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.

Journal of Molecular Biology
Michal R SzymanskiWlodzimierz Bujalowski

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. AD...Continue Reading

References

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Citations

Feb 20, 2013·Biochemistry·Michal R SzymanskiWlodzimierz Bujalowski
Feb 20, 2013·Biochemistry·Michal R SzymanskiWlodzimierz Bujalowski
Jun 7, 2011·Journal of Molecular Biology·Michal R SzymanskiWlodzimierz Bujalowski
Dec 5, 2017·Nucleic Acids Research·Tricia A WindgassenJames L Keck
Dec 30, 2018·The Journal of Biological Chemistry·Tricia A WindgassenJames L Keck
Sep 12, 2018·Proceedings of the National Academy of Sciences of the United States of America·Tricia A WindgassenJames L Keck

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