PROCESSING OF OXIDATIVE DNA LESIONS--TRANSIENT KINETICS

DNA 氧化损伤的处理——瞬态动力学

• 批准号: 6172841
• 负责人: ZAFER HATAHET
• 金额: $ 9.05万
• 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/6172841
• 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.

DNA的完整性不断受到广泛的威胁。 破坏性物质的光谱。 最大的挑战之一是氧化 因为它是由从电离辐射到正常的 细胞代谢并且它与诱变有关， 致癌和衰老。 氧化性DNA损伤是否 永久的生物学后果取决于它们之间的相互作用 不同的细胞成分负责维持 DNA的完整性 这些包括碱基切除修复糖基化酶和DNA 聚合酶 DNA N-糖基化酶面临着在少数 病变，通常，高结构相似性，其同源基地，在一个 大量过量的正常核苷酸 这是进一步复杂化的， 大，重叠底物光谱观察N-糖基化酶，使 病变识别的过程非常有趣。 定义 N-糖基化酶和AP裂解酶的瞬态动力学参数 反应以及病变结构和序列背景的影响 对维修效率的研究将大大有助于发展一个 碱基切除修复作用的综合机理模型 氧化DNA上的酶 也许最重要的决定因素 任何病变的生物学有害潜力是与DNA的相互作用 聚合酶，因为结果通常是永久性的。 做了大量工作 以表征这些相互作用。 我们建议 使用瞬态动力学来描述跨损伤DNA的机制 综合和扩展的综合模型开发的综合 天然模板包括含有氧化损伤的模板。 的 损伤结构、序列背景和DNA特性的影响 聚合酶，如校正病变旁路的持续合成能力， 使用重组相T4 DNA聚合酶全酶解决， 含有几种氧化损伤之一的模板。