Genetic and Molecular Analysis of Yeast DNA Replication

项目摘要/摘要 这项提议的长期目标是了解调节高保真的细胞过程。 真核DNA复制。低保真复制和容易出错的复制后DNA修复可能会导致 染色体非整倍体和突变，可导致衰老、癌症和出生缺陷。这项提议将 使用分子遗传分析来识别这些过程的调节分子，使用单一的- 以真核细胞、酿酒酵母为模型系统。这是遗传和分子的结合 方法将集中在一些重要的细胞周期蛋白激酶，包括DDK(CDC7-Dbf4)，CDK (细胞周期蛋白依赖性激酶)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-末端来阐明MCM DNA解旋酶的功能和调节 末端(Beta)-指和N-末端区域A受DDK调控。我们的假设是N- 末端手指对结合起点很重要，C末端手指对解旋酶很重要 沿着DNA的移位或移动。此外，我们认为MCM复合体的磷酸化 通过DDK使MCM5的A结构域发生结构变化，导致解旋酶激活。我们的系统 依赖于保守的古生菌MCM解旋酶的原子结构和体外生化研究 酵母DNA复制的分子遗传学活体研究。在目标2中，我们将研究Rad53的一个新角色 在DNA复制中。我们目前的假设是，Rad53调节对启动dna非常重要的蛋白质。 复制和在染色质结构中。这种作用不依赖于RAD53蛋白的检查点功能。在这 目的，我们将继续利用我们独特的cdc7-mcm5-rad53-组蛋白H3/H4“互动组”遗传系统来 识别更多相互作用的蛋白质，并将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TLS中的作用来研究DNA复制的保真度和突变的调节 (跨病变合成)。我们的研究表明，DDK既可以调节自发的，也可以调节诱导的 DNA聚合酶(Zeta)诱变。我们的假设是，DDK作为Rev7的“染色质加载器”， Rev3容易出错的DNA聚合酶的一个重要附件。DDK对于复制可能也很重要 DNA损伤和TLS后重新启动。突变分析，全基因组基因筛查， ChIP(染色质免疫沉淀)和亲和力/抗体分离将用于检测其中的许多 假设。我们的研究对人类疾病具有重要意义，因为Rad53(Chk2)和Rad53(Chk2)的人类同源物 TLS聚合酶Pol？(ETA-XPV)在家族性癌易感Li-Fraumeni和 分别为变异型色素性干皮病综合征。

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.