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BIOLOGICAL SCIENCES / BIOCHEMISTRY
Real-time observation of bacteriophage T4 gp41 helicase reveals an unwinding mechanism

Timothée Lionnet^*,, Michelle M. Spiering, Stephen J. Benkovic^,, David Bensimon^*, and Vincent Croquette^*

*Laboratoire de Physique Statistique, Ecole Normale Supérieure, Centre National de la Recherche Scientifique-UMR8550, 24 Rue Lhomond, 75005 Paris, France; and Department of Chemistry, Pennsylvania State University, 104 Chemistry Building, University Park, PA 16802

Contributed by Stephen J. Benkovic, October 17, 2007 (received for review September 29, 2007)

Helicases are enzymes that couple ATP hydrolysis to the unwinding of double-stranded (ds) nucleic acids. The bacteriophage T4 helicase (gp41) is a hexameric helicase that promotes DNA replication within a highly coordinated protein complex termed the replisome. Despite recent progress, the gp41 unwinding mechanism and regulatory interactions within the replisome remain unclear. Here we use a single tethered DNA hairpin as a real-time reporter of gp41-mediated dsDNA unwinding and single-stranded (ss) DNA translocation with 3-base pair (bp) resolution. Although gp41 translocates on ssDNA as fast as the in vivo replication fork (400 bp/s), its unwinding rate extrapolated to zero force is much slower (30 bp/s). Together, our results have two implications: first, gp41 unwinds DNA through a passive mechanism; second, this weak helicase cannot efficiently unwind the T4 genome alone. Our results suggest that important regulations occur within the replisome to achieve rapid and processive replication.

passive mechanism | single molecule | magnetic tweezers | DNA replication | replisome

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/cgi/content/full/0709793104/DC1.

To whom correspondence may be sent at the present address: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. E-mail: tlionnet{at}aecom.yu.edu

To whom correspondence may be addressed at: Department of Chemistry, Pennsylvania State University, 414 Wartik Laboratory, University Park, PA 16802. E-mail: sjb1{at}psu.edu

© 2007 by The National Academy of Sciences of the USA

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