Daftar Isi: Analysis of D-loop stability in DMC1 and RAD51 driven strand invasion reactions of homologous recombination

Daftar Isi:

The initial steps of homologous recombination (HR) involve processing of the DNA ends by exonucleases to generate 3’-ssDNA tails (1-5). Then, a protein of the RecA family (ubiquitous Rad51 and meiosis-specific Dmc1) binds to this ssDNA and promotes invasion of the DNA ends into the homologous duplex DNA (6,7). As a result, joint molecules (D-loops) are formed. It is currently thought that the joint molecules continue down one of two pathways. D-loops destined to being processed by DSBR (double strand break repair) pathway should resist dissociation to generate crossovers, and those destined for SDSA (synthesis-dependent strand annealing) pathway need to dissociate producing non-crossovers. The mechanism that channels recombination intermediates into these HR pathways is unknown. We demonstrated that DMC1-generated D-loops are substantially more resistant to dissociation by branch migration proteins RAD54 and Bloom syndrome helicase (BLM) than those formed by RAD51. We propose that the intrinsic resistance DMC1-generated recombination intermediates helps to ensure formation of crossing over that is required for the faithfull segregation of homologous chromosomes in meiosis. Here we describe the protocols for an analysis of D-loop stability using purified human HR proteins.