TY - JOUR
T1 - Mechanisms of distinctive mismatch tolerance between Rad51 and Dmc1 in homologous recombination
AU - Xu, Jingfei
AU - Zhao, Lingyun
AU - Peng, Sijia
AU - Chu, Huiying
AU - Liang, Rui
AU - Tian, Meng
AU - Connell, Philip P
AU - Li, Guohui
AU - Chen, Chunlai
AU - Wang, Hong-Wei
N1 - 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.
PY - 2021/12/6
Y1 - 2021/12/6
N2 - 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.
AB - 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.
UR - http://hdl.handle.net/10754/673979
UR - https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gkab1141/6454278
U2 - 10.1093/nar/gkab1141
DO - 10.1093/nar/gkab1141
M3 - Article
C2 - 34871438
SN - 0305-1048
JO - Nucleic Acids Research
JF - Nucleic Acids Research
ER -