Reconstituting the Key Steps of the DNA Double-Strand Break Repair In Vitro
Double-stranded DNA breaks (DSB), the most harmful type of DNA lesions, cause cell death and genome instability. Homologous recombination repairs DSB using homologous DNA sequences as templates. Here we describe a set of reactions that lead to reconstitution of the double-stranded DNA break repair process in vitro employing purified human homologous recombination proteins and DNA polymerase η. Reconstitution of critical steps of DSB repair in vitro may help to better understand the mechanisms of recombinational DNA repair and the role of various human homologous recombination proteins in this process.
Key wordsHomologous recombination DNA strand exchange branch migration Holliday junction joint molecules D-loops
This work was supported by the NIH Grant CA100839, MH084119, and the Leukemia and Lymphoma Society Scholar Award 1054-09 (to AVM) and NIH Grant F31 AG033484-01 (to MJR).
- 8.Sambrook, J., and Russell, D.W. (2001) Molecular cloning: a laboratory manual, 3rd Edition (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press).Google Scholar