Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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We identified a minimal domain of human p53 required for the transactivation of a p53 response element in Saccharomyces cerevisiae. This domain contains the central region of p53 sufficient for specific DNA binding, which colocalizes with the region responsible for binding simian virus 40 large T antigen, 53BP1, and 53BP2. Thirty amino acid positions, including natural mutational hot spots (R175, R213, R248, R249, and R273), in the minimal DNA-binding domain were mutated by alanine substitution. Alanine substitutions at positions R213, R248, R249, D281, R282, R283, E286, and N288 affected transactivation but allowed binding to at least one of the three interacting proteins; these amino acids may be involved in amino acid-base pair contacts. Surprisingly, alanine substitution at the mutational hot spot R175 did not affect DNA binding, transactivation, or T-antigen binding, although it nearly eliminated binding to 53BP1 and 53BP2. Mutation of H168 significantly affected only T-antigen binding, and mutation of E285 affected only 53BP1 binding. Thus, we implicate specific residues of p53 in different DNA and protein interactions.
Mol Cell Biol 1994 Dec
PMID:Distinct residues of human p53 implicated in binding to DNA, simian virus 40 large T antigen, 53BP1, and 53BP2. 796 67

53BP1 is a human BRCT protein that was originally identified as a p53-interacting protein by the Saccharomyces cerevisiae two-hybrid screen. Although the carboxyl-terminal BRCT domain shows similarity to Crb2, a DNA damage checkpoint protein in fission yeast, there is no evidence so far that implicates 53BP1 in the checkpoint. We have identified a Xenopus homologue of 53BP1 (XL53BP1). XL53BP1 is associated with chromatin and, in some cells, localized to a few large foci under normal conditions. Gamma-ray irradiation induces increased numbers of the nuclear foci in a dose-dependent manner. The damage-induced 53BP1 foci appear rapidly (in 30 min) after irradiation, and de novo protein synthesis is not required for this response. In human cells, 53BP1 foci colocalize with Mrel1 foci at later stages of the postirradiation period. XL53BP1 is hyperphosphorylated after X-ray irradiation, and inhibitors of ATM-related kinases delay the relocalization and reduce the phosphorylation of XL53BP1 in response to X-irradiation. In AT cells, which lack ATM kinase, the irradiation-induced responses of 53BP1 are similarly affected. These results suggest a role for 53BP1 in the DNA damage response and/or checkpoint control which may involve signaling of damage to p53.
Mol Cell Biol 2001 Mar
PMID:Phosphorylation and rapid relocalization of 53BP1 to nuclear foci upon DNA damage. 1123 9

snm1 mutants of Saccharomyces cerevisiae have been shown to be specifically sensitive to DNA interstrand crosslinking agents but not sensitive to monofunctional alkylating agents, UV, or ionizing radiation. Five homologs of SNM1 have been identified in the mammalian genome and are termed SNM1, SNM1B, Artemis, ELAC2, and CPSF73. To explore the functional role of human Snm1 in response to DNA damage, we characterized the cellular distribution and dynamics of human Snm1 before and after exposure to DNA-damaging agents. Human Snm1 was found to localize to the cell nucleus in three distinct patterns. A particular cell showed diffuse nuclear staining, multiple nuclear foci, or one or two larger bodies confined to the nucleus. Upon exposure to ionizing radiation or an interstrand crosslinking agent, the number of cells exhibiting Snm1 bodies was reduced, while the population of cells with foci increased dramatically. Indirect immunofluorescence studies also indicated that the human Snm1 protein colocalized with 53BP1 before and after exposure to ionizing radiation, and a physical interaction was confirmed by coimmunoprecipitation assays. Furthermore, human Snm1 foci formed after ionizing radiation were largely coincident with foci formed by human Mre11 and to a lesser extent with those formed by BRCA1, but not with those formed by human Rad51. Finally, we mapped a region of human Snm1 of approximately 220 amino acids that was sufficient for focus formation when attached to a nuclear localization signal. Our results indicate a novel function for human Snm1 in the cellular response to double-strand breaks formed by ionizing radiation.
Mol Cell Biol 2002 Dec
PMID:hSnm1 colocalizes and physically associates with 53BP1 before and after DNA damage. 1244 82

