Visualizing homology search with ssDNA curtains
【摘要】：Homologous recombination(HR) mediates the exchange of genetic information between sister or homologous chromatids. During HR, members of the RecA/Rad51 family of recombinases, like scRad51, hRad51, RecA, and scDmc1, must align and pair single-stranded DNA(ssDNA) with a homologous ds DNA template, but the physical basis for these early stages of recombination remain largely undefined. Here we use ssDNA-curtains to visualize Rec A/Rad51 family members as they align and pair homologous DNA sequences in real-time. We find microhomology sampling is the key point to understand homology search:(1) During interrogating ds DNA, Rec A/Rad51 family members enable robust kinetic selection of 8-nt tracts of microhomology, which kinetically confines the search to sites with a high probability of being a homologous target;(2) RecA/Rad51 family members rapidly sample and reject ds DNA with 8-nt tracts of microhomology through a mechanism characterized by its distinctive power-law dependence;(3) Successful pairing with a 9 th 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;(4) ds DNA binding on the presynaptic complex by a short length of microhomology can be disrupted by other ds DNA molecules with an equal or longer microhomology length. This process is termed facilitated exchange, and it depends on the microhomology length and sequence. These findings provide crucial new insights into the physical and evolutionary underpinnings of DNA recombination.