Mechanisms of distinctive mismatch tolerance between Rad51 and Dmc1 in homologous recombination

Jingfei Xu, Lingyun Zhao, Sijia Peng, Huiying Chu, Rui Liang, Meng Tian, Philip P Connell, Guohui Li, Chunlai Chen, Hong-Wei Wang

    Research output: Contribution to journalArticlepeer-review

    13 Scopus citations

    Abstract

    Homologous recombination (HR) is a primary DNA double-strand breaks (DSBs) repair mechanism. The recombinases Rad51 and Dmc1 are highly conserved in the RecA family; Rad51 is mainly responsible for DNA repair in somatic cells during mitosis while Dmc1 only works during meiosis in germ cells. This spatiotemporal difference is probably due to their distinctive mismatch tolerance during HR: Rad51 does not permit HR in the presence of mismatches, whereas Dmc1 can tolerate certain mismatches. Here, the cryo-EM structures of Rad51–DNA and Dmc1–DNA complexes revealed that the major conformational differences between these two proteins are located in their Loop2 regions, which contain invading single-stranded DNA (ssDNA) binding residues and double-stranded DNA (dsDNA) complementary strand binding residues, stabilizing ssDNA and dsDNA in presynaptic and postsynaptic complexes, respectively. By combining molecular dynamic simulation and single-molecule FRET assays, we identified that V273 and D274 in the Loop2 region of human RAD51 (hRAD51), corresponding to P274 and G275 of human DMC1 (hDMC1), are the key residues regulating mismatch tolerance during strand exchange in HR. This HR accuracy control mechanism provides mechanistic insights into the specific roles of Rad51 and Dmc1 in DNA double-strand break repair and may shed light on the regulatory mechanism of genetic recombination in mitosis and meiosis.
    Original languageEnglish (US)
    JournalNucleic Acids Research
    DOIs
    StatePublished - Dec 6 2021

    Bibliographical note

    KAUST Repository Item: Exported on 2021-12-13
    Acknowledgements: We thank P. Sung (University of Texas Health San Antonio) for providing hRAD51 wt construct and Z. Qi (Peking University) for providing ScDMC1 wt construct and for advice on the smFRET work. We thank W. X. Zhao (University of Texas Health San Antonio) for advice on the protein production and biochemical assays. We thank J.L. Lei, Y.J. Xu and T. Yang for their support in cryo-EM and high-performance computation in the National Protein Science Facility (Beijing) at Tsinghua University. We thank X. Li for help in cryo-EM data collection. We thank Prof. H. L. Peng (Peking University) for kindly providing us the graphene grids for ScDmc1 cryo-EM sample preparation. We acknowledge the staff’s help for the protein preparation and identification facility at the Technology Center for Protein Science at Tsinghua University.

    Author Contributions: H.-W.W. conceived the project. H.-W.W., C.C. and J.X. designed the smFRET experiments. H.-W.W. and G.-H.L. designed the molecular dynamic simulation strategy. J.X. generated key research materials of hRAD51 proteins and performed biochemical assays and smFRET assays with help of S.P. and L.Z. performed EM and structural determination. H.C. performed MD simulation. R.L., M.T. and P.P.C. generated research materials of ScDMC1 and hDMC1. J.X., L.Z., H.C., H.-W.W., C.C. and G.-H.L. wrote the manuscript.

    ASJC Scopus subject areas

    • Genetics

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