Escherichia coli replicative helicase PriA protein-single-stranded DNA complex. Stoichiometries, free energy of binding, and cooperativities.
Analyses of interactions of the Escherichia coli replicative helicase, PriA protein, with a single-stranded (ss) DNA have been performed, using the quantitative fluorescence titration technique. The stoichiometry of the PriA helicase.ssDNA complex has been examined in binding experiments with a series of ssDNA oligomers. The total site-size of the PriA.ssDNA complex, i.e. the maximum number of nucleotide residues occluded by the PriA helicase in the complex, is 20 +/- 3 residues per protein monomer. However, the protein can efficiently form a complex with a minimum of 8 nucleotides. Thus, the enzyme has a strong ssDNA-binding site that engages in direct interactions with a significantly smaller number of nucleotides than the total site-size. The ssDNA-binding site is located in the center of the enzyme molecule, with the protein matrix protruding over a distance of approximately 6 nucleotides on both sides of the binding site. The analysis of the binding of two PriA molecules to long oligomers was performed using statistical thermodynamic models that take into account the overlap of potential binding sites, cooperative interactions, and the protein.ssDNA complexes with different stoichiometries. The intrinsic affinity depends little upon the length of the ssDNA. Moreover, the binding is accompanied by weak cooperative interactions.
Jezewska
M J
MJ
Department of Human Biological Chemistry and Genetics, the University of Texas Medical Branch, Galveston, Texas 77555-1053, USA.
Rajendran
S
S
Bujalowski
W
W
D001665:Binding Sites; D004265:DNA Helicases / Q000737:chemistry / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008956:Models, Chemical; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism*; D051716:Replication Protein A; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D051716:Replication Protein A; C030570:oligodeoxyadenylic acid; D004265:DNA Helicases