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Multi-step sequential mechanism of the E coli PriA helicase binding to single stranded nucleic acid



Multi-step sequential mechanism of the E coli PriA helicase binding to single stranded nucleic acid



Biophysical Journal 86(1): 313a, January



The kinetic mechanism of the E. coli replicative helicase PriA protein binding to ssDNA has been studied, using the fluorescence stopped-flow technique. Experiments have been performed with a series of fluorescent ethenoderivatives of ssDNA adenosine-oligomers, differing in the number of nucleotide residues. The PriA helicase monomer binds the ssDNA in a minimum sequential three-step process. In the first bimolecular step, the enzyme associates very fast with the ssDNA without inducing significant conformational changes in the nucleic acid. The dependence of the partial equilibrium constant characterizing the first step, upon the length of the ssDNA strictly reflects the relationship between the size of the DNA-binding site and the number of potential binding sites on the ssDNA. Thus, in the first kinetic intermediate, within the total site-size of 20+-3 nucleotides residues of the helicase-ssDNA complex, only the DNA binding site that encompasses 6.3+-1 residues is directly involved in interactions with the ssDNA. As a result, the successive intramolecular steps are independent of the length of the ssDNA. Moreover, they are accompanied by large changes in the DNA structure. Salt and glycerol effects on the dynamics of the helicase-ssDNA interactions indicate very different nature of the formed intermediates. While the bimolecular step is characterized by a net ion release and water uptake, a net ion uptake and water release accompany the intramolecular transitions. The data indicate that ion binding to specific sites on the protein stabilizes the helicase-nucleic acid complex. Furthermore, magnesium cations and nucleotide cofactor have little effect in the binding of the PriA helicase to ssDNA of different length.

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Accession: 035349597

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