Human DNA Repair Enzymes for Redox and Alkylation Damage

基因遗传和对癌症等与年龄有关的疾病的抵抗力取决于基因的稳定性。 人类基因组稳定的基因组维持反过来又严重依赖于细胞系统， “自发性”DNA损伤这种损害包括水解腐烂损伤，特别是脱碱基位点，如 以及由代谢副产物如自由基和烷基化剂形成的损伤。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糖基化酶去除改变的碱基以产生脱碱基位点，脱碱基位点在BER中进一步加工。 依赖于Ape 1内切核酸酶和DNA聚合酶β的几个步骤。我们的长期目标是 为了确定Ape 1蛋白作为碱基切除修复（BER）的中心参与者的生物学功能， DNA和其他过程，并了解各个分支活动的协调 的BER。过去的努力在定义Ape 1活性的生物化学方面是有价值的，但直到最近， 我们能够通过使用小干扰技术对蛋白质的细胞功能进行遗传测试， RNA（siRNA）。这些数据清楚地支持Ape 1在维持人类细胞活力方面的重要作用。 细胞通过处理内源性过程形成的脱碱基DNA损伤。最近的其他工作表明， sirtuin Sirt 6在调节BER中的新作用，可能通过影响DNA聚合酶的活性 β的由于小鼠中SIRT 6的缺失缩短了寿命并产生了各种相关的表型， 对于过早老化的情况，这些观测结果提供了误码率和正常老化之间的第一个直接联系 过程本文提出的工作建立在这一成功的基础上，以确定Ape 1的细胞功能， 它处理的DNA损伤的起源，以及BER如何被sirtuin调制。三 具体目标将实现我们的目标： 1.建立具有APE 1特异性si-RNA表达调节的人或小鼠细胞系，并检查细胞内APE 1特异性si-RNA的表达。 伴随Ape 1耗竭的生物学效应的动力学; 2.确定DNA糖基化酶（特别是UNG 2、OGG 1、AAG/MPG和NTH 1）的贡献， 氧化损伤是Ape 1缺陷细胞中积累的致死性损伤的来源; 3.直接测试SirtG在DNA修复中的作用以及BER的子途径。

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.