PROCESSING OF OXIDATIVE DNA LESIONS--TRANSIENT KINETICS

DNA 氧化损伤的处理——瞬态动力学

• 批准号: 6376330
• 负责人: ZAFER HATAHET
• 金额: $ 9.32万
• 依托单位: UNIVERSITY OF TEXAS HLTH CTR AT TYLER
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6376330
• 关键词: DNA damage; DNA directed DNA polymerase; DNA repair; DNA replication; Escherichia coli; N glycosidase; bacterial proteins; bacteriophage T4; chemical kinetics; endonuclease; enzyme activity; microorganism culture; nucleic acid sequence; oligonucleotides; oxidative stress; virus protein

DESCRIPTION: The integrity of DNA is continuously threatened by a wide spectru of damaging agents. One of the greatest challenges is oxidation since it is caused by agents ranging from ionizing radiation to normal cellular metabolism and it has been implicated in mutagenesis, carcinogenesis and aging. Whether o not oxidative DNA lesions have permanent biological consequences depends on th interactions between them and the different cellular components responsible fo maintaining the integrity of DNA. These include base excision repair glycosylases and DNA polymerases. DNA N-glycosylases are faced with identifyin a small number of lesions with, often, high structural similarity to their cognate bases in a vast excess of normal nucleotides. This is further complicated by the large, overlapping substrate spectra observed for N-glycosylases, making the process of lesion recognition very interesting. Definition of the transient-state kinetic parameters of the N-glycosylase and AP lyase reactions as well as the influence of lesion structure and sequence context on repair efficiency should contribute significantly to developing a comprehensive mechanistic model for the action of base excision repair enzymes on oxidized DNA. Perhaps the most important determinant of the biologically harmful potential of any lesion is interactions with DNA polymerases since the outcome is often permanent. Much work has been done both in vitro and in vivo to characterize these interactions. We propose to use transient-state kinetics to delineate the mechanism of translesion DNA synthesis and extend the comprehensive model developed for synthesis on natural templates to include oxidative lesion-containing templates. The influence of lesion structure, sequence context and proprieties of the DNA polymerase such as proofreading an processivity on lesion bypass will be addressed using a reconstituted phase T4 DNA polymerase holoenzyme on templates containing one of several oxidative lesions.

描述：DNA的完整性不断受到广泛的威胁 破坏剂的幽灵。最大的挑战之一是氧化。 因为它是由从电离辐射到正常的各种因素引起的 细胞新陈代谢和突变相关， 致癌和衰老。氧化dna损伤是否有 永久性的生物后果取决于它们之间的相互作用 以及不同的细胞成分负责维持 DNA的完整性。其中包括碱基切除修复、糖基酶和DNA。 聚合酶。DNA N-糖基酶在少数几种 通常，与其同源碱基结构高度相似的病变 大量的正常核苷酸。这一点因 观察到N-糖基酶的大的重叠底物光谱，使 病变识别的过程非常有趣。概念的定义 N-糖基酶和AP裂解酶的暂态动力学参数 反应以及病变结构和序列背景的影响 关于维修效率的研究应该对开发一种 基层切除修复作用的综合力学模型 氧化的DNA上的酶。也许最重要的决定因素是 任何损伤的生物危害性都是与DNA的相互作用 聚合酶，因为结果往往是永久性的。已经做了很多工作 在体外和体内，以表征这些相互作用。我们建议 用暂态动力学描述跨损伤DNA的机制 综合和扩展了为综合而开发的综合模型 天然模板，包括含氧化损伤的模板。这个 病变结构、序列背景和DNA特性的影响 聚合酶，如校对病变旁路的进程，将 使用重组相T4 DNA聚合酶全酶 包含几种氧化损伤之一的模板。