Why do eukaryotes have two Rad51/RecA family recombinases?

同源重组（Homologous recombination， HR）是一种重要的DNA修复途径，在有性生殖过程中有助于基因组完整性和遗传多样性的产生。HR是由重组酶这种蛋白质催化的。绝大多数真核生物都有两种重组酶：Rad51和Dmc1，前者可用于身体大多数细胞的DNA修复，后者是产生配子（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，它们也催化相同的基本反应，这就提出了为什么细胞需要这两种重组酶的问题？这个看似简单的问题涉及到真核生物特化功能进化的更广泛的问题，这些问题尚未得到解决。为了帮助解决这个问题，我们将利用最先进的单分子成像方法研究模式生物酿酒酵母（酿酒酵母）中的Rad51和Dmc1。通过研究Rad51和Dmc1这两种关键DNA修复酶之间的异同，特别是它们如何与DNA和其他蛋白质相互作用，这项研究将深入了解真核生物进化出Rad51和Dmc1的原因。这项跨学科的工作也将为学生提供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仅在减数分裂中表达，在减数分裂期间是催化活性重组酶，而Rad51是组成性表达的，被减数分裂特异性调节辅助因子下调。这两种蛋白质被认为是在真核生物早期进化史上的一次基因复制事件中产生的，它们在不同物种之间保持着45%的相同。然而，Rad51和Dmc1都含有对Rad51或Dmc1谱系特异性的氨基酸。最重要的假设是，谱系特异性氨基酸在确定Rad51和Dmc1之间的差异中起着至关重要的作用。将对这些谱系特异性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助因子特异性和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）工具来观察基因重组早期阶段的单个重组酶细丝。这种独特的单分子成像方法通过对多个反应轨迹进行并行成像，可以快速收集单个分子的统计相关信息。由此产生的两种重组酶的详细机制信息将为其在同源重组中的特殊作用的进化提供新的见解。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值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.