Why do eukaryotes have two Rad51/RecA family recombinases?

为什么真核生物有两种 Rad51/RecA 家族重组酶？

• 批准号: 1817315
• 负责人: Eric Greene
• 金额: $ 90万
• 依托单位: Columbia University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1817315&HistoricalAwards=false
• 关键词: Why; do; eukaryotes; two; Rad51

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.

同源重组（HR）是一种重要的DNA修复途径，有助于基因组的完整性和有性生殖过程中遗传多样性的产生。HR由称为重组酶的蛋白质催化。绝大多数真核生物都有两种重组酶：Rad51，可用于身体大多数细胞的DNA修复，以及Dmc 1，这是产生配子（精子和卵子）所必需的。Rad51和Dmc 1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了关于真核生物特殊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，Rad51和Dmc 1从模式生物酿酒酵母（啤酒酵母）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这两种关键的DNA修复酶之间的相似性和差异性，特别是它们如何与DNA和其他蛋白质相互作用，深入了解真核生物为什么会进化出Rad51和Dmc 1。这项跨学科的工作还将为学生提供STEM领域的尖端教育，使他们能够成功地为未来的科学事业做出贡献。Dmc 1仅在减数分裂中表达，并且是减数分裂期间的催化活性重组酶，而组成型表达的Rad51被减数分裂特异性调节辅因子下调。这两种蛋白质被认为是在真核生物早期进化史中的基因复制事件中产生的，并且它们在不同物种之间保持约45%的相同性。然而，Rad51和Dmc 1都含有对Rad51谱系或Dmc 1谱系具有特异性的氨基酸。总体假设是，谱系特异性氨基酸在定义Rad51和Dmc 1之间的差异方面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种重组酶的辅因子特异性和DNA底物相互作用。该研究将利用“DNA Curtains”和全内反射荧光显微镜（TIRFM）工具，在遗传重组的早期阶段可视化单个重组酶丝。这种独特的单分子成像方法能够通过实现多个反应轨迹的并行成像来快速收集来自单个分子的统计相关信息。这两种重组酶的详细机制信息将为同源重组中其专门作用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。

项目成果

Single-molecule visualization of human RECQ5 interactions with single-stranded DNA recombination intermediates.

• DOI: 10.1093/nar/gkaa1184
• 发表时间: 2021-01-11
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Xue C;Molnarova L;Steinfeld JB;Zhao W;Ma C;Spirek M;Kaniecki K;Kwon Y;Beláň O;Krejci K;Boulton SJ;Sung P;Greene EC;Krejci L
• 通讯作者: Krejci L

DNA Curtains Shed Light on Complex Molecular Systems During Homologous Recombination

• DOI: 10.3791/61320
• 发表时间: 2020-06-01
• 期刊: JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
• 影响因子: 1.2
• 作者: Meir, Aviv;Kong, Muwen;Greene, Eric C.
• 通讯作者: Greene, Eric C.

DNA Repair Pathway Choices in CRISPR-Cas9-Mediated Genome Editing.

• DOI: 10.1016/j.tig.2021.02.008
• 发表时间: 2021-07
• 期刊: Trends in genetics : TIG
• 作者: Xue C;Greene EC
• 通讯作者: Greene EC