Understanding the Basis of Fidelity in Eukaryotic Recombinases

为了生存，所有的生物都进化出了各种修复受损的机制 DNA在双链断裂(DSB)中，同源重组(HR)是关键 使用同源DNA作为模板进行修复的修复机制。有丝分裂中的失败 重组可导致人类染色体重排和癌症，如BRCA1所见 而BRCA2突变和减数分裂重组失败可导致不孕、流产和 非整倍体疾病，如唐斯综合征。 在DSB形成后，受损的DNA最终会经过一系列步骤进行处理 使单链DNA突出部分被一种称为重组酶的蛋白质丝所覆盖。这些 然后重组酶搜索基因组以找到同源序列，然后将其用作 修复受损DNA的模板。真核生物有两种重组酶，RAD51和Dmc1。 RAD51是有丝分裂过程中唯一的重组酶，而Dmc1仅在有丝分裂过程中表达 减数分裂。目前还不知道为什么大多数真核生物有两种重组酶。然而，最近的一篇论文 在本实验室发表的文章展示了RAD51和RAD51之间的第一个生化/生物物理差异 Dmc1。当在同源DNA配对过程中出现错配时，Dmc1似乎稳定下来 错配，而RAD51似乎破坏了错配的稳定性。这种不同的响应可以 反映每个重组酶独特的生物学作用：RAD51负责有丝分裂HR，以及 通常使用相同的姐妹染色单体作为修复模板；相比之下，Dmc1必须使用 不同亲本来源的减数分裂HR的同源基因。我们认为Dmc1稳定的能力 不匹配反映了促进承载单个模板的重组的要求 减数分裂过程中的核苷酸多态。 在这个提案中，我们想了解哪些结构元素允许RAD51和 Dmc1对不匹配的行为有所不同。我们将确定两者的DNA结合区 重组酶，确定在每个重组酶中唯一保守的氨基酸 并交换这些元素以制造嵌合蛋白。我们将测试这些嵌合体是否 与野生型相比，蛋白质对错配产生相反的反应。在 提议，我已经证明了我可以创造一个稳定的RAD51嵌合体 不匹配，并将尝试创建可破坏不匹配的Dmc1嵌合体。那我会的 通过将我的嵌合体整合到 酵母基因组和监测体内有丝分裂和减数分裂HR。这项提案将试图提供 对真核重组保真度的进一步认识和意义。

In order to survive, all organisms have evolved various mechanisms for repairing damaged DNA. In the case of double-stranded breaks (DSBs), homologous recombination (HR) is a critical repair mechanism that uses homologous DNA as a template for repair. Failures in mitotic recombination can lead to chromosomal rearrangements and cancer in humans, as seen in BRCA1 and BRCA2 mutations and failures in meiotic recombination can lead to infertility, miscarriage, and aneuploidy disorders, such as Downs Syndrome. Upon formation of a DSB, the damaged DNA is processed through a series of steps eventually leaving single-stranded DNA overhangs covered in a filament of proteins called recombinases. These recombinases then search the genome to find a homologous sequence, which can then be used as a template for repair of the damaged DNA. Eukaryotes have two recombinases, Rad51 and Dmc1. Rad51 is the only recombinase during mitotic HR, whereas, Dmc1 is expressed exclusively during meiosis. It is not known why most eukaryotes have two recombinases. However, a recent paper published in this lab demonstrated the first biochemical/biophysical difference between Rad51 and Dmc1. When presented with a mismatch during homologous DNA pairing, Dmc1 seems to stabilize the mismatch, while Rad51 appears to destabilize the mismatch. This differential response may reflect the unique biological roles of each recombinase: Rad51 is responsible for mitotic HR, and typically utilizes an identical sister chromatid as a template for repair; In contrast, Dmc1 must utilize homologs of different parental origins for meiotic HR. We propose that the ability of Dmc1 to stabilize mismatches reflects a requirement to promote recombination between template bearing single nucleotide polymorphisms during meiosis. In this proposal, we want to understand what are the structural elements that allow Rad51 and Dmc1 to behave differently to mismatches. We will identify DNA-binding regions of the two recombinases, determine amino acids that are uniquely conserved within each of the recombinases and swap these elements in order to make chimeric proteins. We will test whether these chimeric proteins produce the opposite response to mismatches as compared to their wild type forms. In the proposal, I have already demonstrated that I can create a Rad51 chimera that can stabilize mismatches and will attempt to create a Dmc1 chimera that can destabilize mismatches. I will then address the biological significance of mismatch (de)stabilization by incorporating my chimeras into yeast genomes and monitoring mitotic and meiotic HR in vivo. This proposal will attempt to provide further insight into and significance of the fidelity of eukaryotic recombination.