ROLE OF OBF1 IN INITIATION OF YEAST DNA REPLICATION

OBF1 在酵母 DNA 复制启动中的作用

• 批准号: 3306210
• 负责人: SHLOMO EISENBERG
• 金额: $ 20.97万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-30 至 1993-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3306210
• 关键词: DNA replication; DNA replication origin; affinity chromatography; chimeric proteins; eukaryote; fungal genetics; genetic enhancer element; molecular cloning; phosphorylation; polymerase chain reaction; protein kinase; protein structure function; yeasts

DESCRIPTION (Adapted from the applicant's abstract): In eukaryotic cells the mechanism and regulation of DNA replication, a process of fundamental importance for normal cell proliferation, is not understood. The long term objective is to gain a better understanding of this process on the biochemical and genetic level. The investigators believe that progress in this area hinges upon their ability to elucidate the mechanism of initiation of replication at a single replicon. Thus understanding of the structural organization of an origin of replication is necessary and the identification and characterization of the proteins responsible for DNA replication are crucial. The investigators have recently completed a systematic structural analysis of the yeast nuclear origin, the ARS121. The investigators have also purified and characterized the OBF1 protein and cloned its gene. Based on these studies the investigators have suggested that OBF1, a multiple phosphorylated protein in vivo, acts as an enhancer of DNA replication. The investigators objective in the proposal is to elucidate the mechanism of OBF1 function as it pertains to its role in DNA replication. To attain this objective the short term specific aims are: (I) to develop an in vivo functional assay for OBF1 action in replication by (i) constructing and expressing lexA-OBF1 protein fusions and (ii) testing the action of these fusions on chimeric origins containing the bacterial lexA-binding site instead of the OBF1 recognition sequences; (II) To use such an assay to dissect the OBF1 protein in order to identify the origin activation domain. This will be performed by generating truncated OBF1 fused to lexA; (III) to isolate, by affinity chromatography procedures, and clone the genes of the remaining components of the ARS initiation replication apparatus. These will entail fractionation of yeast extracts using specific DNA-cellulose and OBF-1 sepharose affinity procedures; (IV) to identify OBF1 phosphorylated domains and correlate it with the origin activation domain; (V) to identify the OBF1 protein kinase and analyses the phosphorylation process in the context of the cell cycle. By achieving these short term goals, the investigators will be able to resolve and reconstitute in vitro the initiation replication apparatus, which will provide the means for a biochemical analysis of initiation of replication of a eukaryotic replicon. In addition, the investigators will gain a deeper understanding of the regulatory mechanisms that govern the activation of origins of replication in eukaryotic cells.

描述（改编自申请人的摘要）：在真核中 细胞DNA复制的机理和调节 对正常细胞增殖的根本重要性尚不清楚。 长期目标是对此有更好的理解 生化和遗传水平的过程。 调查人员相信 这一领域的进展取决于他们阐明 在单个复制子处复制的机理。 因此 理解复制起源的结构组织 是必要的，蛋白质的识别和表征 负责DNA复制至关重要。 调查人员有 最近完成了酵母核的系统结构分析 起源，ARS121。 调查人员还纯化了 表征了OBF1蛋白并克隆其基因。 基于这些 研究研究者提出了obf1，一个倍数 体内磷酸化的蛋白质是DNA复制的增强剂。 该提案的研究人员目的是阐明 OBF1的机制功能与其在DNA中的作用有关 复制。 为了实现这一目标，短期特定的目的是： （i）开发用于OBF1动作的体内功能测定 通过（i）构建和表达Lexa-Obf1蛋白融合的复制 （ii）测试这些融合对嵌合起源的作用 包含细菌Lexa结合位点，而不是OBF1 识别序列； （ii）使用这种测定法剖析OBF1 蛋白质以识别原点活化域。 这将是 通过生成融合到Lexa的截短的OBF1来执行； （iii）分离， 通过亲和色谱程序，并克隆该基因 ARS启动复制设备的其余部分。 这些 将需要使用特定的DNA-纤维素来分馏酵母提取物 和OBF-1 Sepharose亲和力程序； （iv）识别obf1 磷酸化结构域并将其与原点激活相关联 领域; （v）鉴定OBF1蛋白激酶并分析 在细胞周期的背景下的磷酸化过程。 通过实现 这些短期目标，调查人员将能够解决并 在体外重新构建启动复制设备，这将 提供了对开始的生化分析的手段 真核复制子的复制。 此外，调查人员 将对监管机制有更深入的了解 控制真核细胞中复制起源的激活。