Escherichia coli replicative helicase PriA protein-single-stranded DNA complex

Stoichiometries, free energy of binding, and cooperativities

Maria J. Jezewska, Surendran Rajendran, Wlodzimierz Bujalowski

Research output: Contribution to journalArticle

31 Citations (Scopus)

Abstract

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. tare 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 ≃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.

Original languageEnglish (US)
Pages (from-to)27865-27873
Number of pages9
JournalJournal of Biological Chemistry
Volume275
Issue number36
DOIs
StatePublished - Sep 8 2000

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Single-Stranded DNA
Stoichiometry
Escherichia coli
Free energy
Nucleotides
Binding Sites
Oligomers
Proteins
Molecules
Statistical mechanics
Statistical Models
Enzymes
Titration
Thermodynamics
Monomers
Fluorescence
Experiments

ASJC Scopus subject areas

  • Biochemistry

Cite this

Escherichia coli replicative helicase PriA protein-single-stranded DNA complex : Stoichiometries, free energy of binding, and cooperativities. / Jezewska, Maria J.; Rajendran, Surendran; Bujalowski, Wlodzimierz.

In: Journal of Biological Chemistry, Vol. 275, No. 36, 08.09.2000, p. 27865-27873.

Research output: Contribution to journalArticle

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