Enzymatic Mechanisms of DNA Replication

DNA 复制的酶促机制

• 批准号: 7035384
• 负责人: Judith L CAMPBELL
• 金额: $ 38.86万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-09-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7035384
• 关键词: DNA directed DNA polymerase; DNA repair; DNA replication; X ray crystallography; cell cycle; clinical research; enzyme activity; enzyme mechanism; fluorescence microscopy; fungal proteins; genetic crossing over; green fluorescent proteins; immunoprecipitation; isozymes; laboratory rabbit; monoclonal antibody; point mutation; proliferating cell nuclear antigen; protein localization; protein protein interaction; protein purification; site directed mutagenesis; two dimensional gel electrophoresis

DESCRIPTION (provided by applicant): Genomic approaches and the discovery that E. coli UmuC and DinB, yeast RAD30 and Xeroderma pigmentosum variant (XPV) encode DNA polymerases have led to an explosion in the number of known DNA polymerases. We have discovered, originally via a two hybrid screen, that the essential but non-catalytic C terminal domain of yeast DNA polymerase (pol) epsilon, a polymerase required for DNA replication, repair, and the DNA replication checkpoint, interacts both physically and functionally with one of the most novel of the new polymerases, pol sigma. Yeast pol sigma mutants are defective in completion of S phase, in DNA repair, in chromosome condensation, and in sister chromatid cohesion. The discovery of this interaction lends a new dimension to our continuing search for the precise role of pot epsilon in chromosome dynamics during S phase. We find that pol epsilon mutants are defective in cohesion. The overall goat of our studies is to determine how pol epsilon and pol sigma interact in these various cellular processes. First, to define the in vivo role of the interaction, structure/function analysis will be carried out by using site directed mutations and correlating effects on interaction between the two proteins with phenotypes of resulting mutants, especially with respect to sister chromatid cohesion. Second, we will characterize pol sigma biochemically- definition of substrates, fidelity, processivity, and stimulation by PCNA. A defining aspect of our studies is focus on the interaction between pol sigma and pot epsilon. We will also address pol epsilon's interaction with another cohesion protein,Ctf18, a component of an alternative polymerase clamp loader. The key questions in the nascent field of sister chromatid cohesion are what is the nature of the links between the chromosomes and what is the mechanism by which DNA replication contributes to forming these links. With this in mind, we will determine if pol epsilon is required for establishing cohesion and/or maintenance. We will study the fate of replication forks at sites of cohesion in vivo and the association of polymerases with these sites. We will use chromatin immunoprecipitation, 2D gel electrophoresis of DNA replication intermediates, and microscopic analysis of chromosome dynamics using fluorescence microscopy and the GFP-repressor/operator tagging strategy, techniques set up by us during the previous granting period. Several collaborations are in place to insure progress.

描述（由申请人提供）： 基因组学的方法和发现，E。大肠杆菌UmuC和DinB、酵母RAD 30和着色性干皮病变异体（XPV）编码的DNA聚合酶导致已知DNA聚合酶的数量激增。 我们已经发现，最初通过双杂交筛选，酵母DNA聚合酶（pol）的基本但非催化的C末端结构域，DNA复制，修复和DNA复制检查点所需的聚合酶，与最新的聚合酶之一，pol sigma在物理和功能上相互作用。 酵母pol sigma突变体在S期完成、DNA修复、染色体凝聚和姐妹染色单体凝聚方面有缺陷。 这种相互作用的发现为我们继续寻找在S期染色体动力学中potaxis的确切作用提供了一个新的维度。 我们发现，聚合酶突变体的凝聚力是有缺陷的。 我们研究的总体目标是确定聚合酶和聚合酶在这些不同的细胞过程中如何相互作用。 首先，为了定义相互作用的体内作用，将通过使用定点突变和对两种蛋白质之间的相互作用的相关效应与所得突变体的表型（特别是关于姐妹染色单体凝聚）进行结构/功能分析。 第二，我们将描述polsigma生物化学-底物的定义，保真度，持续合成能力和PCNA的刺激。我们的研究的一个定义方面是专注于polsigma和potsigma之间的相互作用。 我们还将解决聚合酶的相互作用与另一个凝聚力蛋白，Ctf 18，一个替代聚合酶钳加载的组成部分。 在姐妹染色单体凝聚力的新生领域的关键问题是什么是染色体之间的联系的性质是什么，是什么机制，DNA复制有助于形成这些链接。 考虑到这一点，我们将确定是否需要建立内聚性和/或维护。 我们将研究复制叉的命运在网站的凝聚力在体内和协会的聚合酶与这些网站。 我们将使用染色质免疫沉淀，DNA复制中间体的2D凝胶电泳，以及使用荧光显微镜和GFP阻遏物/操纵子标记策略的染色体动力学显微镜分析，这些技术是我们在前一个授予期间建立的。 为确保取得进展，已经开展了几项合作。

项目成果

Role of multifunctional autonomously replicating sequence binding factor 1 in the initiation of DNA replication and transcriptional control in Saccharomyces cerevisiae.

多功能自主复制序列结合因子 1 在酿酒酵母 DNA 复制启动和转录控制中的作用。

• DOI: 10.1128/mcb.12.3.1064-1077.1992
• 发表时间: 1992
• 期刊: Molecular and cellular biology
• 影响因子: 5.3
• 作者: Rhode,PR;Elsasser,S;Campbell,JL
• 通讯作者: Campbell,JL

Cloning of Saccharomyces cerevisiae DNA replication genes: isolation of the CDC8 gene and two genes that compensate for the cdc8-1 mutation.

酿酒酵母 DNA 复制基因的克隆：分离 CDC8 基因和两个补偿 cdc8-1 突变的基因。

• DOI: 10.1128/mcb.3.10.1730-1737.1983
• 发表时间: 1983
• 期刊: Molecular and cellular biology
• 影响因子: 5.3
• 作者: Kuo,CL;Campbell,JL
• 通讯作者: Campbell,JL

Purification of DNA polymerase II stimulatory factor I, a yeast single-stranded DNA-binding protein.

DNA 聚合酶 II 刺激因子 I（一种酵母单链 DNA 结合蛋白）的纯化。

• DOI: 10.1073/pnas.87.2.677
• 发表时间: 1990
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Brown,WC;Smiley,JK;Campbell,JL
• 通讯作者: Campbell,JL

Evidence that yeast SGS1, DNA2, SRS2, and FOB1 interact to maintain rDNA stability.

有证据表明酵母 SGS1、DNA2、SRS2 和 FOB1 相互作用以维持 rDNA 稳定性。

• DOI: 10.1016/j.mrfmmm.2003.08.015
• 发表时间: 2003
• 期刊: Mutation research
• 作者: Weitao,Tao;Budd,Martin;Campbell,JudithL
• 通讯作者: Campbell,JudithL

DNA polymerase III, a second essential DNA polymerase, is encoded by the S. cerevisiae CDC2 gene.

DNA 聚合酶 III 是第二种必需的 DNA 聚合酶，由酿酒酵母 CDC2 基因编码。

• DOI: 10.1016/0092-8674(89)90582-5
• 发表时间: 1989
• 期刊: Cell
• 影响因子: 64.5
• 作者: Sitney,KC;Budd,ME;Campbell,JL
• 通讯作者: Campbell,JL