DNA Sequence Alignment by Microhomology Sampling during Homologous Recombination

Abstract

Summary Homologous recombination (HR) mediates the exchange of genetic information between sister or homologous chromatids. During HR, members of the RecA/Rad51 family of recombinases must somehow search through vast quantities of DNA sequence to align and pair ssDNA with a homologous dsDNA template. Here we use single-molecule imaging to visualize Rad51 as it aligns and pairs homologous DNA sequences in real-time. We show that Rad51 uses a length-based recognition mechanism while interrogating dsDNA, enabling robust kinetic selection of 8-nucleotide (nt) tracts of microhomology, which kinetically confines the search to sites with a high probability of being a homologous target. Successful pairing with a 9th nucleotide coincides with an additional reduction in binding free energy and subsequent strand exchange occurs in precise 3-nt steps, reflecting the base triplet organization of the presynaptic complex. These findings provide crucial new insights into the physical and evolutionary underpinnings of DNA recombination.

On the influence of protein-DNA register during homologous recombination

“…[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).…”

“…19 The dsDNA substrates contained short tracts of microhomology ( 15 -nt) complementary to defined regions of the Rad51/RecA-coated presynaptic ssDNA. Interactions between these dsDNA molecules and the Rad51/RecA presynaptic complexes were monitored by direct visualization with total internal reflection fluorescence microscopy (TIRFM).…”

“…Rad51/RecA presynaptic complexes required at least 8-nt of microhomology to stably capture a dsDNA fragment, in agreement with previous bulk biochemical results, 22 leading to a lifetime on the order of »10 minutes. 19 Importantly, the lifetime measurements are a direct proxy for the stability of the interaction between the presynaptic ssDNA and a given tract of microhomology with the dsDNA. 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.…”

“…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.…”