OBF1 AND INITIATION OF YEAST DNA REPLICATION

OBF1 和酵母 DNA 复制的启动

• 批准号: 2184244
• 负责人: SHLOMO EISENBERG
• 金额: $ 25.77万
• 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 1997-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2184244
• 关键词: DNA binding protein; DNA replication origin; Saccharomyces cerevisiae; chimeric proteins; eukaryote; fungal genetics; gene mutation; genetic enhancer element; genetic mapping; genetic transcription; immunofluorescence technique; laboratory rat; molecular cloning; phosphorylation; protein kinase; protein sequence; protein structure function; tissue /cell culture

In eukaryotic cells the regulation and mechanism of nuclear DNA replication is poorly understood. Our long-term objective is to gain better understanding of this process on the molecular level. The models systems used in these studies is a Saccharomyces cerevisiae origin of replication clone on plasmids. We have made the following major steps pointed towards achieving these goals: (i) we have delineated an yeast chromosomal origin of replication, the ARS121; (ii) we have purified and clone a protein, OBF1(also known as ABF1 and BAF1), that binds specifically to a DNA replication enhancer found in ARS121 and other yeast origins; (iii) we have now reconstituted in vitro an OBF1-dependent, putative multiprotein initiation complex at he ARS121 origin. These studies suggest that the function of OBF1 in initiation of DNA replication is to interact with another factor, OBF2, to promote the ATP-dependent binding of a third factor, CBF; (iv) we have shown that OBF1 is phosphorylated in vivo at multiple serine residues; (v) we have shown cell cycle-specific phosphorylation of a least three sites in the OBF1 protein; (vi) we have identified and purified a multiprotein complex kinase. These findings suggest a possible regulations of OBF1 functions by phosphorylation. The short-term objectives of this proposal are: (i) to elucidate the mechanism of the assembly of the putative initiation complex at the ARS121 origin. This will involve the purification and cloning of OBF2 and CBF and the analysis of protein-DNA and Protein-protein interactions at the ARS121 origin; (ii) to delineate the OBFI domains important for its function in replication; (iii) to understand the regulation of OBF1 function by phosphorylation. This will involve the identification, isolation and cloning of the kinases and phosphorylation. This will involve the identification, isolation and cloning of the kinases and phosphatases. It will also involve both in vitro and vivo analysis of the effect of ser/ala mutations in the OBFI phosphorylation sites on OBFI function replication.

在真核细胞中，核DNA的调节和机制 复制知之甚少。 我们的长期目标是获得 在分子水平上更好地理解这一过程。 模型 这些研究中使用的系统是酿酒酵母的起源 质粒上的复制克隆。 我们已经提出了以下重大步骤，指出了这些 目标：（i）我们描述了复制的酵母染色体起源， ARS121; （ii）我们已经纯化并克隆蛋白质OBF1（也已知 作为ABF1和BAF1），该abf1和baf1特别结合了DNA复制增强子 在ARS121和其他酵母起源中发现； （iii）我们现在已经重组了 体外依赖于OBF1的，推定的多蛋白启动复合物 他ARS121起源。 这些研究表明OBF1在 DNA复制的启动是与另一个因素obf2相互作用 为了促进第三个因素的ATP依赖性结合，CBF； （iv）我们有 表明在多个丝氨酸残基在体内磷酸化。 （v）我们显示了至少三个细胞周期特异性磷酸化 OBF1蛋白中的位点； （vi）我们已经确定并纯化了 多蛋白复合激酶。 这些发现表明 OBF1功能的可能法规通过磷酸化。 该提案的短期目标是：（i）阐明 假定启动复合物的组装机制 ARS121起源。 这将涉及OBF2的纯化和克隆 CBF以及蛋白质-DNA和蛋白质 - 蛋白质相互作用的分析 在ARS121起源； （ii）描绘OBFI领域对 它的复制功能； （iii）了解OBF1的调节 磷酸化功能。 这将涉及身份证明， 激酶和磷酸化的分离和克隆。 这会 涉及激酶的鉴定，隔离和克隆和克隆 磷酸酶。 它还将涉及体外和体内分析 OBFI磷酸化位点中Ser/ALA突变对OBFI的影响 功能复制。