Why do eukaryotes have two Rad51/RecA family recombinases?

同源重组(HR)是有性生殖过程中一条重要的DNA修复途径，有助于基因组的完整性和遗传多样性的产生。HR是由称为重组酶的蛋白质催化的。绝大多数真核生物有两种重组酶：RAD51和Dmc1，RAD51可用于人体大多数细胞的DNA修复，Dmc1是产生配子(精子和卵子)所必需的。RAD51和Dmc1在氨基酸序列水平上密切相关，它们也催化相同的碱性反应，这就提出了一个问题，为什么细胞需要这两种重组酶？这个看似简单的问题触及了更广泛的问题，即真核生物中尚未解决的特殊功能的进化。为了帮助解决这个问题，模式生物酿酒酵母(酿酒酵母)的RAD51和Dmc1将通过最先进的单分子成像方法进行研究。这项研究将通过研究RAD51和Dmc1这两种关键的DNA修复酶之间的异同，特别是它们与DNA和其他蛋白质的相互作用，从而深入了解真核生物为什么同时进化RAD51和Dmc1。这项跨学科的工作还将为学生提供STEM领域的前沿教育，使他们能够在未来成功地为科学事业做出贡献。Dmc1只在减数分裂中表达，是减数分裂过程中具有催化活性的重组酶，而RAD51是结构性表达的，受减数分裂特异的调节辅助因子下调。这两种蛋白质被认为是在真核生物的早期进化史中的一次基因复制事件中产生的，它们在不同物种中保持着约45%的同源性。然而，RAD51和Dmc1都含有RAD51谱系或Dmc1谱系所特有的氨基酸。最重要的假设是，谱系特异的氨基酸在定义RAD51和Dmc1之间的差异方面发挥了关键作用。将对这些谱系特定的氨基酸进行详细分析，以确定它们如何定义每个重组酶的辅因子特异性和DNA底物相互作用。这项研究将利用“DNA窗帘”和全内反射荧光显微镜(TIRFM)工具，在基因重组的早期阶段显示单个重组酶细丝。这种独特的单分子成像方法通过实现多个反应轨迹的并行成像，实现了从单个分子快速收集统计相关信息。由此得到的关于这两个重组酶的详细机械信息将为他们在同源重组中专业角色的演变提供新的见解。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Homologous recombination (HR) is an important DNA repair pathway that contributes to both genome integrity and the generation of genetic diversity during sexual reproduction. HR is catalyzed by proteins called recombinases. The vast majority of eukaryotes have two recombinases: Rad51, which can be used for DNA repair in most cells of the body, and Dmc1, which is required for the production of gametes (sperm and eggs). Rad51 and Dmc1 are closely related at the amino acid sequence level and they also catalyze the same basic reactions, which raises the question of why do cells need both of these recombinases? This seemingly simple question touches on broader questions about the evolution of specialized functions in eukaryotes that are yet to be resolved. To help address this issue, Rad51 and Dmc1 from the model organism Saccharomyces cerevisiae (Brewer's yeast) will be studied by state-of-the-art single-molecule imaging methods. The research will yield insights into why eukaryotes have evolved both Rad51 and Dmc1 by investigating the similarities and differences between these two crucial DNA repair enzymes, in particular how they interact with DNA and with other proteins. This interdisciplinary work will also provide students with cutting-edge education in STEM fields and enable them to successfully contribute to the scientific enterprise in the future. Dmc1 is expressed only in meiosis and is the catalytically active recombinase during meiosis, whereas Rad51, which is constitutively expressed, is downregulated by meiosis-specific regulatory co-factors. The two proteins are thought to have arisen from a gene duplication event during the early evolutionary history of eukaryotes, and they remain ~45% identical to one another across species. However, Rad51 and Dmc1 both contain amino acids that are specific for either the Rad51 lineage or the Dmc1 lineage. The overarching hypothesis is that lineage-specific amino acids play crucial roles in defining the differences between Rad51 and Dmc1. A detailed analysis of these lineage-specific amino acids will be conducted to determine how they define the co-factor specificity and DNA substrate interactions for each recombinase. The research will utilize "DNA Curtains" and total internal reflection fluorescence microscopy (TIRFM) tools to visualize individual recombinase filaments during the early stages of genetic recombination. This unique approach to single molecule imaging enables rapid collection of statistically relevant information from individual molecules by enabling parallel imaging of multiple reaction trajectories. The resulting detailed mechanistic information on both recombinases will provide new insights into evolution of their specialized roles in homologous recombination.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.