Genetic and Molecular Analysis of Yeast DNA Replication

项目总结/摘要 这项提案的长期目标是了解调节高保真度的细胞过程 真核DNA复制低保真度复制和易出错的复制后DNA修复可导致 染色体非整倍性和突变，可导致衰老、癌症和出生缺陷。这项建议会 采用分子遗传分析，以确定这些过程的调节分子，使用单一的， 细胞的真核生物，酿酒酵母作为模型系统。这种结合了基因和分子 该方法将集中在一些重要的细胞周期蛋白激酶，其中包括DDK（Cdc 7-Dbf 4），CDK （细胞周期蛋白依赖性激酶）和Rad 53检查点激酶。提出了三个具体目标。在目标#1中，我们 将阐明MCM DNA解旋酶的功能和调节，通过研究N-末端和C-末端， 末端（β）-指和N-末端区域A结构域由DDK调节。我们的假设是N- 末端指对于结合起点是重要的，C末端指对于解旋酶是重要的 沿着DNA沿着移位或移动。此外，我们提出MCM复合物的磷酸化 通过DDK在Mcm 5的A结构域中产生结构变化，导致解旋酶活化。我们的系统 依赖于保守的大肠杆菌MCM解旋酶的原子结构和生物化学体外研究， 酵母中DNA复制的分子遗传学体内研究。在目标2中，我们将研究Rad 53的新作用 DNA复制我们目前的假设是Rad 53调节对DNA起始重要的蛋白质 复制和染色质结构。这种作用不依赖于Rad 53蛋白的检查点功能。在这 我们将继续利用我们独特的cdc 7-mcm 5-rad 53-组蛋白H3/H4“相互作用组”遗传系统， 鉴定更多相互作用的蛋白质，并分析突变体中染色质的变化。在目标3中，我们 通过研究DDK在TLS中的作用，探讨DNA复制的保真度和突变的调控 （trans-lesion synthesis）。我们的研究表明，DDK调节自发和诱导 DNA聚合酶（zeta）诱变。我们的假设是DDK作为Rev 7的“染色质装载器”， 是Rev 3易错DNA聚合酶的重要附件。DDK对复制也很重要 DNA损伤和TLS后重新启动。结合突变分析，全基因组遗传筛选， ChIP（染色质免疫沉淀）和亲和/抗体分离将用于测试其中的许多 假设我们的研究对于人类疾病具有重要意义，因为Rad 53（Chk 2）和 TLS聚合酶Pol <$（eta-XPV）在家族性癌症易感性Li-Fraumeni中发生突变， 着色性干皮病变异综合征。

Project Summary/Abstract The long-term objective of this proposal is to understand the cellular processes that regulate high-fidelity eukaryotic DNA replication. Low fidelity replication and error-prone post-replication DNA repair can result in chromosomal aneuploidy and mutations, which can cause aging, cancer and birth defects. This proposal will employ molecular genetic analysis to identify the regulatory molecules of these processes using the single- celled eukaryote, Saccharomyces cerevisiae as a model system. This combined genetic and molecular approach will focus on a number of important cell cycle protein kinases, which include DDK (Cdc7-Dbf4), CDK (cyclin-dependent kinase) and the Rad53 checkpoint kinase. Three specific aims are proposed. In aim #1, we will elucidate the function and regulation of the MCM DNA helicase by investigating both the N- terminal and C- terminal ¿ (Beta)-fingers and the N-terminal region A domain regulated by DDK. Our hypothesis is that the N- terminal ¿-fingers are important for binding origins and the C-terminal fingers are important for helicase translocation or movement along the DNA. Furthermore, we propose that phosphorylation of the MCM complex by DDK produces a structural change in the A domain of Mcm5 resulting in helicase activation. Our system relies on atomic structural and biochemical in vitro studies of the conserved Archaeal MCM helicases and molecular genetic in vivo studies of DNA replication in yeast. In aim 2, we will investigate a novel role of Rad53 in DNA Replication. Our current hypothesis is that Rad53 regulates proteins important for the initiation of DNA replication and in chromatin structure. This role is independent of Rad53 protein's checkpoint function. In this aim, we will continue to exploit our unique cdc7-mcm5-rad53-histone H3/H4 "interactome" genetic system to identify more interacting proteins and will also analyze changes in chromatin in our mutants. In aim 3, we investigate the fidelity of DNA replication and the regulation of mutagenesis by studying the role of DDK in TLS (trans-lesion synthesis). Our studies have shown that DDK regulates both spontaneous and induced mutagenesis by DNA polymerase ¿ (zeta). Our hypothesis is that DDK acts as a "chromatin loader" of Rev7, an important accessory of the Rev3 error-prone DNA polymerase ¿. DDK may also be important for replication restart after DNA damage and TLS. A combination of mutational analysis, whole-genome genetic screens, ChIP (chromatin immuno-precipitation), and affinity/antibody isolation will be used to test many of these hypotheses. Our studies have significance for human disease as the human homologues of Rad53 (Chk2) and the TLS polymerase Pol¿ (eta-XPV) are mutated in the familial cancer-predisposing Li-Fraumeni and Xeroderma pigmentosum variant syndromes, respectively.