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

Nongkhlaw M, Dutta P, Hockensmith JW, Komath SS, Muthuswami R - Nucleic Acids Res. (2009)

Bottom Line: 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.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).

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India.

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).

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Stern–Volmer plots. ADAAD was titrated with acrylamide in absence and presence of ATP and DNA. (Filled square) protein alone; (open square) in presence of saturating concentration of ATP; (filled circle) in presence of saturating concentration of slDNA; (open circle) in presence of saturating concentrations of slDNA and ATP; (filled triangle) in presence of ssDNA; and (open triangle) in presence of saturating concentration of ssDNA and ATP.
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Figure 5: Stern–Volmer plots. ADAAD was titrated with acrylamide in absence and presence of ATP and DNA. (Filled square) protein alone; (open square) in presence of saturating concentration of ATP; (filled circle) in presence of saturating concentration of slDNA; (open circle) in presence of saturating concentrations of slDNA and ATP; (filled triangle) in presence of ssDNA; and (open triangle) in presence of saturating concentration of ssDNA and ATP.

Mentions: Titrating ADAAD with acrylamide resulted in significant fluorescence quenching (∼70%). The Stern–Volmer (SV) plots (Figure 5) for this quencher were biphasic, indicating that not all 13 Trp residues of the protein were equally accessible. As can also be seen from Figure 5 and Supplementary Table 6, one fraction of Trp residues was relatively easily accessed by acrylamide (KSV1 = 9.76 ± 0.10 M−1) while another set was less accessible (KSV2 = 6.70 ± 1.00 M−1). From modified SV plots, the fraction accessible to the quencher (fa) was estimated to be ∼95% with a SV constant for the accessible fraction (Ka) being 9.95 ± 0.05 M−1. In the presence of ATP, both KSV1 and KSV2 were reduced, as were the Ka values (Figure 5, Supplementary Table 6).Figure 5.


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

Nongkhlaw M, Dutta P, Hockensmith JW, Komath SS, Muthuswami R - Nucleic Acids Res. (2009)

Stern–Volmer plots. ADAAD was titrated with acrylamide in absence and presence of ATP and DNA. (Filled square) protein alone; (open square) in presence of saturating concentration of ATP; (filled circle) in presence of saturating concentration of slDNA; (open circle) in presence of saturating concentrations of slDNA and ATP; (filled triangle) in presence of ssDNA; and (open triangle) in presence of saturating concentration of ssDNA and ATP.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 5: Stern–Volmer plots. ADAAD was titrated with acrylamide in absence and presence of ATP and DNA. (Filled square) protein alone; (open square) in presence of saturating concentration of ATP; (filled circle) in presence of saturating concentration of slDNA; (open circle) in presence of saturating concentrations of slDNA and ATP; (filled triangle) in presence of ssDNA; and (open triangle) in presence of saturating concentration of ssDNA and ATP.
Mentions: Titrating ADAAD with acrylamide resulted in significant fluorescence quenching (∼70%). The Stern–Volmer (SV) plots (Figure 5) for this quencher were biphasic, indicating that not all 13 Trp residues of the protein were equally accessible. As can also be seen from Figure 5 and Supplementary Table 6, one fraction of Trp residues was relatively easily accessed by acrylamide (KSV1 = 9.76 ± 0.10 M−1) while another set was less accessible (KSV2 = 6.70 ± 1.00 M−1). From modified SV plots, the fraction accessible to the quencher (fa) was estimated to be ∼95% with a SV constant for the accessible fraction (Ka) being 9.95 ± 0.05 M−1. In the presence of ATP, both KSV1 and KSV2 were reduced, as were the Ka values (Figure 5, Supplementary Table 6).Figure 5.

Bottom Line: 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.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).

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India.

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).

Show MeSH
Related in: MedlinePlus