Analysis of the activities of RAD54, a SWI2/SNF2 protein, using a specific small-molecule inhibitor.
RAD54, an important homologous recombination protein, is a member of the SWI2/SNF2 family of ATPase-dependent DNA translocases. In vitro, RAD54 stimulates RAD51-mediated DNA strand exchange and promotes branch migration of Holliday junctions. It is thought that an ATPase-dependent DNA translocation is required for both of these RAD54 activities. Here we identified, by high-throughput screening, a specific RAD54 inhibitor, streptonigrin (SN), and used it to investigate the mechanisms of RAD54 activities. We found that SN specifically targets the RAD54 ATPase, but not DNA binding, through direct interaction with RAD54 and generation of reactive oxygen species. Consistent with the dependence of branch migration (BM) on the ATPase-dependent DNA translocation of RAD54, SN inhibited RAD54 BM. Surprisingly, the ability of RAD54 to stimulate RAD51 DNA strand exchange was not significantly affected by SN, indicating a relatively smaller role of RAD54 DNA translocation in this process. Thus, the use of SN enabled us to identify important differences in the effect of the RAD54 ATPase and DNA translocation on two major activities of RAD54, BM of Holliday junctions and stimulation of DNA pairing.
|Year of Publication||
J Biol Chem
2013 Nov 01
|PubMed Central ID||
MH097512 / MH / NIMH NIH HHS / United States
R03 MH097512 / MH / NIMH NIH HHS / United States
CA100839 / CA / NCI NIH HHS / United States
DA033981 / DA / NIDA NIH HHS / United States
R03 DA033981 / DA / NIDA NIH HHS / United States
R01 CA100839 / CA / NCI NIH HHS / United States
R56 CA100839 / CA / NCI NIH HHS / United States
1R21AI087388 / AI / NIAID NIH HHS / United States
R21 AI087388 / AI / NIAID NIH HHS / United States