The Escherichia coli PriA helicase-double-stranded DNA complex: Location of the strong DNA-binding subsite on the helicase domain of the protein and the affinity control by the two nucleotide-binding sites of the enzyme

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

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9 Scopus citations

Abstract

The Escherichia coli PriA helicase complex with the double-stranded DNA (dsDNA), the location of the strong DNA-binding subsite, and the effect of the nucleotide cofactors, bound to the strong and weak nucleotide-binding site of the enzyme on the dsDNA affinity, have been analyzed using the fluorescence titration, analytical ultracentrifugation, and photo-cross-linking techniques. The total site size of the PriA-dsDNA complex is only 5 ± 1 bp, that is, dramatically lower than 20 ± 3 nucleotides occluded in the enzyme-single-stranded DNA (ssDNA) complex. The helicase associates with the dsDNA using its strong ssDNA-binding subsite in an orientation very different from the complex with the ssDNA. The strong DNA-binding subsite of the enzyme is located on the helicase domain of the PriA protein. The dsDNA intrinsic affinity is considerably higher than the ssDNA affinity and the binding process is accompanied by a significant positive cooperativity. Association of cofactors with strong and weak nucleotide-binding sites of the protein profoundly affects the intrinsic affinity and the cooperativity, without affecting the stoichiometry. ATP analog binding to either site diminishes the intrinsic affinity but preserves the cooperativity. ADP binding to the strong site leads to a dramatic increase of the cooperativity and only slightly affects the affinity, while saturation of both sites with ADP strongly increases the affinity and eliminates the cooperativity. Thus, the coordinated action of both nucleotide-binding sites on the PriA-dsDNA interactions depends on the structure of the phosphate group. The significance of these results for the enzyme activities in recognizing primosome assembly sites or the ssDNA gaps is discussed.

Original languageEnglish (US)
Pages (from-to)344-362
Number of pages19
JournalJournal of Molecular Biology
Volume402
Issue number2
DOIs
StatePublished - Sep 2010

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Keywords

  • Allosteric interactions
  • DNA binding
  • Helicases
  • Motor proteins
  • PriA protein

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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