Translesion synthesis by yeast DNA polymerases

描述(申请人提供)：我们的长期目标是描述真核细胞用来克服DNA损伤引起的复制障碍的机制。本项目的具体目的是阐明酿酒酵母DNA聚合酶ETA和n跨损伤合成(TLS)的机制。在目标1中，我们将进行生化和遗传学研究，以检验这一假设，即Rev1 DNA合成活性与Poleta_合作，在鸟嘌呤的活性N位复制来自内源代谢的过去高度扭曲的DNA加合物。目的2将验证Rev1通过与增殖细胞核抗原的相互作用，在Poleta靶向病变部位中发挥关键作用的想法。在目标3中，作为Poln、Rev1和Rev7相关多蛋白组合的组成部分的蛋白质将被纯化，相互作用的蛋白质也将被纯化。生化和遗传学研究将确定新发现的蛋白质因素在TLS中的作用。在目标4中，体内和体外研究将检测Poln、Rev1、Rev3、Rev7和其他蛋白质的泛素化，并确定Rad6-Rad18和Mms2-Ubcl3泛素结合酶复合体在这些泛素化事件中的作用。在目标5中，将确定Poleta和Poln进入在病变部位停滞的复制复合体的机制。我们的研究将对Poleta和Poln跨病变合成的机制产生重要的见解。这一结果将与癌症生物学高度相关，因为复制过程中绕过DNA损伤的方式对基因组稳定性有重大影响，事实上，人类Poln的失活会导致癌症倾向综合征，这是着色性干皮病的变种。

DESCRIPTION (provided by applicant): Our long-term objective is to delineate the mechanisms eukaryotic cells employ to overcome replication blockage caused by DNA lesions. The specific aims of this project are to elucidate the mechanisms of translesion synthesis (TLS) by DNA polymerases eta and n in the yeast Saccharomyces cerevisiae. In Aim 1, we will carry out biochemical and genetic studies to examine the hypothesis that the Rev1 DNA synthetic activity co-operates with Poleta_ to replicate past highly distorting DNA adducts originating from endogenous metabolism at the reactive N position of guanine. Aim 2 will test the idea that Rev1, via its interaction with PCNA, plays a crucial role in the targeting of Poleta to the lesion site. In Aim 3, proteins that are components of Poln, Revl, and Rev7 associated multi-protein assemblies will be purified, as will interacting proteins. Biochemical and genetic studies will define the roles of the newly identified protein factors in TLS. In Aim 4, in vivo and in vitro studies will examine the ubiquitination of Poln, Rev1, Rev3, Rev7, and other proteins, and ascertain the roles of the Rad6-Radl8 and Mms2-Ubcl3 ubiquitin conjugating enzyme complexes in these ubiquitination events. In Aim 5, the mechanism by which Poleta and Poln enter into the replication complex stalled at a lesion site will be determined. Our studies should yield important insights into the mechanisms of translesion synthesis by Poleta and Poln. The results will be highly relevant for cancer biology, as the manner in which DNA lesions are bypassed during replication has a major impact on genome stability, and, in fact, inactivation of Poln in humans leads to the cancer prone syndrome, the variant form of xeroderma pigmentosum.