53BP1 is a p53 binding protein of unknown function that binds to the central DNA-binding domain of p53. It relocates to the sites of DNA strand breaks in response to DNA damage and is a putative substrate of the ataxia telangiectasia-mutated (ATM) kinase. To study the biological role of 53BP1, we disrupted the 53BP1 gene in the mouse. We show that, similar to ATM(-/-) mice, 53BP1-deficient mice were growth retarded, immune deficient, radiation sensitive, and cancer prone. 53BP1(-/-) cells show a slight S-phase checkpoint defect and prolonged G(2)/M arrest after treatment with ionizing radiation. Moreover, 53BP1(-/-) cells feature a defective DNA damage response with impaired Chk2 activation. These data indicate that 53BP1 acts downstream of ATM and upstream of Chk2 in the DNA damage response pathway and is involved in tumor suppression.
Mol Cell Biol 2003 Apr
PMID:p53 Binding protein 53BP1 is required for DNA damage responses and tumor suppression in mice. 1264 Jan 36

The fission yeast checkpoint protein Crb2, related to budding yeast Rad9 and human 53BP1 and BRCA1, has been suggested to act as an adapter protein facilitating the phosphorylation of specific substrates by Rad3-Rad26 kinase. To further understand its role in checkpoint signaling, we examined its localization in live cells by using fluorescence microscopy. In response to DNA damage, Crb2 localizes to distinct nuclear foci, which represent sites of DNA double-strand breaks (DSBs). Crb2 colocalizes with Rad22 at persistent foci, suggesting that Crb2 is retained at sites of DNA damage during repair. Damage-induced Crb2 foci still form in cells defective in Rad1, Rad3, and Rad17 complexes, but these foci do not persist as long as in wild-type cells. Our results suggest that Crb2 functions at the sites of DNA damage, and its regulated persistent localization at damage sites may be involved in facilitating DNA repair and/or maintaining the checkpoint arrest while DNA repair is under way.
Mol Cell Biol 2003 Sep
PMID:Retention but not recruitment of Crb2 at double-strand breaks requires Rad1 and Rad3 complexes. 1291 37

Many conventional anticancer treatments kill cells irrespective of whether they are normal or cancerous, so patients suffer from adverse side effects due to the loss of healthy cells. Anticancer insights derived from cell cycle research has given birth to the idea of cell cycle G2 checkpoint abrogation as a cancer cell specific therapy, based on the discovery that many cancer cells have a defective G1 checkpoint resulting in a dependence on the G2 checkpoint during cell replication. Damaged DNA in humans is detected by sensor proteins (such as hHUS1, hRAD1, hRAD9, hRAD17, and hRAD26) that transmit a signal via ATR to CHK1, or by another sensor complex (that may include gammaH2AX, 53BP1, BRCA1, NBS1, hMRE11, and hRAD50), the signal of which is relayed by ATM to CHK2. Most of the damage signals originated by the sensor complexes for the G2 checkpoint are conducted to CDC25C, the activity of which is modulated by 14-3-3. There are also less extensively explored pathways involving p53, p38, PCNA, HDAC, PP2A, PLK1, WEE1, CDC25B, and CDC25A. This review will examine the available inhibitors of CHK1 (Staurosporin, UCN-01, Go6976, SB-218078, ICP-1, and CEP-3891), both CHK1 and CHK2 (TAT-S216A and debromohymenialdisine), CHK2 (CEP-6367), WEE1 (PD0166285), and PP2A (okadaic acid and fostriecin), as well as the unknown checkpoint inhibitors 13-hydroxy-15-ozoapathin and the isogranulatimides. Among these targets, CHK1 seems to be the most suitable target for therapeutic G2 abrogation to date, although an unexplored target such as 14-3-3 or the strategy of targeting multiple proteins at once may be of interest in the future.
Mol Cancer Ther 2004 Apr
PMID:G2 checkpoint abrogators as anticancer drugs. 1507 95

Mammalian ATR and ATM checkpoint kinases modulate chromatin structures near DNA breaks by phosphorylating a serine residue in the carboxy-terminal tail SQE motif of histone H2AX. Histone H2A is similarly regulated in Saccharomyces cerevisiae. The phosphorylated forms of H2AX and H2A, known as gamma-H2AX and gamma-H2A, are thought to be important for DNA repair, although their evolutionarily conserved roles are unknown. Here, we investigate gamma-H2A in the fission yeast Schizosaccharomyces pombe. We show that formation of gamma-H2A redundantly requires the ATR/ATM-related kinases Rad3 and Tel1. Mutation of the SQE motif to AQE (H2A-AQE) in the two histone H2A genes caused sensitivity to a wide range of genotoxic agents, increased spontaneous DNA damage, and impaired checkpoint maintenance. The H2A-AQE mutations displayed a striking synergistic interaction with rad22Delta (Rad52 homolog) in ionizing radiation (IR) survival. These phenotypes correlated with defective phosphorylation of the checkpoint proteins Crb2 and Chk1 and a failure to recruit large amounts of Crb2 to damaged DNA. Surprisingly, the H2A-AQE mutations substantially suppressed the IR hypersensitivity of crb2Delta cells by a mechanism that required the RecQ-like DNA helicase Rqh1. We propose that gamma-H2A modulates checkpoint and DNA repair through large-scale recruitment of Crb2 to damaged DNA. This function correlates with evidence that gamma-H2AX regulates recruitment of several BRCA1 carboxyl terminus domain-containing proteins (NBS1, 53BP1, MDC1/NFBD1, and BRCA1) in mammals.
Mol Cell Biol 2004 Jul
PMID:Histone H2A phosphorylation controls Crb2 recruitment at DNA breaks, maintains checkpoint arrest, and influences DNA repair in fission yeast. 1522 25

