Biochemical Analysis of Genetic Recombination in Yeast

描述(申请人提供)：本研究的长期目标 计划是了解同源重组的分子机制 在真核生物中。同源重组在这一过程中起着两个重要作用。 大多数生物的生命周期。它被要求修复DNA中的致命损伤， 例如双链断裂，这对配对和 减数分裂过程中同源染色体的分离。这些因素的重要性 功能的证据是增加的突变，有丝分裂和减数分裂 在没有重组的情况下的非整倍体。因为许多易患癌症的人类 综合征与基因组不稳定性的增加有关，对 重组的机制很可能对理解 这些疾病。我们的目标是确定需要的基因和蛋白质 酿酒酵母中的同源重组。手术切除 产生单链DNA的DNA末端是 重组。Mre11p是一种核酸酶，需要处理 减数分裂特异的双链断裂(DSB)，但Mre11核酸酶活性 在有丝分裂细胞中处理HO诱导的DSB不是必需的。核酸酶 将使用基因筛查或通过以下方式识别mre11的冗余 筛选酵母GST融合蛋白文库。Mre11建筑群将是 从有丝分裂和减数分裂细胞中纯化鉴定新的相关因子 与Mre11结合，并用于生化鉴定。我们开发了一个殖民地 有丝分裂重组相关基因的颜色扇形分析。 利用这一方法，我们鉴定了RAD51非依赖的重组途径 这需要Rad52的同源物，Rad59。RAD51--独立重组 通过两条主要途径发生，即断裂诱导复制(BIR)和单链复制 退火(SSA)。RAD59在BIR和寡核苷酸导向基因中的作用 目标将被确定。RAD59残基对DNA修复和 RAD51-独立重组将被确定。学习与研究 RAD52/RAD59复合体将阐述复合体在链退火中的作用 和链入侵启动DNA合成。其他在人体内起作用的基因 RAD51非依赖的重组途径将通过使用 菌落颜色扇形分析及其产物与Rad59的相互作用 双混合屏幕

DESCRIPTION (provided by applicant): The long-term objective of this research program is to understand the molecular mechanisms of homologous recombination in eukaryotes. Homologous recombination plays two essential roles during the life cycle of most organisms. It is required to repair lethal lesions in DNA, such as double-strand breaks, and it is essential for the pairing and segregation of homologous chromosomes during meiosis. The importance of these functions is evidenced by increased mutagenesis, and mitotic and meiotic aneuploidy in the absence of recombination. Since many human cancer-prone syndromes are associated with increased genome instability, an understanding of the mechanisms of recombination is likely to be important in understanding these diseases. Our goals are to identify the genes and proteins required for homologous recombination in the yeast, Saccharomyces cerevisiae. The resection of DNA ends to generate single-stranded DNA is an essential step in recombination. Mre11p is a nuclease and is required to process meiosis-specific double-strand breaks (DSBs), but the Mre11 nuclease activity is not required for processing HO-induced DSBs in mitotic cells. Nucleases that are redundant with Mre11 will be identified using genetic screens or by screening a library of yeast GSTfusion proteins. The Mre11 complex will be purified from mitotic and meiotic cells to identify novel associated factors with Mre11 and for biochemical characterization. We developed a colony color-sectoring assay to identify genes involved in mitotic recombination. Using this assay we characterized a RAD51-independent recombination pathway that requires the Rad52 homologue, Rad59. RAD51-independent recombination occurs by two major pathways, break-induced replication (BIR) and single-strand annealing (SSA). The role of RAD59 in BIR and oligonucleotide-directed gene targeting will be determined. Residues of Rad59 important for DNA repair and RAD51-independent recombination will be identified. Studies with the Rad52/Rad59 complex will address the role of the complex in strand annealing and strand invasion to prime DNA synthesis. Other genes that function in the RAD51 -independent recombination pathway will be identified using the colony-color sectoring assay and by interaction of their products with Rad59 in a two hybrid screen