Base triplet stepping by the Rad51/RecA family of recombinases

Abstract

DNA strand exchange plays a central role in genetic recombination across all kingdoms of life, but the physical basis for these reactions remains poorly defined. Using single-molecule imaging, we found that bacterial RecA and eukaryotic Rad51 and Dmc1 all stabilize strand exchange intermediates in precise three-nucleotide steps. Each step coincides with an energetic signature (0.3 kBT) that is conserved from bacteria to humans. Triplet recognition is strictly dependent on correct Watson-Crick pairing. Rad51, RecA, and Dmc1 can all step over mismatches, but only Dmc1 can stabilize mismatched triplets. This finding provides insight into why eukaryotes have evolved a meiosis-specific recombinase. We propose that canonical Watson-Crick base triplets serve as the fundamental unit of pairing interactions during DNA recombination.

Two distinct conformational states define the interaction of human RAD 51‐ ATP with single‐stranded DNA

“…Although much information is now available for the structure of the RecA/RAD51 nucleoprotein filaments (Chen et al , 2008; Lee et al , 2015; Prentiss et al , 2015), our understanding of the mechanistic basis for their unique ability to promote strand exchange between homologous DNA sequences remains incomplete. To provide further insight into hRAD51 function and investigate its apparent ability to adopt different conformations in the polymeric state, we obtained a crystal structure of the hRAD51‐ATP filament (Appendix Fig S5A–C and Table S1).…”

On the influence of protein-DNA register during homologous recombination

“…The 3-nt stepping patterns were also readily recapitulated using molecular dynamics or Monte Carlo simulations of a course-grained model for RS-DNA in the absence of any proteins, strongly suggesting that the base triplet architecture of RS-DNA itself was responsible for the observed triplet stepping. 18 A crucial implication of these findings is that only fully paired base triplets are capable of forming stable pairing interactions within the context of RS-DNA (Fig. 1).…”

“…Remarkably, the dsDNA binding stability exhibited a distinctive 3-nt stepping pattern when the length of the microhomology was increased from 8-nt to 15-nt in single nucleotide increments. 18,19 That is, the measured stability of the interactions increased in quantized units for each set of 3 homologous nucleotides. Similar 3-nt stepping was observed for human and S. cerevisiae Rad51, human and S. cerevisiae Dmc1 (a meiosis-specific Rad51 paralog), and E. coli RecA, suggesting that it is conserved across the entire Rad51/RecA family.…”

“…[10][11][12][13][14][15] We have developed the single-molecule DNA curtains method to establish a more detailed understanding of the molecular mechanisms that contribute to homologous DNA recombination. [16][17][18][19] For studies of recombination, we use ssDNA curtains, where long ssDNA molecules are tethered to a lipid bilayer on the surface of a microfluidic sample chamber and then organized into "curtains" using a series of strategically positioned barriers to lipid diffusion; details describing the preparation of DNA curtains can be found in references. [16][17][18][19] The ssDNA molecules are coated with a GFP-tagged version of replication protein A (RPA), which is a ubiquitous ssDNA-binding protein found in eukaryotes.…”

“…[16][17][18][19] For studies of recombination, we use ssDNA curtains, where long ssDNA molecules are tethered to a lipid bilayer on the surface of a microfluidic sample chamber and then organized into "curtains" using a series of strategically positioned barriers to lipid diffusion; details describing the preparation of DNA curtains can be found in references. [16][17][18][19] The ssDNA molecules are coated with a GFP-tagged version of replication protein A (RPA), which is a ubiquitous ssDNA-binding protein found in eukaryotes. 20 The RPA-ssDNA can then be used as a substrate to assemble presynaptic complexes by addition of the appropriate Rad51/ RecA protein along with the required nucleotide cofactor (ATP, AMP-PNP, or ATPgS).…”