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Dynamic look at DNA unwinding by a replicative helicase

  1. Leemor Joshua-Tora,b,c,2
  1. aW. M. Keck Structural Biology Laboratory,
  2. bHoward Hughes Medical Institute, and
  3. cCold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724; and
  4. dCenter for Biophysics and Computational Biology and
  5. eDepartment of Physics and Center for the Physics of Living Cells, University of Illinois at Urbana–Champaign, Urbana, IL 61801

  1. Edited by Stephen J. Benkovic, The Pennsylvania State University, University Park, PA, and approved January 28, 2014 (received for review December 2, 2013)

Significance

Precise replication of the genome is essential for maintaining the integrity of genetic information in all forms of life. A key step in this process is the unwinding of the DNA double helix, a subject of intensive research and debate. We took a multifaceted approach to study fundamental mechanisms of helicase function, using the E1 helicase from papillomavirus. Our findings reveal that E1 employs a strand exclusion mechanism to unwind DNA with the N-terminal side leading at the replication fork. Intriguingly, DNA unwinding by E1 is modulated by the origin-recognition domain, suggesting a previously unsuspected role for this domain in regulating helicase activity.

Abstract

A prerequisite for DNA replication is the unwinding of duplex DNA catalyzed by a replicative hexameric helicase. Despite a growing body of research, key elements of helicase mechanism remain under substantial debate. In particular, the number of DNA strands encircled by the helicase ring during unwinding and the ring orientation at the replication fork completely contrast in contemporary mechanistic models. Here we use single-molecule and ensemble assays to address these questions for the papillomavirus E1 helicase. We find that E1 unwinds DNA with a strand-exclusion mechanism, with the N-terminal side of the helicase ring facing the replication fork. We show that E1 generates strikingly heterogeneous unwinding patterns stemming from varying degrees of repetitive movements, which is modulated by the DNA-binding domain. Together, our studies reveal previously unrecognized dynamic facets of replicative helicase unwinding mechanisms.

Footnotes

  • ↵1Present address: Department of Structural Biology, St Jude Children’s Research Hospital, Memphis, TN 38105.

  • ↵2To whom correspondence may be addressed. E-mail: tjha{at}illinois.edu or leemor{at}cshl.edu.

  • Author contributions: S.-J.L., E.J.E., A.S., T.H., and L.J. designed research; S.-J.L., S. Syed, E.J.E., S. Schuck, and A.S. performed research; S.-J.L. contributed new reagents/analytic tools; S.-J.L., S. Syed, E.J.E., S. Schuck, A.S., T.H., and L.J. analyzed data; and S.-J.L., E.J.E., A.S., T.H., and L.J. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission.

  • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1322254111/-/DCSupplemental.

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