Joint Molecule Formation During Recombination

描述（由申请人提供）：当断裂或受损的染色体使用同源染色体作为修复模板时，发生同源重组。脱附是DNA复制和基因组稳定性的基础，并且是减数分裂期间染色体分离所必需的。我们的长期目标是了解重组的机制和调控。基本的重组反应是通过断裂的染色体和同源模板之间的配对和链交换形成联合分子（JM）中间体。DNA核酸酶和解旋酶的多样性在重组的每个步骤中发挥作用，并将体内功能分配给特定的酶，以及理解它们在重组过程中如何相互作用仍然是具有挑战性的问题。本研究将探讨核酸酶和解旋酶在JM代谢中的体内作用。 具体目标1。表征霍利迪结解析的途径。我们已经确定了五个活动负责基本上所有的JM决议和交换过程中减数分裂。这些包括错配修复因子，Exo 1，Mlh 1和Mlh 3; XPF家族核酸酶，Mus 81-Mms 4; Slx 4，它形成两种不同的核酸酶复合物;最近鉴定的HJ解离酶，Yen 1;和RecQ解旋酶，Sgs 1，它在复合物中起解离dHJ的作用，而不是解决它们。分子和遗传学方法将被用来分配特定的角色，这些活动在体内和测试特定模型的减数分裂JM决议。已经开发了一种测定系统，其首次允许检测在有丝分裂DSB修复期间体内形成的JM。这种独特的工具将被用来确定JM解决有丝分裂DSB修复因子的作用。 具体目标2。确定DNA解旋酶在调节关节分子代谢中的作用。 三个DNA解旋酶，Sgs 1，Srs 2和Mph 1似乎独立地发挥作用，以抑制在有丝分裂DSB修复过程中的交换。然而，在减数分裂期间，这些反交换因子必须在交换位点被抑制，但可能需要完成非交换重组。分子检测将用于确定这些解旋酶在减数分裂和有丝分裂DSB修复过程中调节JM形成的作用。一个特定的模型减数分裂JM形成和解旋酶在这个过程中的作用将被检查。我们还将测试Srs 2在减数分裂过程中被所谓的“介体”蛋白阻止破坏RecA蛋白Rad 51和Dmc 1的细丝的想法。最后，我们将研究另外两种解旋酶的分子作用：Hrq 1，最近发现的RecQ 4的真菌同源物，在Rothmund-Thomson综合征中突变;和解旋酶/核酸酶Dna 2，在有丝分裂DSB修复过程中有缺陷的交叉重组与不育，妊娠流产和遗传疾病有关。在体细胞中与癌症特别相关。了解同源重组的分子过程将有助于我们更好地了解这些疾病的病因。 公共卫生相关性：同源重组是有性生殖和染色体修复所必需的。这一过程的缺陷与人类不育、流产和遗传疾病，特别是癌症有关。对同源重组机制和调控的深入了解将有助于我们更好地理解这些疾病的病因并设计新的治疗方法。

DESCRIPTION (provided by applicant): Homologous recombination occurs when a broken or damaged chromosome uses a homologous chromosome as repair template. Recombination underpins DNA replication and genome stability, and is essential for chromosome segregation during meiosis. Our long-term goal is to understand the mechanism and regulation of recombination. The fundamental recombination reaction is the formation of Joint Molecule (JM) intermediates via pairing and strand-exchange between a broken chromosome and a homologous template. A multiplicity of DNA nucleases and helicases function during every step of recombination and assigning in vivo functions to specific enzymes, and understanding how they interact during recombination remain challenging issues. This proposal will investigate the in vivo roles of nucleases and helicases in JM metabolism. Specific Aim 1. To Characterize the Pathways of Holliday Junction Resolution. We have identified five activities responsible for essentially all JM resolution and crossing-over during meiosis. These include mismatch repair factors, Exo1, Mlh1 and Mlh3; XPF-family nuclease, Mus81-Mms4; Slx4, which forms two distinct nuclease complexes; the recently identified HJ resolvase, Yen1; and RecQ helicase, Sgs1, which functions in a complex to dissociate dHJs rather than resolve them. Molecular and genetic approaches will be used to assign specific roles to these activities in vivo and test specific models of meiotic JM resolution. An assays system has been developed that, for the first time, allows detection of JMs formed in vivo during mitotic DSB-repair. This unique tool will be used to determine the roles of JM resolving factors in mitotic DSB-repair. Specific Aim 2. To Determine the Roles of DNA Helicases in Regulating Joint Molecule Metabolism. Three DNA helicases, Sgs1, Srs2 and Mph1 appear to function independently to suppress crossing-over during mitotic DSB-repair. However, during meiosis, these anti-crossover factors must be inhibited at sites of crossing-over, but may be required to complete non-crossover recombination. Molecular assays will be used to determine the roles of these helicases in regulating JM formation during meiosis and during mitotic DSB-repair. A specific model of meiotic JM formation and the roles of helicases in this process will be examined. We will also test the idea that Srs2 is prevented from disrupting filaments of RecA proteins, Rad51 and Dmc1, during meiosis by the so-called "mediator" proteins. Finally, we will examine the molecular roles of two additional helicases: Hrq1, a recently identified fungal homolog of RecQ4, which is mutated in Rothmund-Thomson syndrome; and the helicase/nuclease Dna2, which is defective for crossing-over during mitotic DSB-repair Defective recombination is associated with infertility, pregnancy miscarriage and genetic disease. In somatic cells is especially relevant for cancer. An understanding of the molecular processes of homologous recombination will help us better understand the etiology of these disorders. PUBLIC HEALTH RELEVANCE: Homologous recombination is required for sexual reproduction and chromosome repair. Defects in this process are linked to human infertility, miscarriage and genetic diseases, especially cancer. A greater understanding of the mechanism and regulation of homologous recombination will help us better understand the etiology of these diseases and design novel therapies.