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 MgCl2 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.
