Authors
Dmitry Bugreev and Alexander Mazin
Summary
Double-stranded DNA breaks (DSBs), the most harmful DNA lesions, cause cell death and genome instability. Homologous recombination (HR) is a major pathway that repairs double-stranded DNA breaks (DSBs)1. The salient feature of the HR mechanism is that it uses homologous DNA sequences as a template to achieve accurate repair of DSBs. In eukaryotes, the initial steps of HR involve processing of broken DNA by exonucleases to generate ssDNA tails, binding of Rad51 protein (RecA homolog) to these tails to form the Rad51 nucleoprotein filament, and searching by this filament for the homologous DNA template to form joint molecules (D-loops) (Fig. 1). Once joint molecules are formed, the 3’-ssDNA tails of a broken chromosome are extended by DNA polymerase, restoring the lost information. Afterward, the joint molecules dissociate, leading to re-joining of the broken chromosome through the synthesis-dependent strand annealing (SDSA) pathway2. Recently, by reconstituting the process of DSB repair in vitro we demonstrated that Rad54 protein can promote dissociation of D-loops3. Here we describe the protocols for this DSB repair reconstitution using purified human HR proteins and DNA polymerase η.Further details
The protocol was published on Protocol Exchange in 2007. To see the entire protocol, click on the source link.Advertisement
Stats
- Recommendations n/a n/a positive of 0 vote(s)
- Views 253
- Comments 0