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Interaction of different oligomeric states of hexameric DNA-helicase RepA with single-stranded DNA studied by analytical ultracentrifugation

Item Type:Article
Title:Interaction of different oligomeric states of hexameric DNA-helicase RepA with single-stranded DNA studied by analytical ultracentrifugation
Creators Name:Xu, H. and Frank, J. and Holzwarth, J.F. and Saenger, W. and Behlke, J.
Abstract:Analytical ultracentrifugation was used to determine the molecular mass, M, of hexameric DNA-helicase RepA at pH 5.8 and 7.6. At pH 7.6, a molecular mass of 179.5 ± 2.6 kDa was found, consistent with the known hexameric state of RepA, (RepA) 6. At pH 5.8, (RepA) 6 associates to form a dimer with a molecular mass of 366.2 ± 4.1 kDa. Analytical ultracentrifugation was also applied to characterize the interaction of single-stranded DNA (ssDNA) with the two different oligomeric states of (RepA) 6 at pH 5.8 and 7.6. The dissociation constants, K(d), for the equilibrium binding of (dA) 30 to the (RepA) 6 dimer at pH 5.8 and to (RepA) 6 at pH 7.6 were determined at 10°C in the presence of 0.5 mM ATPγS, 10 mM MgCl 2 and 60 mM NaCl as K(d5.8) = 0.94 ± 0.13 μM at pH 5.8 and K(d7.6) = 25.4 ± 6.4 μM at pH 7.6. The stoichiometries, n, for the two complexes (dA) 30/(RepA) 6 dimer and (dA) 30/(RepA) 6 at pH 5.8 and 7.6 were calculated from the corresponding binding curves. At pH 5.8 one (dA) 30 molecule was bound per (RepA) 6 dimer, while at pH 7.6 one (dA) 30 molecule was bound to one (RepA) 6. Binding curves were compatible with a single ssDNA binding site present on the (RepA) 6 dimer and on (RepA) 6, respectively, with no indication of cooperativity. (RepA) 6 tends to form larger aggregates under acidic conditions (pH < 6.0) which are optimal for ssDNA binding. In contrast, at pH 5.8 in the presence of 60 mM NaCl, only the (RepA) 6 dimer was observed both in the absence and presence of (dA) 30.
Keywords:Analytical Ultracentrifugation, Equilibrium Binding, RepA, Single-Stranded DNA, Stoichiometry
Source:FEBS Letters
ISSN:0014-5793
Publisher:Elsevier (The Netherlands)
Volume:482
Number:3
Page Range:180-184
Date:1 January 2000
PubMed:View item in PubMed

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