Quantitative analysis of binding of single-stranded DNA by Escherichia coli DnaB helicase and the DnaB x DnaC complex.
DnaB helicase is responsible for unwinding duplex DNA during chromosomal DNA replication and is an essential component of the DNA replication apparatus in Escherichia coli. We have analyzed the mechanism of binding of single-stranded DNA (ssDNA) by the DnaB x DnaC complex and DnaB helicase. Binding of ssDNA to DnaB helicase was significantly modulated by nucleotide cofactors, and the modulation was distinctly different for its complex with DnaC. DnaB helicase bound ssDNA with a high affinity [Kd = (5.09 +/- 0.32) x 10(-8) M] only in the presence of ATPgammaS, a nonhydrolyzable analogue of ATP, but not other nucleotides. The binding was sensitive to ionic strength but not to changes in temperature in the range of 30-37 degrees C. On the other hand, ssDNA binding in the presence of ADP was weaker than that observed with ATPgammaS, and the binding was insensitive to ionic strength. DnaC protein hexamerizes to form a 1:1 complex with the DnaB hexamer and loads it onto the ssDNA by forming a DnaB6 x DnaC6 dodecameric complex. Our results demonstrate that the DnaB6 x DnaC6 complex bound ssDNA with a high affinity [Kd = (6.26 +/- 0.65) x 10(-8) M] in the presence of ATP, unlike the DnaB hexamer. In the presence of ATPgammaS or ADP, binding of ssDNA by the DnaB6 x DnaC6 complex was a lower-affinity process. In summary, our results suggest that in the presence of ATP in vivo, the DnaB6 x DnaC6 complex should be more efficient in binding DNA as well as in loading DnaB onto the ssDNA than DnaB helicase itself.
Biswas
Subhasis B
SB
Department of Molecular Biology, School of Medicine and Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey, Stratford, New Jersey 08084, USA. biswassb@umdnj.edu
Biswas-Fiss
Esther E
EE
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D004265:DNA Helicases / Q000737:chemistry / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D053488:DnaB Helicases; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D019793:Fluorescein / Q000378:metabolism; D009690:Nucleic Acid Conformation / Q000187:drug effects; D009994:Osmolar Concentration; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D013379:Substrate Specificity / Q000187:drug effects; D013696:Temperature
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; C071702:DnaC protein, E coli; D029968:Escherichia coli Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D004265:DNA Helicases; D053488:DnaB Helicases; D019793:Fluorescein