Translesion synthesis by yeast DNA polymerases

描述（由申请人提供）：我们的长期目标是描述真核细胞用于克服由DNA损伤引起的复制阻断的机制。本项目的具体目标是阐明酿酒酵母中DNA聚合酶eta和n的跨损伤合成（TLS）机制。在目的1中，我们将进行生物化学和遗传学研究，以检验Rev 1 DNA合成活性与Poleta_合作复制源自鸟嘌呤活性N位内源性代谢的过去高度扭曲的DNA加合物的假设。目的2将测试Rev 1通过其与PCNA的相互作用在Poleta靶向病变部位中起关键作用的想法。在目标3中，作为Poln、Revl和Rev 7相关多蛋白组装体的组分的蛋白质将被纯化，相互作用蛋白质也将被纯化。生物化学和遗传学研究将确定新发现的蛋白质因子在TLS中的作用。在目标4中，体内和体外研究将检查Poln、Rev 1、Rev 3、Rev 7和其他蛋白的泛素化，并确定Rad 6-Rad 18和Mms 2-Ubcl 3泛素缀合酶复合物在这些泛素化事件中的作用。在目标5中，将确定Poleta和Poln进入停滞在病变部位的复制复合物的机制。我们的研究应产生重要的见解translesion合成Poleta和Poln的机制。这些结果将与癌症生物学高度相关，因为在复制过程中绕过DNA损伤的方式对基因组稳定性有重大影响，事实上，人类Poln的失活导致癌症倾向综合征，即着色性干皮病的变体。

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.