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

Journal of Molecular Biology
M J JezewskaW Bujalowski

Abstract

Interactions between the replicative RepA helicase hexamer of plasmid RSF1010 with the single-stranded DNA (ssDNA) have been studied, using the quantitative fluorescence titration, analytical sedimentation velocity, and sedimentation equilibrium techniques. Experiments were performed with fluorescein-labeled ssDNA oligomers. Studies with unmodified ssDNA oligomers were accomplished using the macromolecular competition titration method. Analyses of RepA helicase interactions with a series of the ssDNA provide direct evidence that the total site-size of the RepA hexamer-ssDNA complex is 19 +/- 1 nucleotide residues. The total ssDNA-binding site of the hexamer has a heterogeneous structure. Part of the total binding site constitutes the proper ssDNA-binding site of the enzyme, an area that possesses strong ssDNA-binding capability and encompasses only 8 +/- 1 residues of the ssDNA. The statistical effect on the macroscopic binding constant for the proper ssDNA-binding site indicates that it is structurally separated from the remaining part of the total ssDNA-binding site. Engagement in interactions with the ssDNA is accompanied by net ion release. Moreover, the proper ssDNA-binding site shows little base specificity. On the other ...Continue Reading

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Citations

Feb 4, 2011·The Journal of Biological Chemistry·Yuan ChengMatthew R Redinbo
Mar 21, 2007·Molecular BioSystems·Alessandro Vindigni
May 4, 2007·Biochemistry·Susan S DignamJohn David Dignam

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