DNA REPLICATION IN SACCHAROMYCES CEREVISIAE

酿酒酵母中的 DNA 复制

• 批准号: 6385598
• 负责人: CAROL S. NEWLON
• 金额: $ 58.59万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-04-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6385598
• 关键词: DNA replication; DNA replication origin; Saccharomyces cerevisiae; binding sites; chromosome deletion; chromosomes; fungal genetics; genetic recombination; genetic regulatory element; genetic screening; molecular cloning; nucleic acid structure

The long-term objective of this work is to understand how a eukaryotic chromosome replicates. To this end, a replication map of chromosome III of the yeast, Saccharomyces cerevisiae, has been prepared. This map includes the positions of ARS elements, detected by their ability to promote the autonomous replication of plasmids, the positions of DNA replication origins, detected by 2D gel analysis of replication intermediates, and the positions of DNA replication termination. This proposal addresses three major issues concerning the replication and maintenance of chromosome III. First, both genetic and molecular approaches will be used to study the mechanism(s) by which a derivative of this chromosome lacking active chromosomal replicators. Second, the mechanism(s) by which chromosomal replicator activity is influenced by chromosomal context will be studied by examining the temporal pattern of replication of the chromosome and by using several genetic and molecular approaches designed to map the determinants of replicator activity. Finally, the structure of several chromosome III replicators will be further defined using both genetic and biochemical approaches. The binding sites for the replicator initiator protein, ORC, will be identified, and novel proteins that bind to replicators will be sought with a genetic screen. Replication timing determinants in ARS301 will be identified, and the role of ARS304 in the function of the recombination enhancer that activates HML as a donor of information in mating type switching will be analyzed. These studies will provide new insights into a fundamental cellular process, chromosome replication. Knowledge gained from the genetically tractable yeast system is applicable to larger eukaryotes, and guides the approaches to these experimentally difficult systems. The proposed studies are also likely to lead to a further understanding of chromosome rearrangements and the reactivation of DNA replication that are characteristics of malignant cells.

这项工作的长期目标是了解真核染色体如何复制。 为此，已经制备了酵母菌III染色体III的复制图，即酿酒酵母。 该地图包括ARS元素的位置，这些位置是由它们促进质粒自主复制的能力，DNA复制起源的位置，通过对复制中间体的2D凝胶分析以及DNA复制终止的位置检测到的。该提案解决了有关复制和维护III的三个主要问题。 首先，遗传和分子方法都将用于研究该染色体缺乏活性染色体复制剂的衍生物的机制。 其次，通过检查染色体复制的时间模式以及使用旨在映射复制剂活性的决定因素的几种遗传和分子方法，将研究染色体复制剂活性受染色体环境影响的机制。 最后，将使用遗传和生化方法进一步定义几个染色体III染色体的结构。 将鉴定复制器引发剂蛋白ORC的结合位点，并通过遗传筛选来寻求与复制剂结合的新型蛋白质。 将确定ARS301中的复制正时决定因素，并将分析ARS304在重组增强子功能中的作用，该重组增强子的功能将分析HML作为交配类型切换中信息供体的供体。这些研究将为基本细胞过程，染色体复制提供新的见解。 从遗传上的酵母系统中获得的知识适用于较大的真核生物，并指导这些实验困难系统的方法。 拟议的研究还可能导致对染色体重排的进一步理解，而DNA复制的重新激活是恶性细胞的特征。