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

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 þ 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 þ 4.1 kDa. Analytical ultracentrifugation was also applied to characterize the interaction of singlestranded 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 10³C in the presence of 0.5 mM ATPQ QS, 10 mM MgCl 2 and 60 mM NaCl as K d5X8 = 0.94 þ 0.13 W WM at pH 5.8 and K d7X6 = 25.4 þ 6.4 W WM 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 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 . ß

Interactions of the RepA Helicase Hexamer of Plasmid RSF1010 with the ssDNA. Quantitative Analysis of Stoichiometries, Intrinsic Affinities, Cooperativities, and Heterogeneity of the Total ssDNA-binding Site

“…The protein is a DNA replicative helicase that is essential for the RFS1010 plasmid replication in bacterial cells, unwinding the double-stranded DNA (dsDNA) in a 5 0 /3 0 direction. [13][14][15][16][17] The molecular mass of the RepA monomer is 29,896 Da, i.e. it is one of the smallest known helicases.…”

“…[13][14][15][16][17] In neutral and slightly alkaline conditions, the protein exclusively exists as a very stable hexamer. At pH!6, the condition applied in crystallographic studies, the RepA helicase hexamers associate into a dodecamer, possibly, corresponding to the dodecamer seen in the crystal.…”

“…However, at lower pH, the protein tends to form tubular oligomers of the hexameric unit. [13][14][15][16][17] Both the hexamer and the dodecamer have helicase activity, although significantly higher efficiency of dsDNA unwinding has been found at lower pH where the dodecamer structure is predominant. 13,14 Functional homology and high stability of the RepA hexamer resembles the behavior of the hexamer of the E. coli DnaB helicase.…”

“…the dimer of hexamers. 16,17 It has been suggested that a single dodecamer unit binds to ssDNA oligomers, differing in the length of the ssDNA from 12 to 45 nucleotide residues. 17 Surprisingly, no cooperativity in the binding process was found in these studies, in spite of the fact that the protein forms large tubular aggregates even in the absence of the DNA in the same conditions.…”

“…13,14 Analysis of interactions of the hexamer with the ssDNA binding was hindered by the proposed low affinity, beyond the resolution of applied techniques. 16,17 Fundamental quantities of the interaction of the RepA hexamer with the ssDNA such as the site-size of the protein-ssDNA complex, intrinsic affinity, base specificity, cooperativity of the binding process, and the effect of solution conditions are still unknown.…”