MECHANISM OF BASE EXCISION REPAIR IN HIGHER EUKARYOTES

高等真核生物碱基切除修复机制

• 批准号: 2752159
• 负责人: YOSHIHIRO MATSUMOTO
• 金额: $ 24.05万
• 依托单位: FOX CHASE CANCER CENTER
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 2004-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2752159
• 关键词: DNA damage; DNA directed DNA polymerase; DNA repair; X ray crystallography; chemical elimination; endonuclease; enzyme activity; enzyme mechanism; intermolecular interaction; molecular cloning; nucleic acid sequence; oligonucleotides; phosphoester ligase; proliferating cell nuclear antigen; site directed mutagenesis; stereochemistry; tissue /cell culture

Base excision repair is a major mechanism to correct DNA damage, such as modified bases and apurinic/apyrimidinic (AP) sites. These lesions are among the most abundant lesions generated through normal cellular metabolisms and are also induced by environmental agents including many anti-cancer drugs and ionizing radiation. In addition, many of these lesions are mutagenic if not repaired. Thus, understanding the mechanism of base excision repair is an urgent subject for reducing the cancer risk and also for improving cancer treatments. The long-term objective of this research project is to define the fundamental reaction of base excision repair in higher eukaryotes. The first specific aim is to elucidate the excision mechanism of a 5'- incised AP site by DNA polymerase beta (pol beta) from structural and functional perspectives. Three approaches will be made toward this goal: 1) functional analyses of a series of site-directed mutations introduced into residues in the N-terminal 8-kDa domain of pol beta which is responsible for the excision activity; 2) stereochemical analyses of the DNA substrate and the product of this excision reaction; 3) crystallographic analyses of the 8-kDa domain/DNA complexes. Since the 8-kDa domain contains a helix-hairpin-helix motif, a DNA-binding motif which has been found in numbers of proteins involved in DNA transaction, the information obtained from this study will also contribute to understanding the interaction of these proteins with DNA. The second specific aim in this project is to dissect a protein complex assembled on an AP site during the PCNA-dependent repair reaction which presumably serves as an alternative pathway of base excision repair in higher eukaryotes in addition to the pol beta-dependent pathway. Since previous studies suggest the importance of the structure of the DNA substrate (circular DNA versus linear DNA) in the PCNA-dependent reaction in vitro, the effect of end-capping of linear DNA on the assembly of the PCNA-dependent repair complex will be evaluated. Furthermore, the roles of replication factor C (RF-C) and PCNA in recognition of a 5'-incised AP site and interaction with DNA, DNA polymerase delta, flap endonuclease 1 (FEN1) and DNA ligase in the repair complex will be characterized in a reconstituted system. Some of the protein factors employed in the PCNA-dependent AP site repair are also involved in DNA replication and nucleotide excision repair. However, since the 5'-incised AP site is a unique DNA structure in this repair system, this study will add a new insight to the functions of these versatile proteins.

基础切除修复是纠正DNA损伤的主要机制，例如 作为修改的碱基和肾上腺素/apyrimidinic（AP）位点。这些病变 是通过正常细胞产生的最丰富的病变之一 代谢，也是由包括许多人在内的环境因素诱导的 抗癌药物和电离辐射。 此外，其中许多 如果不修复，病变是诱变的。 因此，了解 基础切除修复机理是降低的紧急主题 癌症风险以及改善癌症治疗的风险。 长期 该研究项目的目的是定义基本反应 较高真核生物的基础切除修复。 第一个具体目的是阐明5'-的切除机制 通过结构和 功能观点。 将采用三种方法 目标：1）一系列站点定向突变的功能分析 引入POL Beta的N末端8 kDa域中的残留物 这是负责切除活动的原因； 2）立体化学 DNA底物的分析和该切除反应的乘积； 3）8 kDa结构域/DNA复合物的晶体学分析。自从 8 kDa结构域包含一个螺旋速率螺旋图案，一个DNA结合 在参与DNA的蛋白质数量中发现的基序 交易，从这项研究中获得的信息也将 有助于理解这些蛋白与DNA的相互作用。 该项目的第二个特定目的是剖析蛋白质复合物 在PCNA依赖性修复反应中，在AP位点组装，该反应 大概是基础切除修复的替代途径 除了POL Beta依赖性途径外，较高的真核生物。 自从 先前的研究表明DNA结构的重要性 PCNA依赖性的底物（圆形DNA与线性DNA） 反应在体外，线性DNA终端限制对 将评估PCNA依赖性修复复合物的组装。 此外，复制因子C（RF-C）和PCNA的作用 识别5'构的AP位点以及与DNA，DNA相互作用 聚合酶三角洲，皮瓣核酸内切酶1（FEN1）和DNA连接酶 修复复合物将在重构系统中进行表征。 一些 PCNA依赖性AP位点修复中使用的蛋白质因子是 还参与了DNA复制和核苷酸切除修复。 但是，由于5'构成的AP位点是独特的DNA结构 维修系统，这项研究将为功能增添新的见解 这些多功能蛋白。