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。重组是DNA复制和基因组稳定的基础，也是减数分裂过程中染色体分离的必要条件。我们的长期目标是了解重组的机制和调控。基本的重组反应是在断裂的染色体和同源模板之间通过配对和链交换形成联合分子（Joint Molecule， JM）中间体。多种DNA核酸酶和解旋酶在重组的每一步都发挥作用，并将体内功能分配给特定的酶，以及了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。具体目标表征假日路口分解的路径。我们已经确定了在减数分裂期间负责所有JM分解和交叉的五种活动。其中包括错配修复因子，Exo1， Mlh1和Mlh3；xpf家族核酸酶Mus81-Mms4；Slx4，形成两种不同的核酸酶复合物；最近鉴定的HJ解析瓶，燕城1号；RecQ解旋酶Sgs1在复合体中起解离dhj而不是溶解它们的作用。分子和遗传方法将用于在体内分配这些活动的特定作用，并测试减数分裂JM分辨率的特定模型。一种检测系统已经开发出来，这是第一次允许检测有丝分裂dsb修复过程中体内形成的JMs。这种独特的工具将用于确定JM解决因子在有丝分裂dsb修复中的作用。具体目标2。探讨DNA解旋酶在调节关节分子代谢中的作用。三种DNA解旋酶，Sgs1， Srs2和Mph1似乎独立地抑制有丝分裂dsb修复过程中的交叉。然而，在减数分裂期间，这些抗交叉因子必须在交叉位点被抑制，但可能需要完成非交叉重组。分子分析将用于确定这些解旋酶在减数分裂和有丝分裂dsb修复期间调节JM形成中的作用。减数分裂JM形成的具体模型和解旋酶在这一过程中的作用将被检查。我们还将测试Srs2在减数分裂期间被所谓的“中介”蛋白阻止破坏RecA蛋白Rad51和Dmc1的细丝的想法。最后，我们将研究另外两个解旋酶的分子作用：Hrq1，最近发现的RecQ4的真菌同源物，在rothmond - thomson综合征中发生突变；在有丝分裂dsb修复过程中，解旋酶/核酸酶Dna2在交叉上存在缺陷，有缺陷的重组与不孕、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。

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.