Dataset Information

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.

SUBMITTER: Deakyne JS

PROVIDER: S-EPMC3814753 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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Analysis of the activities of RAD54, a SWI2/SNF2 protein, using a specific small-molecule inhibitor.

Deakyne Julianna S JS Huang Fei F Negri Joseph J Tolliday Nicola N Cocklin Simon S Mazin Alexander V AV

The Journal of biological chemistry 20130916 44

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 act ...[more]

PMID: 24043618

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Project description:Free-living bacteria have regulatory systems that can quickly reprogram gene transcription in response to changes in cellular environment. The RapA ATPase, a prokaryotic homolog of the eukaryote Swi2/Snf2 chromatin remodeling complex, may facilitate such reprogramming, but the mechanisms by which it does so is unclear. We used multi-wavelength single-molecule fluorescence microscopy in vitro to examine RapA function in the E. coli transcription cycle. In our experiments, RapA at < 5 nM concentration did not appear to alter transcription initiation, elongation, or intrinsic termination. Instead, we directly observed a single RapA molecule bind specifically to the kinetically stable post-termination complex (PTC) -- consisting of core RNA polymerase (RNAP) bound to dsDNA -- and efficiently remove RNAP from DNA within seconds in an ATP-hydrolysis-dependent reaction. Kinetic analysis elucidates the process through which RapA locates the PTC and the key mechanistic intermediates that bind and hydrolyze ATP. This study defines how RapA participates in the transcription cycle between termination and initiation and suggests that RapA helps set the balance between global RNAP recycling and local transcription re-initiation in proteobacterial genomes.SignificanceRNA synthesis is an essential conduit of genetic information in all organisms. After transcribing an RNA, the bacterial RNA polymerase (RNAP) must be reused to make subsequent RNAs, but the steps that enable RNAP reuse are unclear. We directly observed the dynamics of individual molecules of fluorescently labeled RNAP and the enzyme RapA as they colocalized with DNA during and after RNA synthesis. Our studies show that RapA uses ATP hydrolysis to remove RNAP from DNA after the RNA is released from RNAP and reveal essential features of the mechanism by which this removal occurs. These studies fill in key missing pieces in our current understanding of the events that occur after RNA is released and that enable RNAP reuse.

Dataset Information

Analysis of the activities of RAD54, a SWI2/SNF2 protein, using a specific small-molecule inhibitor.

Publications

Analysis of the activities of RAD54, a SWI2/SNF2 protein, using a specific small-molecule inhibitor.

Similar Datasets

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