Interactions of Escherichia coli primary replicative helicase DnaB protein with single-stranded DNA. The nucleic acid does not wrap around the protein hexamer.
The interactions of the Escherichia coli primary replicative helicase DnaB protein with single-stranded (ss) DNA have been studied using the thermodynamically rigorous fluorescence titration technique, which allowed us to obtain absolute stoichiometries of the formed complexes and interaction parameters without any assumptions about the relationship between the observed signal change and the degree of binding. Binding of the DnaB protein to the ssDNA fluorescent derivative poly(d epsilon A) is accompanied by a strong increase of the nucleic acid fluorescence. We show that, in the presence of the ATP nonhydrolyzable analog AMP-PNP, the DnaB helicase binds polymer ssDNA with the site-size of 20 +/- 3 nucleotides per protein hexamer. This stoichiometry has been fully confirmed in the binding experiments with ssDNA oligomers of 40 and 20 residues in length. Two DnaB hexamers bind to 40-mer, and one DnaB hexamer binds to 20-mer. Thermodynamic studies of the 20-mer binding to the DnaB hexamer show that the hexamer has a single, strong binding site for ssDNA. Moreover, photo-cross-linking experiments indicate that only a single subunit is primarily in contact with ssDNA. This surprisingly very low site-size of the large hexameric helicase--ssDNA complex, the existence of only a single, strong ssDNA binding site on the hexamer, and the results of photo-cross-linking experiments preclude the possibility of extensive wrapping of the ssDNA around the hexamer and formation of the complex in which all six protomers are simultaneously bound to ss nucleic acid.(ABSTRACT TRUNCATED AT 250 WORDS)
Bujalowski
W
W
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch at Galveston 77555-1053, USA.
Jezewska
M J
MJ
D001426:Bacterial Proteins / Q000378:metabolism*; D004265:DNA Helicases / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D053488:DnaB Helicases; D004926:Escherichia coli / Q000378:metabolism*; D010777:Photochemistry; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D004265:DNA Helicases; D053488:DnaB Helicases