Abstract
DMC1 and RAD51 are conserved recombinases that catalyze homologous recombination. DMC1 and RAD51 share similar properties in DNA binding, DNA-stimulated ATP hydrolysis, and catalysis of homologous DNA strand exchange. A large body of evidence indicates that attenuation of ATP hydrolysis leads to stabilization of the RAD51-ssDNA presynaptic filament and enhancement of DNA strand exchange. However, the functional relationship of ATPase activity, presynaptic filament stability, and DMC1-mediated homologous DNA strand exchange has remained largely unexplored. To address this important question, we have constructed several mutant variants of human DMC1 and characterized them biochemically to gain mechanistic insights. Two mutations, K132R and D223N, that change key residues in the Walker A and B nucleotide-binding motifs ablate ATP binding and render DMC1 inactive. On the other hand, the nucleotide-binding cap D317K mutant binds ATP normally but shows significantly attenuated ATPase activity and, accordingly, forms a highly stable presynaptic filament. Surprisingly, unlike RAD51, presynaptic filament stabilization achieved via ATP hydrolysis attenuation does not lead to any enhancement of DMC1-catalyzed homologous DNA pairing and strand exchange. This conclusion is further supported by examining wild-type DMC1 with non-hydrolyzable ATP analogues. Thus, our results reveal an important mechanistic difference between RAD51 and DMC1.
Highlights
DMC1 and RAD51 recombinases catalyze ATP-dependent DNA strand exchange via the presynaptic filament
Construction of DMC1 Mutants— ATP is required for DMC1-mediated homologous DNA pairing and strand exchange [34], the role of ATP hydrolysis in the reaction remains uncertain
ATP hydrolysis based on the following rationale. (a) We changed Asp-317 in the ATP cap in DMC1 to Lys (Fig. 1A, panel I) because mutating the analogous residue in RAD51 has no effect on ATP binding ability but greatly attenuates ATP hydrolysis [30]. (b) Lys-132 in DMC1, part of the Walker A motif involved in ATP binding, was changed to Arg because the equivalent mutation in other ATPases [28] often affects ATP hydrolysis without a significant effect on ATP binding (Fig. 1A, panel II). (c) Asp-223, part of the Walker B motif in DMC1, was mutated to Asn (Fig. 1A, panel III) because the equivalent mutation in several AAAϩ ATPases ablates ATP hydrolysis without a strong effect on ATP binding (36 –38)
Summary
DMC1 and RAD51 recombinases catalyze ATP-dependent DNA strand exchange via the presynaptic filament. The functional relationship of ATPase activity, presynaptic filament stability, and DMC1-mediated homologous DNA strand exchange has remained largely unexplored. To address this important question, we have constructed several mutant variants of human DMC1 and characterized them biochemically to gain mechanistic insights. Unlike RAD51, presynaptic filament stabilization achieved via ATP hydrolysis attenuation does not lead to any enhancement of DMC1-catalyzed homologous DNA pairing and strand exchange. This conclusion is further supported by examining wild-type DMC1 with non-hydrolyzable ATP analogues. Our results reveal an important mechanistic difference between RAD51 and DMC1
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
