NEW HUMAN DNA REPAIR ENDONUCLEASE

新人类 DNA 修复核酸内切酶

• 批准号: 6318462
• 负责人: ALFONSO BELLACOSA
• 金额: $ 8.65万
• 依托单位: FOX CHASE CANCER CENTER
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6318462
• 关键词: DNA repair; cell line; chemical binding; cytosine; endonuclease; gene expression; genetic mapping; human tissue; immunoprecipitation; molecular cloning; mutant; nucleic acid methylation; nucleic acid sequence; protein localization; protein purification; protein structure function; site directed mutagenesis; southern blotting; tissue /cell culture; transfection; western blottings

DESCRIPTION: (adapted from the investigator's abstract) The maintenance of genomic integrity relies on the efficacy of DNA repair systems. These systems counteract the mutational burden imposed on DNA by exogenous attacks, endogenous reactive species, and errors originating during replication. Failure of DNA surveillance and repair mechanisms leads to an increase in the mutation rate, and this, in turn, results in predisposition to cancer. A prominent role in mutational avoidance and genomic stability is performed by the DNA mismatch repair system. This system handles base pair mismatches, short insertions/deletions and recombination-derived heteroduplexes. Patients with Hereditary Non-Polyposis Colorectal Cancer (HNPCC) carry a germline mutation in genes involved in DNA mismatch repair (h MSH2, h MLH1, GTBP /hMSH6, hPMS2 and hPMS1). These genes encode human homologues of the E. coli mismatch repair proteins MutS and MutL. In the bacterial system, a third protein, the single-strand endonuclease MutH, performs the crucial function of strand recognition, incising the newly synthesized DNA strand carrying the mutation. The new strand is identified by virtue of the transient lack of adenine methylation at GATC sites. To date, eukaryotic homologues of MutH, i.e. eukaryotic mismatch repair endonucleases, have not been identified, and the molecular determinants of strand discrimination in eukaryotic cells - which lack GATC methylation - have remained elusive. By employing the yeast interaction trap with hMLH1 as bait , MED1 (mismatch repair endonuclease1), a novel human gene encoding a protein with homology to bacterial endonucleases, was cloned. Sequence analysis of MED1 suggests a possible mechanism of strand recognition based on cytosine methylation at CpG sites. For its interaction with hMLH1 and homology to bacterial DNA repair proteins, MED1 is a putative mismatch repair protein and might be a long sought eukaryotic functional homologue of MutH. Since mismatch repair genes are mutated in HNPCC and sporadic cancers with microsatellite instability, MED1 is a candidate gene for cancer genetic testing. Based in these observations, experiments are proposed to address: 1) the biochemical properties of MED1; 2) its functional role in DNA repair. These studies may provide new insights into the mechanisms of eukaryotic mismatch repair and further the link between defective DNA repair and cancer.

描述：（改编自研究者的摘要）维护 基因组完整性依赖于 DNA 修复系统的功效。 这些 系统抵消外源对 DNA 造成的突变负担 攻击、内源性反应物种以及源自过程中的错误 复制。 DNA 监视和修复机制的失败会导致 突变率增加，这反过来又导致易感性 到癌症。 在突变避免和基因组稳定性方面发挥着重要作用 由 DNA 错配修复系统执行。 该系统处理基础 配对错配、短插入/缺失和重组衍生 异源双链体。 遗传性非息肉病性结直肠癌患者 (HNPCC) 在参与 DNA 错配修复的基因中携带种系突变 (h MSH2、h MLH1、GTBP /hMSH6、hPMS2 和 hPMS1)。 这些基因编码人类 大肠杆菌错配修复蛋白 MutS 和 MutL 的同源物。 在 细菌系统，第三种蛋白质，单链核酸内切酶 MutH， 执行链识别的关键功能，切割新的 合成的DNA链携带突变。 新链被识别 由于 GATC 位点暂时缺乏腺嘌呤甲基化。 到 日期，MutH 的真核同源物，即真核错配修复 核酸内切酶尚未被鉴定，其分子决定因素 真核细胞中的链辨别 - 缺乏 GATC 甲基化 - 仍然难以捉摸。 通过使用酵母与 hMLH1 相互作用陷阱 作为诱饵，MED1（错配修复核酸内切酶1），一种编码新型人类基因 克隆了与细菌核酸内切酶同源的蛋白质。 顺序 MED1 的分析提出了一种基于链识别的可能机制 CpG 位点的胞嘧啶甲基化。 与 hMLH1 和 与细菌 DNA 修复蛋白同源，MED1 是假定的错配 修复蛋白，可能是长期寻找的真核功能同源物 穆特H。 由于错配修复基因在 HNPCC 和散发性癌症中发生突变 MED1具有微卫星不稳定性，是癌症遗传的候选基因 测试。 根据这些观察结果，建议进行实验来解决： 1）MED1的生化特性； 2）其在DNA修复中的功能作用。 这些研究可能为真核生物机制提供新的见解 错配修复并进一步研究缺陷 DNA 修复与 癌症。