Genetic and Molecular Analysis of Yeast DNA Replication

酵母 DNA 复制的遗传和分子分析

• 批准号: 7908226
• 负责人: ROBERT A SCLAFANI
• 金额: $ 23.67万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7908226
• 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; cancer risk; chromosome replication; genetic analysis; helicase; human disease; in vivo; mutant; novel; public health relevance; recombinational repair

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.

描述（由申请人提供）：该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。低富度复制和容易出错的恢复后DNA修复会导致染色体非整倍性和突变，这会导致衰老，癌症和先天缺陷。该建议将采用分子遗传分析，以使用单细胞真核生物酿酒酵母作为模型系统来鉴定这些过程的调节分子。这种组合的遗传和分子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（Cyclin依赖性激酶）和RAD53检查点激酶。提出了三个具体目标。在AIM＃1中，我们将通过研究由DDK调节的N端和N端区域A域和N末端区域A域A和N端区域A域A域（beta）2（Beta）2（beta）2（beta） - 末端2（beta） - 末端2（beta）。我们的假设是，N末端2-Finger对于结合起源很重要，C末端手指对于解旋酶易位或沿DNA运动很重要。此外，我们提出，DDK对MCM复合物的磷酸化产生了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研究，对酵母中DNA复制的体内研究中保守的古细菌MCM解旋酶和分子遗传学研究。在AIM 2中，我们将研究RAD53在DNA复制中的新作用。我们目前的假设是RAD53调节蛋白质对于启动DNA复制和染色质结构重要。该角色与Rad53蛋白的检查点功能无关。在此目标中，我们将继续利用我们独特的CDC7-MCM5-RAD53-isHistone H3/H4“ Interactome”遗传系统，以识别更多相互作用的蛋白质，并将分析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（Trans-Lesion合成）来研究DNA复制的忠诚度和诱变的调节。我们的研究表明，DDK通过DNA聚合酶6（Zeta）调节自发和诱导的诱变。我们的假设是DDK充当Rev7的“染色质加载器”，这是Rev3易误后DNA聚合酶6的重要附件。DDK对于在DNA损伤和TLS后重新启动复制也可能很重要。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检验许多这些假设。我们的研究对人类疾病具有重要意义，因为RAD53（CHK2）和TLS聚合酶POL7（ETA-XPV）的人类同源物分别在家族性癌性li-fraumeni和静脉疾病中突变。 公共卫生相关性：由于拟议的研究集中于染色体复制的忠诚度和遗传突变的产生，因此它们与人类健康相关，就癌症，衰老和先天缺陷而言。已知该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。