Multistep sequential mechanism of Escherichia coli helicase PriA protein-ssDNA interactions. Kinetics and energetics of the active ssDNA-searching site of the enzyme

Roberto Galletto, Maria J. Jezewska, Wlodzimierz Bujalowski

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Abstract

Kinetics of the Escherichia coli PriA helicase interactions with the ssDNA has been studied, using the fluorescence stopped-flow technique. Experiments have been performed with a series of fluorescent etheno derivatives of ssDNA adenosine oligomers, differing in the number of nucleotide residues. The PriA helicase binds the ssDNA in the sequential process defined by k1 k2 k3 PriA + ssDNA ↔ (P)1 ↔ (P) 2 ↔ (P)3 k-1 k-2 k-3 In the first step, the enzyme associates fast with the ssDNA without inducing conformational changes in the DNA. The dependence of the partial equilibrium constant, characterizing the first step, upon the length of the ssDNA strictly reflects the statistical relationship between the size of the DNA-binding site and the number of potential binding sites on the ssDNA. Only the DNA-binding site that encompasses 6.3 ± 1 residues is directly involved in interactions. The site is located on a structural domain allowing the enzyme to efficiently search and recognize small patches of the ssDNA. Intramolecular steps are independent of the ssDNA length and accompanied by changes in the DNA structure. Salt and glycerol effects on the studied kinetics indicate a very different nature of the intermediates. While the bimolecular step is characterized by net ion release and water uptake, net ion uptake and water release accompany intramolecular transitions. Specific ion binding stabilizes the helicase-ssDNA complex in (P)2 and (P)3 intermediates. However, magnesium and AMP-PNP do not affect the mechanism of enzyme-ssDNA interactions. The sequential character of the mechanism indicates that the enzyme does not exist in a preequilibrium conformational transition prior to the DNA binding.

Original languageEnglish (US)
Pages (from-to)11002-11016
Number of pages15
JournalBiochemistry
Volume43
Issue number34
DOIs
StatePublished - Aug 31 2004

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ASJC Scopus subject areas

  • Biochemistry

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