Genetic and Molecular Analysis of Yeast DNA Replication

描述(由申请人提供)：这项提案的长期目标是了解调控高保真真核DNA复制的细胞过程。低保真复制和容易出错的复制后DNA修复会导致染色体非整倍体和突变，这可能会导致衰老、癌症和出生缺陷。这项建议将使用单细胞真核生物酿酒酵母作为模型系统，使用分子遗传分析来识别这些过程的调控分子。这种遗传和分子相结合的方法将重点放在一些重要的细胞周期蛋白激酶上，包括DDK(CDC7-Dbf4)、CDK(周期蛋白依赖激酶)和Rad53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N-末端和C-末端2(β)-指以及受DDK调控的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-末端的2个手指对于结合起点是重要的，而C-末端的手指对于解旋酶的移位或沿DNA的移动是重要的。此外，我们认为DDK对MCM复合体的磷酸化会导致MCM5的A结构域的结构变化，从而导致解旋酶的激活。我们的系统依赖于保守的古生菌MCM解旋酶的体外原子结构和生化研究，以及在酵母中DNA复制的分子遗传学体内研究。在目标2中，我们将研究Rad53在DNA复制中的一个新角色。我们目前的假设是，Rad53调节对启动DNA复制和染色质结构至关重要的蛋白质。这种作用不依赖于RAD53蛋白的检查点功能。为此，我们将继续利用我们独特的cdc7-mcm5-rad53-组蛋白H3/H4“相互作用组”遗传系统来识别更多相互作用的蛋白质，并分析我们突变体中染色质的变化。在目标3中，我们通过研究DDK在跨损伤合成(TLS)中的作用来研究DNA复制的保真度和突变的调节。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Rev7的“染色质加载器”，Rev7是容易出错的DNA聚合酶6的重要附件。DDK对于DNA损伤和TLS后的复制重启也可能是重要的。突变分析、全基因组遗传筛选、染色质免疫沉淀芯片和亲和力/抗体分离将用于检验其中许多假说。我们的研究对人类疾病具有重要意义，因为人类Rad53同源物(Chk2)和TLS聚合酶Pol7(ETA-XPV)分别在家族性癌症易感Li-Fraumeni和色素性干皮病变异综合征中发生突变。 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，因此它们与癌症、衰老和出生缺陷方面的人类健康相关。在这个酵母模型系统中研究的几个人类基因的缺陷被认为会增加癌症风险。

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.