Mechanisms of Double-strand Break Repair in Yeast

描述：拟议研究的长期目标是了解 DNA双链断裂修复的细胞机制 真核生物。通过同源重组修复双链断裂对于 保持基因组的完整性和染色体的准确分离 在减数分裂期间。DNA修复途径中的缺陷与几个 人类的癌症易感综合征。最近的研究表明，人类的乳房 肿瘤基因BRCA1和BRCA2在同源重组途径中通过 它们与RAD51的相关性和细胞株的表型特征 亚形等位基因。因此，了解DSB修复的机制将 对于理解这些人类的病理很重要。在真核生物中， 重组修复途径的组成部分是高度保守的，并且 RAD51蛋白通过催化突触和链在DSB修复中起关键作用 同源双链之间的交换。然而，RAD51需要许多其他 蛋白质，包括复制因子A(RPA)、Rad52、RadS4、Rad55和Rad57， 以完成链交换。这项提议的主要目标是进一步 研究RAD51及其两种辅助蛋白Rad55和RAD55的作用 RAD57，在这个过程中。我们已经分离出一类新的RAD51等位基因 部分抑制了rad55和rad57菌株的DNA修复缺陷。这些 将接受遗传和生化分析以了解其机制 为了镇压。直接确定Rad55和Rad57在 在RAD51催化的链交换的刺激下，这两种蛋白都将得到纯化 体外测定DNA结合、ATPase和链交换活性 在RAD51和RPA存在或不存在的情况下。使用基因替换技术 在RAD51突变体中高效地出现了双链线状片段。 我们建议研究基因打靶的遗传控制，作用于 靶基因的转录，以及使用各种靶向的机制 向量。使用基因替换进行靶向基因破坏已经 应用于基因治疗，因此对其机制的理解是 根本性的重要性。

DESCRIPTION:The long-term objective of the proposed research is to understand the cellular mechanisms for the repair of DNA double strand breaks (DSBs) in eukaryotes. The repair of DSBs by homologous recombination is essential to maintain genome integrity and for the accurate segregation of chromosomes during meiosis. Defects in DNA repair pathways are associated with several cancer-prone syndromes in humans. Recent studies implicate the human breast cancer genes, BRCAI and BRCA2, in the homologous recombination pathway through their association with Rad51 and phenotypic characterization of cell lines with hypomorphic alleles. Therefore, understanding the mechanisms of DSB repair will be important for understanding these human pathologies. In eukaryotes, components of the recombinational repair pathway are highly conserved, and the Rad51 protein plays a key role in DSB repair by catalyzing synapsis and strand exchange between homologous duplexes. However, Rad51 requires a number of other proteins, including replication factor A (RPA), Rad52, RadS4, Rad55 and Rad57, to accomplish strand exchange. The major goal of this proposal is to further characterize the role of Rad51 and two of the accessory proteins, Rad55 and Rad57, in this process. We have isolated a novel class of rad51 alleles that partially suppress the DNA repair defects of rad55 and rad57 strains. These will be subject to genetic and biochemical analyses to understand the mechanism for suppression. To directly determine the role of Rad55 and Rad57 in stimulation of Rad5 1-catalyzed strand exchange, both proteins will be purified and characterized in vitro for DNA binding, ATPase and strand exchange activity in the presence or absence of Rad5 1 and RPA. Gene replacement using double-stranded linear fragments occurs with high efficiency in rad51 mutants. We propose to investigate the genetic control of gene targeting, role of transcription of the target locus, and mechanisms using a variety of targeting vectors. The use of gene replacement for targeted gene disruption has application for gene therapy, thus an understanding of the mechanism is of fundamental importance.