title: Analysis of the activities of RAD54, a SWI2/SNF2 protein, using a specific small-molecule inhibitor.
abstract: 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.
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19102 and.
authors.forename: Julianna S
authors.initials: JS
authors.lastname: Deakyne
authors.forename: Fei
authors.initials: F
authors.lastname: Huang
authors.forename: Joseph
authors.initials: J
authors.lastname: Negri
authors.forename: Nicola
authors.initials: N
authors.lastname: Tolliday
authors.forename: Simon
authors.initials: S
authors.lastname: Cocklin
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Mazin
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D004265:DNA Helicases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D045643:DNA Repair Enzymes / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D045566:DNA, Cruciform / Q000235:genetics / Q000378:metabolism; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism*; D059765:Homologous Recombination / Q000187:drug effects / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D013308:Streptonigrin / Q000494:pharmacology*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D000903:Antibiotics, Antineoplastic; D045566:DNA, Cruciform; D004271:DNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D013308:Streptonigrin; D000251:Adenosine Triphosphatases; C075468:RAD54 protein, S cerevisiae; C499333:SNF2 protein, S cerevisiae; D004265:DNA Helicases; D045643:DNA Repair Enzymes
keywords: ATPases; DNA Binding Protein; DNA Repair; Homologous Recombination; Small Molecules
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