MECHANISM OF DNA RECOMBINATION AND REPAIR IN YEAST SACCHAROMYCES CEREVISIAE

酿酒酵母 DNA 重组与修复机制

• 批准号: 3965288
• 负责人: A SUGINO
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3965288
• 关键词: DNA directed DNA polymerase; DNA repair; Escherichia coli; Saccharomyces; antibody; antigens; chromosome deletion; computer data analysis; gene expression; genetic library; genetic manipulation; genetic mapping; genetic recombination; immunochemistry; molecular cloning; nucleic acid sequence; oligopeptides; plasmids; tissue /cell culture

Proteins binding to single-stranded DNA are expected to participate in DNA recombination and repair as well as DNA replication. Thus, three different single-stranded-DNA-binding proteins (SSB) have been purified from the yeast Saccharomyces cerevisiae and antibodies have been raised against them. Using the antibodies as probes, their genes have been identified and cloned from a Lambdagt11 yeast DNA library. Deletions of the genes were then constructed, the wild-type genes were replaced by the disrupted genes, and the resulting phenotypes were studied. 20kd SSB is essential for cell viability. However, C-terminal region of the 20kd SSB is still functional, but the cell is UV-sensitive. This strongly indicates that 20kd SSB is required for DNA repair and recombination as well as DNA replication. The RAD52 gene product is required for DNA recombination and repair in yeast. The gene has been cloned and its nucleotide sequence determined by other groups. However, this important gene product has not yet identified and purified. By aid of a computer we identified several possible antigenic regions in the RAD52 gene. The oligopeptides covering the antigenic regions were chemically synthesized and conjugated to BSA and antibodies were raised against the conjugates. In addition, several fusion plasmids of the RAD52 gene and either the LambdapL promoter or the yeast Alpha-mating type pheromon leader sequence or the yeast ADH promotor will be constructed in order to overproduce RAD52 protein in E. coli and yeast. Finally, one of the excision repair genes of yeast (RAD18), which also has been expected to be a subunit of yeast DNA polymerase I, has been cloned and characterized.

与单链DNA结合的蛋白质有望参与DNA 重组和修复以及DNA复制。 因此，三种不同的 单链DNA结合蛋白（SSB）已从 酵母酿酒酵母和抗体已经被提出， 他们 使用抗体作为探针，它们的基因已经被鉴定， 克隆自Lambdagt 11酵母DNA文库。 基因的缺失是 然后构建，野生型基因被破坏的基因取代， 并研究了所得的表型。 20 kd SSB是细胞所必需的 生存能力 然而，20 kd SSB的C-末端区域仍然具有功能， 但细胞对紫外线敏感 这强烈表明20 kd SSB是 这是DNA修复和重组以及DNA复制所必需的。 的 RAD 52基因产物是酵母中DNA重组和修复所必需的。 该基因已被克隆，其核苷酸序列已由其他人确定。 组 然而，这一重要的基因产物尚未确定， 提纯 借助计算机，我们鉴定了几种可能的抗原性。 RAD 52基因的一部分。 覆盖抗原的寡肽 化学合成区域并与BSA和抗体缀合 对共轭物的反应。 此外，几种融合质粒 RAD 52基因和LambdapL启动子或酵母 α-交配型信息素前导序列或酵母ADH启动子将 目的是在大肠杆菌中高效表达RAD 52蛋白。大肠杆菌和酵母菌。 最后，酵母的切除修复基因之一（RAD 18），其也具有 被认为是酵母DNA聚合酶I的一个亚基，已被克隆 和表征了