Genetic and Molecular Analysis of Yeast DNA Replication

酵母 DNA 复制的遗传和分子分析

• 批准号: 8245889
• 负责人: ROBERT A SCLAFANI
• 金额: $ 31.6万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-09-06 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8245889
• 关键词: Aging; Aneuploidy; Antibody Affinity; Binding; Biochemical; Biological Models; C-terminal; Cell Cycle Proteins; Cell physiology; Cells; Chromatin; Chromatin Structure; Complex; Congenital Abnormality; Cyclin-Dependent Kinases; DNA; DNA Damage; DNA Repair; DNA biosynthesis; DNA polymerase zeta; DNA replication origin; DNA-Directed DNA Polymerase; Defect; Eukaryota; Evolution; Fingers; Funding; Gene Mutation; Genes; Genetic; Genetic Recombination; Genetic Screening; Genome; Health; Hereditary Malignant Neoplasm; Histone H3; Homologous Gene; Human; In Vitro; Knowledge; Lesion; Longitudinal Studies; Malignant Neoplasms; Measures; Meiotic Recombination; Metabolism; Molecular; Molecular Analysis; Molecular Genetics; Movement; Mutagenesis; Mutate; Mutation; N-terminal; Phosphorylation; Phosphotransferases; Physiological; Polymerase; Precipitation; Process; Production; Protein Kinase; Protein Region; Proteins; Regulation; Replication Error; Replication Initiation; Role; Saccharomyces cerevisiae; Saccharomycetales; Spo11 protein; Structure; Syndrome; System; Testing; Variant; Xeroderma Pigmentosum; Yeast Model System; Yeasts; abstracting; cancer risk; chromosome replication; genetic analysis; helicase; human disease; in vivo; mutant; novel; Aging; Aneuploidy; Antibody Affinity; Binding; Biochemical; Biological Models; C-terminal; Cell Cycle Proteins; Cell physiology; Cells; Chromatin; Chromatin Structure; Complex; Congenital Abnormality; Cyclin-Dependent Kinases; DNA; DNA Damage; DNA Repair; DNA biosynthesis; DNA polymerase zeta; DNA replication origin; DNA-Directed DNA Polymerase; Defect; Eukaryota; Evolution; Fingers; Funding; Gene Mutation; Genes; Genetic; Genetic Recombination; Genetic Screening; Genome; Health; Hereditary Malignant Neoplasm; Histone H3; Homologous Gene; Human; In Vitro; Knowledge; Lesion; Longitudinal Studies; Malignant Neoplasms; Measures; Meiotic Recombination; Metabolism; Molecular; Molecular Analysis; Molecular Genetics; Movement; Mutagenesis; Mutate; Mutation; N-terminal; Phosphorylation; Phosphotransferases; Physiological; Polymerase; Precipitation; Process; Production; Protein Kinase; Protein Region; Proteins; Regulation; Replication Error; Replication Initiation; Role; Saccharomyces cerevisiae; Saccharomycetales; Spo11 protein; Structure; Syndrome; System; Testing; Variant; Xeroderma Pigmentosum; Yeast Model System; Yeasts; abstracting; cancer risk; chromosome replication; genetic analysis; helicase; human disease; in vivo; mutant; novel

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.

项目摘要/摘要 该提议的长期目标是了解调节高保真性的细胞过程 真核DNA复制。低忠诚度复制和容易出错的复制后DNA修复可能会导致 染色体非整倍性和突变，会导致衰老，癌症和先天缺陷。该提议将 员工分子遗传分析，使用单个过程来鉴定这些过程的调节分子 细胞真核生物，酿酒酵母作为模型系统。这种遗传和分子的结合 方法将集中于许多重要的细胞周期蛋白激酶，其中包括DDK（CDC7-DBF4），CDK （依赖细胞周期蛋白的激酶）和RAD53检查点激酶。提出了三个具体目标。在AIM＃1中，我们 将通过研究N末端和C-来阐明MCM DNA解旋酶的功能和调节 末端�（beta） - fingers和N末端区域A域由DDK调节。我们的假设是n- 端子�-Finger对于结合起源很重要，C末端手指对于解旋酶很重要 沿着DNA的易位或运动。此外，我们提出了MCM复合物的磷酸化 由DDK产生MCM5 A域的结构变化，从而导致解旋酶激活。我们的系统 依赖于原子结构和生化的体外研究，对成本的古细菌MCM解旋酶和 分子遗传在酵母中DNA复制的体内研究。在AIM 2中，我们将研究RAD53的新作用 在DNA复制中。我们目前的假设是RAD53调节对DNA倡议重要的蛋白质 复制和染色质结构。该角色与Rad53蛋白的检查点功能无关。在这个 目的，我们将继续探索我们独特的CDC7-MCM5-RAD53-ESTONE H3/H4“ Interactome”遗传系统 确定更多相互作用的蛋白质，还将分析我们突变体中染色质的变化。在AIM 3中，我们 通过研究DDK在TLS中的作用，研究DNA复制的保真度和诱变的调节 （跨性质合成）。我们的研究表明，DDK调节了赞助和诱导 DNA聚合酶（Zeta）的诱变。我们的假设是DDK充当Rev7的“染色质加载器”， 易经Rev3错误的DNA聚合酶»的重要配件。 DDK对于复制也可能很重要 DNA损伤和TLS后重新启动。突变分析，全基因组遗传筛选的结合， CHIP（染色质免疫培训）和亲和力/抗体隔离将用于测试其中许多 假设。我们的研究对人类疾病具有重要意义，作为RAD53（CHK2）和 TLS聚合酶pol¿（ETA-XPV）在癌症癌的li-fraumeni和 心虫色素化变体综合征分别。