Human DNA Repair Enzymes for Redox and Alkylation Damage

基因遗传和对癌症等与年龄相关的疾病的抵抗力取决于 人类基因组。稳定的基因组维持反过来又关键地依赖于细胞系统 “自发的”DNA损伤。这种损害包括水解性腐烂损害，特别是碱性部位，如 以及由代谢副产物形成的损伤，如自由基和烷化剂。现有数据 表明碱基切除修复(BER)系统最有可能处理这种内源性DNA损伤。 在BER中，DNA糖基酶去除改变的碱基以产生碱性位点，这些碱性位点在 几个步骤依赖于APE1内切酶和DNA聚合酶β。我们的长期目标是 一直在确定APE1蛋白作为碱基切除修复(BER)核心角色的生物学功能 DNA和可能的其他过程，并了解各分支机构活动的协调 误码率。过去的努力在确定APE1活性的生物化学方面是有价值的，但直到最近才有所发现 我们能够通过使用小干扰技术对蛋白质的细胞功能进行基因测试 核糖核酸(SiRNA)。这些数据清楚地支持了APE1在维持人类生存能力方面的重要作用 通过处理由内源性过程形成的基本DNA损伤来处理细胞。最近的其他工作指出， Sirtuin SIRT6在调节误码率中的新作用，可能是通过影响DNA聚合酶的活性 贝塔。因为SIRT6基因的缺失会缩短小鼠的寿命并产生各种相关的表型 对于过早衰老，这些观察提供了BER和正常衰老之间的第一个直接联系 进程。这里提出的工作建立在这一成功的基础上，以定义APE1和Ape1的细胞功能 它所处理的DNA损伤的来源，以及Sirtuin如何调节误码率。三 具体目标将针对我们的目标： 1.建立可调控表达APE1特异性si-RNA的人或小鼠细胞系 APE1耗竭时的生物效应动力学； 2.确定DNA糖基酶(特别是UNG2、OGG1、AAG/MPG和NTH1)的贡献以及 氧化损伤是APE1缺陷细胞中积累的致命性损伤的来源； 3.直接检测SirtG在DNA修复中的作用及BER的亚通路。

Genetic inheritance, and resistance to age-related diseases such as cancer, depend on the stability of the human genome. Stable genome maintenance in turn depends critically on cellular systems that process "spontaneous" DNA damage. Such damage includes hydrolytic decay lesions, in particular abasic sites, as well as lesions formed by metabolic by-products such as free radicals and alkylating agents. Existing data indicate that the base excision repair (BER) system is most likely to deal with this endogenous DNA damage. In BER, DNA glycosylases remove altered bases to generate abasic sites, which are further processed in several steps dependent on the Ape1 endonuclease and DNA polymerase beta. Our long-term goal has been to ascertain the biological functions of Ape1 protein as a central player in base excision repair (BER) of DNA and perhaps other processes, and to understand the coordination of activities in the various branches of BER. Past efforts have been valuable in defining the biochemistry of Ape1 activity, but only recently have we been able to conduct genetic tests of the protein's cellular function through the use of small-interfering RNA (siRNA). These data clearly support an essential role of Ape1 in maintaining the viability of human cells by processing abasic DNA damage formed by endogenous processes. Other recent work points to a novel role for the sirtuin Sirt6 in modulating BER, possibly through effects on the activity of DNA polymerase beta. Since deletion of the SIRT6 in mice shortens lifespan and generates various phenotypes associated with premature aging, these observations provide the first direct link between BER and the normal aging process. The work proposed here builds on this success in order to define the cellular function of Ape1 and the origins of the DNA damage that it processes, and how the BER can be modulated by a sirtuin. Three specific aims will address our goals: 1. Establish human or mouse cell lines with regulated expression of APE1-specific si-RNA and examine the kinetics of the biological effects accompanying Ape1 depletion; 2. Determine the contribution of DNA glycosylases (in particular UNG2, OGG1, AAG/MPG, and NTH1) and of oxidative damage as sources of the lethal lesions that accumulate in Ape1-deficient cells; 3. Directly test the role of SirtG in DNA repair and the sub-pathways of BER.