Spindle poisons represent an important class of anticancer drugs that act by interfering with microtubule polymerization and dynamics and thereby induce mitotic checkpoints and apoptosis. Here we show that mammalian SNM1 functions in an early mitotic stress checkpoint that is distinct from the well-characterized spindle checkpoint that regulates the metaphase-to-anaphase transition. Specifically, we found that compared to wild-type cells, Snm1-deficient mouse embryonic fibroblasts exposed to spindle poisons exhibited elevated levels of micronucleus formation, decreased mitotic delay, a failure to arrest in mitosis prior to chromosome condensation, supernumerary centrosomes, and decreased viability. In addition, we show that both Snm1 and 53BP1, previously shown to interact, coimmunoprecipitate with components of the anaphase-promoting complex (APC)/cyclosome. These findings suggest that Snm1 is a component of a mitotic stress checkpoint that negatively targets the APC prior to chromosome condensation.
Mol Cell Biol 2004 Dec
PMID:Deficiency in SNM1 abolishes an early mitotic checkpoint induced by spindle stress. 1554 52

The hereditary disorder ataxia telangiectasia (A-T) is associated with striking cellular radiosensitivity that cannot be attributed to the characterized cell cycle checkpoint defects. By epistasis analysis, we show that ataxia telangiectasia mutated protein (ATM) and Artemis, the protein defective in patients with RS-SCID, function in a common double-strand break (DSB) repair pathway that also requires H2AX, 53BP1, Nbs1, Mre11, and DNA-PK. We show that radiation-induced Artemis hyperphosphorylation is ATM dependent. The DSB repair process requires Artemis nuclease activity and rejoins approximately 10% of radiation-induced DSBs. Our findings are consistent with a model in which ATM is required for Artemis-dependent processing of double-stranded ends with damaged termini. We demonstrate that Artemis is a downstream component of the ATM signaling pathway required uniquely for the DSB repair function but dispensable for ATM-dependent cell cycle checkpoint arrest. The significant radiosensitivity of Artemis-deficient cells demonstrates the importance of this component of DSB repair to survival.
Mol Cell 2004 Dec 03
PMID:A pathway of double-strand break rejoining dependent upon ATM, Artemis, and proteins locating to gamma-H2AX foci. 1557 27

We screened radiation-sensitive yeast mutants for DNA damage checkpoint defects and identified Dot1, the conserved histone H3 Lys 79 methyltransferase. DOT1 deletion mutants (dot1Delta) are G1 and intra-S phase checkpoint defective after ionizing radiation but remain competent for G2/M arrest. Mutations that affect Dot1 function such as Rad6-Bre1/Paf1 pathway gene deletions or mutation of H2B Lys 123 or H3 Lys 79 share dot1Delta checkpoint defects. Whereas dot1Delta alone confers minimal DNA damage sensitivity, combining dot1Delta with histone methyltransferase mutations set1Delta and set2Delta markedly enhances lethality. Interestingly, set1Delta and set2Delta mutants remain G1 checkpoint competent, but set1Delta displays a mild S phase checkpoint defect. In human cells, H3 Lys 79 methylation by hDOT1L likely mediates recruitment of the signaling protein 53BP1 via its paired tudor domains to double-strand breaks (DSBs). Consistent with this paradigm, loss of Dot1 prevents activation of the yeast 53BP1 ortholog Rad9 or Chk2 homolog Rad53 and decreases binding of Rad9 to DSBs after DNA damage. Mutation of Rad9 to alter tudor domain binding to methylated Lys 79 phenocopies the dot1Delta checkpoint defect and blocks Rad53 phosphorylation. These results indicate a key role for chromatin and methylation of histone H3 Lys 79 in yeast DNA damage signaling.
Mol Cell Biol 2005 Oct
PMID:Role of Dot1-dependent histone H3 methylation in G1 and S phase DNA damage checkpoint functions of Rad9. 1616 26


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