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.
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