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Intermediates revealed in the kinetic mechanism for DNA unwinding by a monomeric helicase

Nature Structural & Molecular Biology volume 13, pages 242–249 (2006)Cite this article

Abstract

Helicases unwind dsDNA during replication, repair and recombination in an ATP-dependent reaction. The mechanism for helicase activity can be studied using oligonucleotide substrates to measure formation of single-stranded (ss) DNA from double-stranded (ds) DNA. This assay provides an 'all-or-nothing' readout because partially unwound intermediates are not detected. We have determined conditions under which an intermediate in the reaction cycle of Dda helicase can be detected by trapping a partially unwound substrate. The appearance of this intermediate supports a model in which each ssDNA product interacts with the helicase after unwinding has occurred. Kinetic analysis indicates that the intermediate appears during a slow step in the reaction cycle that is flanked by faster steps for unwinding. These observations demonstrate a complex mechanism containing nonuniform steps for a monomeric helicase. The potential biological significance of such a mechanism is discussed.

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Figure 1: The kinetic mechanism describing helicase action.
Figure 2: Dda-catalyzed unwinding of various lengths of dsDNA, performed under single-turnover conditions.
Figure 3: Use of MeP-modified DNA substrates to directly measure the length of dsDNA that melts spontaneously downstream of an active helicase.
Figure 4: An intermediate appears in helicase-catalyzed DNA unwinding of longer substrates in the presence of increased amounts of reannealing trap.
Figure 5: Increasing the ssDNA overhang to accommodate multiple Dda molecules does not prevent formation of the intermediate in helicase-catalyzed DNA unwinding.
Figure 6: Kinetic simulation of two possible mechanisms for Dda-catalyzed DNA unwinding.
Figure 7: Expanded kinetic mechanism for DNA unwinding by the monomeric form of Dda helicase.

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Acknowledgements

We would like to thank W. Bujalowski and A.L. Lucius for helpful discussions regarding general aspects of data analysis for DNA-unwinding reactions, with special thanks to A.L. Lucius for providing the initial Scientist script describing the inverse Laplace transform of Scheme 1. We would also like to thank M.K. Levin for helpful discussions. This work was supported by US National Institutes of Health grant R01 GM59400 (to K.D.R.) and the University of Arkansas for Medical Sciences Committee for Allocation of Graduate Student Research Funds (R.L.E.). Core facility support was provided by US National Institutes of Health grant P20 RR15569 (to F. Millet, P.I.).

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  1. Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, 4301 W. Markham St. Slot 516, Little Rock, 72205, Arkansas, USA

    Robert L Eoff & Kevin D Raney

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Correspondence to Kevin D Raney.

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Supplementary information

Supplementary Fig. 1

Position of the reannealing trap does not affect the appearance of the peak in ssDNA product. (PDF 72 kb)

Supplementary Fig. 2

Lowering the concentration of ATP slows the observed rate of helicase-catalyzed unwinding and delays the appearance of the intermediate. (PDF 54 kb)

Supplementary Fig. 3

Decreasing the concentration of ATP decreases the observed unwinding rate but does not affect the burst amplitude of ssDNA product. (PDF 53 kb)

Supplementary Fig. 4

Comparison of simulated eight-step time course with experimental unwinding data at low [ATP]. (PDF 60 kb)

Supplementary Fig. 5

Dda-catalyzed unwinding of a 120:60–mer results in the appearance of the trappable intermediate. (PDF 70 kb)

Supplementary Methods (PDF 26 kb)

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Eoff, R., Raney, K. Intermediates revealed in the kinetic mechanism for DNA unwinding by a monomeric helicase. Nat Struct Mol Biol 13, 242–249 (2006). https://doi.org/10.1038/nsmb1055

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