) is a rare autosomal recessive disorder characterized by congenital abnormalities, progressive bone marrow failure, and cancer susceptibility. FA cells are hypersensitive to DNA crosslinking agents. FA is a genetically heterogeneous disease with at least 11 complementation groups. The eight cloned FA proteins interact in a common pathway with established DNA-damage-response proteins, including BRCA1 and ATM. Six FA proteins (A, C, E, F, G, and L) regulate the monoubiquitination of FANCD2 after DNA damage by crosslinking agents, which targets FANCD2 to BRCA1 nuclear foci containing BRCA2 (FANCD1) and RAD51. Some forms of hexavalent chromium [Cr(VI)] are implicated as respiratory carcinogens and induce several types of DNA lesions, including DNA interstrand crosslinks. We have shown that FA-A fibroblasts are hypersensitive to both Cr(VI)-induced apoptosis and clonogenic lethality. Here we show that Cr(VI) treatment induced monoubiquitination of FANCD2 in normal human fibroblasts, providing the first molecular evidence of Cr(VI)-induced activation of the FA pathway. FA-A fibroblasts demonstrated no FANCD2 monoubiquitination, in keeping with the requirement of FA-A for this modification. We also found that Cr(VI) treatment induced significantly more S-phase-dependent DNA double strand breaks (DSBs), as measured by gamma-H2AX expression, in FA-A fibroblasts compared to normal cells. However, and notably, DSBs were repaired equally in both normal and FA-A fibroblasts during recovery from Cr(VI) treatment. While previous research on FA has defined the genetic causes of this disease, it is critical in terms of individual risk assessment to address how cells from FA patients respond to genotoxic insult.
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PMID:FANCD2 monoubiquitination and activation by hexavalent chromium [Cr(VI)] exposure: activation is not required for repair of Cr(VI)-induced DSBs. 1689 75


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