Elucidating the mechanism of DNA-dependent ATP hydrolysis mediated by DNA-dependent ATPase A, a member of the SWI2/SNF2 protein family

Abstract

The active DNA-dependent ATPase A domain (ADAAD), a member of the SWI2/SNF2 family, has been shown to bind DNA in a structure-specific manner, recognizing DNA molecules possessing double-stranded to single-stranded transition regions leading to ATP hydrolysis. Extending these studies we have delineated the structural requirements of the DNA effector for ADAAD and have shown that the single-stranded and double-stranded regions both contribute to binding affinity while the double-stranded region additionally plays a role in determining the rate of ATP hydrolysis. We have also investigated the mechanism of interaction of DNA and ATP with ADAAD and shown that each can interact independently with ADAAD in the absence of the other. Furthermore, the protein can bind to dsDNA as well as ssDNA molecules. However, the conformation change induced by the ssDNA is different from the conformational change induced by stem-loop DNA (slDNA), thereby providing an explanation for the observed ATP hydrolysis only in the presence of the double-stranded:single-stranded transition (i.e. slDNA).

Global Epigenetic Changes Induced by SWI2/SNF2 Inhibitors Characterize Neomycin-Resistant Mammalian Cells

“…These derivatives, ADAADiK and ADAADiN respectively, were purified and titrated with protein while monitoring the quenching of the intrinsic tryptophan fluorescence. Analysis of the data demonstrated that both ADAADiN and ADAADiK bind to ADAAD in the absence of either ATP or stem-loop DNA (slDNA), a well-characterized DNA effector of ADAAD [13]. The data fit well to a one-site saturation model, suggesting that the ADAADi-ADAAD interaction was predominantly via a single site, and the K d was calculated to be 35.8±5.0 nM for ADAADiN and 21.9±4.2 nM for ADAADiK (Figure S1A and S1B; Table 1).…”

“…His-ADAAD, used for binding studies, was purified as described previously [13]. For mapping and CD studies, ADAAD as well as deletion constructs were expressed as GST-fusion protein in BL21 (DE3) cells and purified as described previously [14].…”

“…Fluorescence was measured at 25°C using Cary-Varian spectrofluorimeter as described previously [13]. Stem-loop DNA (5′- GCGCAATTGCGCTCGACGATTTTTTAGCGCAATTGCGC-3′), synthesized by Sigma-Aldrich, was used in these studies.…”

“…Stem-loop DNA (5′- GCGCAATTGCGCTCGACGATTTTTTAGCGCAATTGCGC-3′), synthesized by Sigma-Aldrich, was used in these studies. The intrinsic fluorescence quenching data obtained with acrylamide was analyzed using Stern-Volmer plots as described previously [13], [14].…”

“…Far UV CD spectra between 200–260 nm were obtained using 0.1 mg/ml protein as described previously [13], [14].…”