DNA synthesis provides the driving force to accelerate DNA unwinding by a helicase

Abstract

Helicases are molecular motors that use the energy of nucleoside 5′-triphosphate (NTP) hydrolysis to translocate along a nucleic acid strand and catalyse reactions such as DNA unwinding. The ring-shaped helicase1 of bacteriophage T7 translocates along single-stranded (ss)DNA at a speed of 130 bases per second2; however, T7 helicase slows down nearly tenfold when unwinding the strands of duplex DNA3. Here, we report that T7 DNA polymerase, which is unable to catalyse strand displacement DNA synthesis by itself, can increase the unwinding rate to 114 base pairs per second, bringing the helicase up to similar speeds compared to its translocation along ssDNA. The helicase rate of stimulation depends upon the DNA synthesis rate and does not rely on specific interactions between T7 DNA polymerase and the carboxy-terminal residues of T7 helicase. Efficient duplex DNA synthesis is achieved only by the combined action of the helicase and polymerase. The strand displacement DNA synthesis by the DNA polymerase depends on the unwinding activity of the helicase, which provides ssDNA template. The rapid trapping of the ssDNA bases by the DNA synthesis activity of the polymerase in turn drives the helicase to move forward through duplex DNA at speeds similar to those observed along ssDNA.

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Figure 1: DNA synthesis by T7 DNA polymerase.
Figure 2: The unwinding rate of T7 helicase depends on the speed of DNA synthesis.
Figure 3: DNA synthesis by T7 DNA polymerase and T7 helicase.
Figure 4: DNA synthesis by T4 and T7 DNA polymerases and T7 helicase.

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Acknowledgements

We thank M. O'Donnell, A. Berdis, N. Andraos, C. C. Richardson and M. Salas for the gift of proteins, and C. M. Drain for critical reading of the manuscript. This research was supported by an NIH grant to S.S.P.

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Correspondence to Smita S. Patel.

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Supplementary information

Supplementary Figure 1

The effect of ssDNA-binding proteins on the unwinding activity of T7 helicase. (JPG 19 kb)

Supplementary Legend

This file contains the legend for Supplementary Fig. 1. (DOC 21 kb)

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Stano, N., Jeong, Y., Donmez, I. et al. DNA synthesis provides the driving force to accelerate DNA unwinding by a helicase. Nature 435, 370–373 (2005). https://doi.org/10.1038/nature03615

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