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Protein displacement by an assembly of helicase molecules aligned along single-stranded DNA

Nature Structural & Molecular Biology volume 11, pages 531–538 (2004)Cite this article

Abstract

Helicases are molecular motors that unwind double-stranded DNA or RNA. In addition to unwinding nucleic acids, an important function of these enzymes seems to be the disruption of protein-nucleic acid interactions. Bacteriophage T4 Dda helicase can displace proteins bound to DNA, including streptavidin bound to biotinylated oligonucleotides. We investigated the mechanism of streptavidin displacement by varying the length of the oligonucleotide substrate. We found that a monomeric form of Dda catalyzed streptavidin displacement; however, the activity increased when multiple helicase molecules bound to the biotinylated oligonucleotide. The activity does not result from cooperative binding of Dda to the oligonucleotide. Rather, the increase in activity is a consequence of the directional bias in translocation of individual helicase monomers. Such a bias leads to protein-protein interactions when the lead monomer stalls owing to the presence of the streptavidin block.

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Figure 1: Determination of the number of nucleotides bound per Dda molecule.
Figure 2: Illustration of streptavidin displacement reaction.
Figure 3: Streptavidin displacement increases as the length of the 3′-biotinylated oligonucleotides increases under single-turnover conditions.
Figure 4: Models for enhanced activity of an assembly of Dda molecules during streptavidin displacement.
Figure 5: Displacement of streptavidin by saturating concentrations of Dda in the absence of a protein trap.
Figure 6: Dda-catalyzed streptavidin displacement observed from the 15-mer oligonucleotide.
Figure 7: Equilibrium binding and dissociation rate constants for the 15-mer.
Figure 8: Model for helicase-catalyzed displacement of streptavidin from biotin (B)-labeled oligonucleotides.

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Acknowledgements

This work was supported by research grant GM59400 (K.D.R) from the US National Institutes of Health (NIH) and the University of Arkansas for Medical Sciences graduate student research fund (A.K.B.). Core lab support for DNA synthesis and characterization was provided by NIH COBRE grant P20 RR15569. We thank C. Cameron for careful reading of the manuscript and R. Eoff for assistance with the stopped-flow experiments.

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  1. Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, 72205, Arkansas, USA

    Alicia K Byrd & Kevin D Raney

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Correspondence to Kevin D Raney.

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Byrd, A., Raney, K. Protein displacement by an assembly of helicase molecules aligned along single-stranded DNA. Nat Struct Mol Biol 11, 531–538 (2004). https://doi.org/10.1038/nsmb774

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