Chin-Dian Wei, Hao-Yen Chang, Chia-Hua Lu, Chih-Chun Chang, Asako Furukohri, Stephen Mwaniki, Akira Shinohara, Peter Chi, Hung-Wen Li
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Mei5–Sae3 stabilizes Dmc1 nucleating clusters for efficient Dmc1 assembly on RPA-coated single-stranded DNA
Interhomolog recombination in meiosis requires a meiosis-specific recombinase, Dmc1. In Saccharomyces cerevisiae, the Mei5–Sae3 complex facilitates the loading of Dmc1 onto the replication protein A (RPA)-coated single-stranded DNA (ssDNA) to form nucleoprotein filaments. In vivo, Dmc1 and Mei5–Sae3 are interdependent in their colocalization on the chromosomes. However, the mechanistic role of Mei5–Sae3 in mediating Dmc1 activity remains unclear. We used single-molecule fluorescence resonance energy transfer and colocalization single-molecule spectroscopy experiments to elucidate how Mei5–Sae3 stimulates Dmc1 assembly on ssDNA and RPA-coated ssDNA. We showed that Mei5–Sae3 stabilized Dmc1 nucleating clusters with two to three molecules on naked DNA by preferentially reducing Dmc1 dissociation rates. Mei5–Sae3 also stimulated Dmc1 assembly on RPA-coated DNA. Using green fluorescent protein-labeled RPA, we showed the coexistence of an intermediate with Dmc1 and RPA on ssDNA before RPA dissociation. Moreover, the displacement efficiency of RPA depended on Dmc1 concentration, and its dependence was positively correlated with the stability of Dmc1 clusters on short ssDNA. These findings suggest a molecular model that Mei5–Sae3 mediates Dmc1 binding on RPA-coated ssDNA by stabilizing Dmc1 nucleating clusters, thus altering RPA dynamics on DNA to promote RPA dissociation.
期刊介绍:
Nucleic Acids Research (NAR) is a scientific journal that publishes research on various aspects of nucleic acids and proteins involved in nucleic acid metabolism and interactions. It covers areas such as chemistry and synthetic biology, computational biology, gene regulation, chromatin and epigenetics, genome integrity, repair and replication, genomics, molecular biology, nucleic acid enzymes, RNA, and structural biology. The journal also includes a Survey and Summary section for brief reviews. Additionally, each year, the first issue is dedicated to biological databases, and an issue in July focuses on web-based software resources for the biological community. Nucleic Acids Research is indexed by several services including Abstracts on Hygiene and Communicable Diseases, Animal Breeding Abstracts, Agricultural Engineering Abstracts, Agbiotech News and Information, BIOSIS Previews, CAB Abstracts, and EMBASE.