MECHANISM OF DNA RECOMBINATION AND REPAIR IN YEAST SACCHAROMYCES CEREVISIAE

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结合蛋白已从酵母中纯化 酿酒酵母和针对它们的抗体已经被提出。 利用这些抗体作为探针，已经鉴定并克隆了它们的基因 来自Lambdagtll酵母DNA文库。 这些基因的缺失， 构建后，野生型基因被破坏的基因取代， 研究所得表型。 需要RAD 52基因产物 用于酵母中的DNA重组和修复。 该基因已被克隆， 其核苷酸序列由其它组决定。 但这 重要的基因产物尚未鉴定和纯化。 借助 通过计算机，我们在RAD 52中鉴定了几个可能的抗原区域， 基因 覆盖抗原区域的寡肽被化学修饰。 合成并与BSA缀合，并产生针对 共轭 此外，几个融合质粒的RAD 52基因和 LambdapL启动子或酵母α-交配型信息素前导序列 序列或酵母ADH启动子将被构建，以便 在E.大肠杆菌和酵母菌。 最后，体外DNA 重组系统酵母Delta-Delta序列已经开发。 这 系统需要ATP、MgCl 2和超螺旋质粒DNA，所述超螺旋质粒DNA至少含有 两个delta序列;它在一个delta序列附近或在一个delta序列处产生双链断裂 但不切割缺乏δ序列的质粒DNA。

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 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 Lambdagtll yeast DNA library. Deletions of these genes were then constructed, the wild-type genes were replaced by the disrupted genes, and the resulting phenotypes were studied. 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, an in vitro DNA recombination system yeast Delta-Delta sequences has been developed. This system requires ATP, MgCl2, and supercoiled plasmid DNA containing at least two delta sequences; it generates a double-strand break near or at the one of delta sequences but does not cleave plasmid DNA lacking delta sequences.