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

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.

Citation
Xu, J., Zhao, L., Peng, S., Chu, H., Liang, R., Tian, M., … Wang, H.-W. (2021). Mechanisms of distinctive mismatch tolerance between Rad51 and Dmc1 in homologous recombination. Nucleic Acids Research. doi:10.1093/nar/gkab1141

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.
FUNDING Natural Natural Science Foundation of China [31825009 to H.-W.W.; 31700654 to L.Z.; 21922704, 22061160466, 21877069 to C.C.; 21625302, 21933010 to G.-H.L.]; Beijing Normal University [310421124 to J.X.]. Funding for open access charge: National Natural Science Foundation of China [31825009 to H.-W.W.].

Publisher
Oxford University Press (OUP)

Journal
Nucleic Acids Research

DOI
10.1093/nar/gkab1141

Additional Links
https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gkab1141/6454278

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