Escherichia coli DnaB helicase-DnaC protein complex

Allosteric effects of the nucleotides on the nucleic acid binding and the kinetic mechanism of NTP hydrolysis. 3

Anasuya Roychowdhury, Michal R. Szymanski, Maria J. Jezewska, Wlodzimierz Bujalowski

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Allosteric interactions between the DNA- and NTP-binding sites of the Escherichia coli DnaB helicase engaged in the DnaB-DnaC complex and the mechanism of NTP hydrolysis by the complex have been examined using the fluorescence titration, analytical ultracentrifugation, and rapid quench-flow technique. Surprisingly, the ssDNA affinity of the DnaB-DnaC complex is independent of the structure of the phosphate group of the cofactor bound to the helicase. Thus, the DnaC protein eliminates the antagonistic allosteric effect of NTP and NDP on the ssDNA affinity of the enzyme. The protein changes the engagement of the DNA-binding subsites of the helicase in interactions with the nucleic acid, depending on the structure of the phosphate group of the present nucleotide cofactor and profoundly affects the structure of the bound DNA. Moreover, the ssDNA affinity of the helicase in the DnaB-DnaC complex is under the control of the nucleotide-binding site of the DnaC protein. The protein does not affect the NTP hydrolysis mechanism of the helicase. Nevertheless, the rate of the chemical step is diminished in the DnaB-DnaC complex. In the tertiary DnaB-DnaC-ssDNA complex, the ssDNA changes the internal dynamics between intermediates of the pyrimidine cofactor, in a manner independent of the base composition of the DNA, while the hydrolysis step of the purine cofactor is specifically stimulated by the homoadenosine ssDNA. The significance of these results for functional activities of the DnaB-DnaC complex is discussed.

Original languageEnglish (US)
Pages (from-to)6747-6763
Number of pages17
JournalBiochemistry
Volume48
Issue number29
DOIs
StatePublished - Jul 28 2009

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DnaB Helicases
Escherichia coli
Nucleic Acids
Hydrolysis
Nucleotides
Kinetics
DNA
Proteins
Phosphates
Binding Sites
Ultracentrifugation
Base Composition
Titration
Fluorescence
Enzymes
Chemical analysis

ASJC Scopus subject areas

  • Biochemistry

Cite this

Escherichia coli DnaB helicase-DnaC protein complex : Allosteric effects of the nucleotides on the nucleic acid binding and the kinetic mechanism of NTP hydrolysis. 3. / Roychowdhury, Anasuya; Szymanski, Michal R.; Jezewska, Maria J.; Bujalowski, Wlodzimierz.

In: Biochemistry, Vol. 48, No. 29, 28.07.2009, p. 6747-6763.

Research output: Contribution to journalArticle

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