Understanding the Basis of Fidelity in Eukaryotic Recombinases

了解真核重组酶保真度的基础

• 批准号: 9544168
• 负责人: Justin B Steinfeld
• 金额: $ 4.05万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9544168
• 关键词: Address; Alleles; Amino Acids; Aneuploidy; BRCA1 Mutation; BRCA2 Mutation; Behavior; Binding; Biochemical; Biochemical Genetics; Biological; Biological Assay; Biophysics; Cells; Chimera organism; Chimeric Proteins; Chromosomal Rearrangement; Chromosomes; DNA; DNA Binding; DNA Binding Domain; DNA Damage; DNA Repair; DNA Sequence; Defect; Disease; Double Strand Break Repair; Down Syndrome; Elements; Engineering; Ensure; Eukaryota; Failure; Family; Filament; Genetic Recombination; Genome; Goals; Growth; Homologous Gene; Human; Individual; Infertility; Investigation; Lead; Life; Link; Maintenance; Malignant Neoplasms; Mediating; Meiosis; Meiotic Recombination; Mitosis; Mitotic; Mitotic Recombination; Monitor; Mutation; Organism; Paper; Pathway interactions; Process; Property; Proteins; Publishing; Rad51 recombinase; Reproduction; Role; Saccharomyces cerevisiae; Sampling; Sequence Homologs; Series; Single Nucleotide Polymorphism; Single-Stranded DNA; Sister Chromatid; Spontaneous abortion; Testing; Triplet Multiple Birth; Trisomy; Yeasts; biophysical properties; design; ds-DNA; experimental study; genetic approach; genetic information; genome integrity; homologous recombination; in vivo; insight; recombinase; repaired; response; single molecule

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.

为了生存，所有生物都进化了修复受损的各种机制 脱氧核糖核酸。在双链休息（DSB）的情况下，同源重组（HR）是关键 修复机制，使用同源DNA作为修复模板。有丝分裂的失败 重组可以导致人类​​的染色体重排和癌症，如BRCA1所示 减数分裂重组的BRCA2突变和失败会导致不育症，流产和 非整倍性疾病，例如唐斯综合症。 DSB形成后，最终通过一系列步骤处理受损的DNA 留下单链的DNA悬垂，被称为重组酶的蛋白细丝覆盖。这些 然后，重组酶搜索基因组以找到同源序列，然后可以用作 用于修复受损DNA的模板。真核生物有两个重组酶RAD51和DMC1。 RAD51是有丝分裂HR期间唯一的重组酶，而DMC1仅在 减数分裂。尚不清楚为什么大多数真核生物都有两个重组酶。但是，最近的论文 在本实验室中发表的证明了Rad51和 DMC1。当在同源DNA配对期间出现不匹配时，DMC1似乎稳定 不匹配，而RAD51似乎破坏了不匹配的稳定。这种差异反应可能 反映每个重组酶的独特生物学作用：RAD51负责有丝分裂HR，并且 通常，将相同的姐妹染色质被用作修复模板；相反，DMC1必须使用 减数分裂HR的不同父母起源的同源物。我们建议DMC1稳定的能力 不匹配反映了促进单个模板之间重组的要求 减数分裂过程中的核苷酸多态性。 在此提案中，我们想了解什么是允许Rad51和 DMC1的行为与不匹配不同。我们将确定两者的DNA结合区域 重组酶，确定在每个重物组织酶中独特保守的氨基酸 并交换这些元素以制成嵌合蛋白。我们将测试这些嵌合 与野生型形式相比，蛋白质与错配产生相反的反应。在 提案，我已经证明我可以创建一个可以稳定的Rad51嵌合体 不匹配，并将尝试创建一种DMC1嵌合体，该嵌合体会破坏不匹配的不匹配。然后我会 通过将我的嵌合体纳入不匹配（DE）稳定的生物学意义（DE） 酵母基因组和体内有丝分裂和减数分裂HR。该建议将尝试提供 对真核重组的保真度的进一步了解和意义。