NEW HUMAN DNA REPAIR ENDONUCLEASE

新人类 DNA 修复核酸内切酶

• 批准号: 2673245
• 负责人: ALFONSO BELLACOSA
• 金额: $ 11.11万
• 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/2673245
• 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：（改编自研究者摘要） 基因组完整性依赖于DNA修复系统的功效。 这些 系统抵消了外源基因对DNA造成的突变负担， 攻击，内源性活性物质，以及 复制的 DNA监视和修复机制的失败导致了 突变率的增加，这反过来又会导致易感性 到癌症 在避免突变和基因组稳定性方面的突出作用 是由DNA错配修复系统完成的 该系统处理基础 配对错配、短插入/缺失和重组衍生的 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 （hMSH 2、hMLH 1、GTBP /hMSH 6、hPMS 2和hPMS 1）。 这些基因编码人类 E.大肠杆菌错配修复蛋白MutS和MutL。 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 携带突变的合成DNA链。 新的链被识别 这是由于GATC位点上腺嘌呤甲基化的短暂缺失。 到 date，MutH的真核同源物，即真核错配修复 核酸内切酶，还没有被确定，和分子决定因素， 在缺乏GATC甲基化的真核细胞中， 仍然难以捉摸。 通过使用酵母相互作用陷阱与hMLH 1 作为诱饵，MED 1（错配修复核酸内切酶1），一种新的人类基因编码 克隆了一种与细菌内切核酸酶具有同源性的蛋白。 序列 MED 1的分析表明，基于链识别的可能机制 在CpG位点的胞嘧啶甲基化。 对于其与hMLH 1的相互作用， 与细菌DNA修复蛋白同源，MED 1是一个假定的错配 修复蛋白，可能是一个长期寻找的真核生物功能同源物， MutH。 由于错配修复基因在HNPCC和散发性癌症中发生突变， MED 1是一个具有微卫星不稳定性的肿瘤遗传学候选基因， 试验. 根据这些观察，建议进行实验，以解决： 1)MED 1的生物化学性质; 2）其在DNA修复中的功能作用。 这些研究可能为真核生物的机制提供新的见解。 错配修复和进一步的DNA修复缺陷之间的联系， 癌