Rapid Exchange of A:T Base Pairs Is Essential for Recognition of DNA Homology by Human Rad51 Recombination Protein

Abstract

Human Rad51 belongs to a ubiquitous family of proteins that enable a single strand to recognize homology in duplex DNA, and thereby to initiate genetic exchanges and DNA repair, but the mechanism of recognition remains unknown. Kinetic analysis by fluorescence resonance energy transfer combined with the study of base substitutions and base mismatches reveals that recognition of homology, helix destabilization, exchange of base pairs, and initiation of strand exchange are integral parts of a rapid, concerted mechanism in which A:T base pairs play a critical role. Exchange of base pairs is essential for recognition of homology, and physical evidence indicates that such an exchange occurs early enough to mediate recognition.

Role of heteroduplex joints in the functional interactions between human Rad51 and wild-type p53

“…Over a period of 1 ± 2 h at 378C 10 ± 20% of the 32 P-labeled oligonucleotide 1 were displaced from the labeled duplex substrate by hRad51 (Figure 1a, lanes 6 ± 8). This represents a strand transfer activity which was expected from the GC content of about 40% (Gupta et al, 1999b). Spontaneous release was negligibly low (Figure 1a, lanes 4 and 5).…”

The synaptic activity of HsDmc1, a human recombination protein specific to meiosis

“…The single-stranded 83-mer oligonucleotides used to form the filaments are designated as (Ϫ) strand and the respective complementary strands as (ϩ) strand. The sequences of G16(Ϫ), G26(Ϫ), and G37(Ϫ) have been described earlier (20). The duplex oligonucleotides used in this study were made by thermal annealing of two complementary strands.…”

“…The His-tagged Dmc1 protein hydrolyzed ATP in a DNA-dependent manner with a catalytic rate constant (k cat ) of 0.7 mols͞mol͞min, similar to the k cat for nontagged protein, described before (27). In previous studies, we have used assays based on FRET to distinguish and kinetically characterize two phases of recombination reactions catalyzed by members of the RecA family of proteins (20,30,31). These phases are the recognition of homology and the exchange of strands.…”

“…Solution of the structure of the single strand has shown that the bases, whose axial spacing is 50% greater than in B-form DNA, are stacked alternately with the deoxyribose moieties of the phosphodiester backbone, a configuration that links the angular displacement of bases to a change in the pucker of the sugar (19). Recent studies of human Rad51 indicate that the filament formed on single-stranded DNA provides a catalytic surface, which, on collision with duplex DNA, promotes the opening of base pairs to check for homology at the point of contact anywhere along the filament (20).…”

Probing the structure of RecA–DNA filaments. Advantages of a fluorescent guanine analog

“…Recent studies of strand exchange mediated by RecA and Rad51, a eukaryotic RecA homolog, have been carried out in the Radding laboratory using DNA substrates with multiple 2AP residues. 49,74,75 In our hands, while 2AP-containing oligonucleotides produce fluorescence changes upon binding RecA, the changes have been difficult to interpret rigorously due to background tryptophan fluorescence, light scattering, and heterogeneity in the oligonucleotide substrates. Indeed, we have observed the significant accumulation of non-full-length oligomers during both synthesis and storage of oligonucleotides having 2AP at multiple positions, 51 presumably as a result of the hydrolytic proclivity of the 2AP heterocycle.…”