A research article titled “Cryo-EM structures of human recombinase RAD51 filaments in the catalysis of DNA strand exchange” has been published online in Nature Structural & Molecular Biology on December 12th, 2016. This work is from Prof. Hong-Wei Wang’s group in the School of Life Sciences at Tsinghua University, in collaboration with Prof. Patrick Sung’s group at Yale University. The structures in this work revealed both conserved and distinct structural features of the RAD51-DNA complexes in comparison with its prokaryotic counterpart. Importantly, they have also captured the structure of an arrested synaptic complex. The results provide insights into the molecular mechanism of DNA homology search and strand exchange processes.
Homologous recombination (HR) plays essential roles in genetic integrity maintenance by eliminating DNA lesions such as double stranded breaks, inter-strand crosslinks, and collapsed replication forks. HR is also indispensable for meiosis wherein it helps ensuring the proper segregation of homologous chromosomes as well as contributing to the generation of genetic diversity. In eukaryotic cellsŁ¬upon ATP binding, recombinase RAD51 protomers assemble into a right-handed helical filament on ssDNA (the invading strand) called the presynaptic filament. The presynaptic complex engages and samples duplex DNA to search for a homologous region in the latter. Successful homology search is followed by limited base pairing between the invading strand and the complementary strand in the dsDNA. This HR stage has been termed synaptic complex formation. Then, more extensive DNA strand exchange occurs, and the newly formed DNA joint is bound by the recombinase filament in the postsynaptic complex. This complex is subsequently resolved with the disassembly of the recombinase filament and the recruitment of a DNA polymerase to extend the 3’ end of the invading DNA strand. However, owing to a lack of structural information of intermediate states, it has remained unknown how the DNA strands in the homologous dsDNA become separated in the synaptic phase of the DNA strand exchange reaction.
In this study, the authors have carried out cryo-EM analyses on human RAD51 in different stages of the DNA strand exchange process. These endeavors have enabled them to determine the structures of presynaptic and postsynaptic complexes of RAD51 at near atomic resolution. Human RAD51 is seen to engage ssDNA and dsDNA in nucleotide triplet clusters as first reported in the bacterial ortholog RecA. RAD51 protomers form the helical assembly mainly via three interfaces, with the most important of which being dependent on an ATP molecule sandwiched between two adjacent protomers, in an arrangement that is also present in other recombinase orthologs such as RecA.
Importantly, with specially designed DNA substrates and a newly developed algorithm, this work also captures the synaptic complex where DNA strand exchange becomes arrested upon encountering a heterologous region. In this arrested state, the displaced strand remains within the vicinity of the synaptic complex, likely accounting for the extra densities in the 3D reconstruction of ~5 Å resolution. This structure is consistent with the premise that a “secondary DNA binding site” in the RAD51 filament facilitates strand separation in the duplex DNA partner. The cryo-EM structures capturing different stages of the DNA strand exchange process and the companion conceptual models provide the framework to delineate how various factors regulate RAD51-dependent homologous recombination.
Fig (a) The atomic model of the presynaptic complex and (b) The atomic model of the presynaptic complex. (c) Arrested state in Rad51-mediated DNA strand exchange are shown, with six consecutive protomers labeled by numbers. (d) A hypothetical model of DNA molecule transition during the synaptic reaction.
Co-first authors of this research article are Ph.D. student Jingfei Xu at the Tsinghua-Peking Joint Center of Life Sciences, Advanced Innovation Fellow Dr. Lingyun Zhao at the Beijing Advanced Innovation Center for Structural Biology at Tsinghua University, both from Prof. Hong-Wei Wang’s group, Dr. Yuanyuan Xu and Dr. Weixing Zhao from Prof. Patrick Sung’s group at Yale University. Data collection and computation were supported by the National Facility for Protein Sciences (Beijing). This work was supported by research funds from the National Science Foundation of China, the Key Research and Development Program of MOST, the Beijing Municipal Science & Technology Commission and the US National Institutes of Health.