Abstract
Both high-fidelity and mismatch-tolerant recombination, catalyzed by RAD51 and DMC1 recombinases, respectively, are indispensable for genomic integrity. Here, we use cryo-EM, MD simulation and functional analysis to elucidate the structural basis for the mismatch tolerance of DMC1. Structural analysis of DMC1 presynaptic and postsynaptic complexes suggested that the lineage-specific Loop 1 Gln244 (Met243 in RAD51) may help stabilize DNA backbone, whereas Loop 2 Pro274 and Gly275 (Val273/Asp274 in RAD51) may provide an open “triplet gate” for mismatch tolerance. In support, DMC1-Q244M displayed marked increase in DNA dynamics, leading to unobservable DNA map. MD simulation showed highly dispersive mismatched DNA ensemble in RAD51 but well-converged DNA in DMC1 and RAD51-V273P/D274G. Replacing Loop 1 or Loop 2 residues in DMC1 with RAD51 counterparts enhanced DMC1 fidelity, while reciprocal mutations in RAD51 attenuated its fidelity. Our results show that three Loop 1/Loop 2 residues jointly enact contrasting fidelities of DNA recombinases.
Highlights
Both high-fidelity and mismatch-tolerant recombination, catalyzed by RAD51 and DMC1 recombinases, respectively, are indispensable for genomic integrity
The strand invasion products mediated by RAD51 and DMC1 filaments are both stabilized in three-nucleotide steps[27,28]
A tight gate and a loose backbone support contribute to the high fidelity of RAD51, whereas a loose gate and a tight backbone support contribute to the mismatch tolerance of DMC1
Summary
Both high-fidelity and mismatch-tolerant recombination, catalyzed by RAD51 and DMC1 recombinases, respectively, are indispensable for genomic integrity. We further performed MD simulations to compare homologous and mismatched structures for the three postsynaptic filaments where the structure with bound dsDNA was available to provide starting coordinates: DMC1, RAD51, and RAD51-VpDg. The RMSD values of the position differences of DNA were calculated throughout the MD simulations to evaluate the mismatch tolerability of each protein, based on their RMSD distributions (Supplementary Fig. 7).
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