Genetic and Molecular Analysis of Yeast DNA Replication

描述（由申请人提供）：本提案的长期目标是了解调节高保真真核DNA复制的细胞过程。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，这可导致衰老、癌症和出生缺陷。本研究将以单细胞真核生物酿酒酵母为模型系统，利用分子遗传学分析方法来鉴定这些过程的调控分子。这种结合遗传和分子的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，我们将通过研究由DDK调节的N-末端和C-末端2（β）-指和N-末端区域A结构域来阐明MCM DNA解旋酶的功能和调节。我们的假设是，N-末端2-指对于结合起点是重要的，并且C-末端指对于解旋酶移位或沿DNA沿着移动是重要的。此外，我们建议，磷酸化的MCM复合物的DDK产生的结构变化中的A域的Mcm 5导致解旋酶激活。我们的系统依赖于保守的酵母MCM解旋酶的原子结构和生物化学体外研究以及酵母中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在DNA复制中的新作用。我们目前的假设是，Rad 53调节蛋白质的DNA复制的启动和染色质结构的重要。这种作用不依赖于Rad 53蛋白的检查点功能。在这个目标中，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”遗传系统来鉴定更多的相互作用蛋白，并分析我们突变体中染色质的变化。目的3：通过研究DDK在TLS（trans-lesion synthesis）中的作用，研究DNA复制的保真度和突变的调控。我们的研究表明，DDK调节DNA聚合酶6（zeta）的自发和诱导突变。我们的假设是DDK作为Rev 7的“染色质装载器”，Rev 3易错DNA聚合酶6的重要附件。DDK对DNA损伤和TLS后的复制重启也很重要。突变分析，全基因组遗传筛选，ChIP（染色质免疫沉淀）和亲和力/抗体分离的组合将用于测试这些假设中的许多。我们的研究对人类疾病具有重要意义，因为Rad 53（Chk 2）和TLS聚合酶Pol 7（eta-XPV）的人类同源物分别在家族性癌症易感性Li-Fraumeni和着色性干皮病变异综合征中突变。 公共卫生相关性：由于拟议的研究重点是染色体复制的保真度和基因突变的产生，它们与癌症、衰老和出生缺陷等人类健康有关。在这个酵母模型系统中研究的几个人类基因的缺陷已知会增加癌症风险。

DESCRIPTION (provided by applicant): 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 2 (Beta)-fingers and the N-terminal region A domain regulated by DDK. Our hypothesis is that the N- terminal 2-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 6 (zeta). Our hypothesis is that DDK acts as a "chromatin loader" of Rev7, an important accessory of the Rev3 error-prone DNA polymerase 6. 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 Pol7 (eta-XPV) are mutated in the familial cancer-predisposing Li-Fraumeni and Xeroderma pigmentosum variant syndromes, respectively. PUBLIC HEALTH RELEVANCE: Because the proposed studies focus on the fidelity of chromosome replication and the production of genetic mutations, they have relevance to human health with respect to cancer, aging and birth defects. Defects in the several of the human genes studied in this yeast model system are known to increase cancer risk.