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Published online on May 5, 2008
Proc. Natl. Acad. Sci. USA, 10.1073/pnas.0800113105


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BIOPHYSICS
Dynamics of phosphodiester synthesis by DNA ligase

Aurélien Crut*,{dagger}, Pravin A. Nair{ddagger}, Daniel A. Koster*,§, Stewart Shuman{ddagger}, and Nynke H. Dekker*,¶

*Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands; and {ddagger}Molecular Biology Program, Sloan–Kettering Institute, New York, NY 10021

Edited by Gregory A. Petsko, Brandeis University, Waltham, MA, and approved March 11, 2008 (received for review January 5, 2008)

Abstract

Ligases are essential actors in DNA replication, recombination, and repair by virtue of their ability to seal breaks in the phosphodiester backbone. Ligation proceeds through a nicked DNA-adenylate intermediate (AppDNA), which must be sealed quickly to avoid creating a potentially toxic lesion. Here, we take advantage of ligase-catalyzed AMP-dependent incision of a single supercoiled DNA molecule to observe the step of phosphodiester synthesis in real time. An exponentially distributed number of supercoils was relaxed per successful incision-resealing event, from which we deduce the torque-dependent ligation probability per DNA swivel. Premature dissociation of ligase from nicked DNA-adenylate accounted for {approx}10% of the observed events. The ability of ligase to form a C-shaped protein clamp around DNA is a key determinant of ligation probability per turn and the stability of the ligase-AppDNA intermediate. The estimated rate of phosphodiester synthesis by DNA ligase (400 s–1) is similar to the high rates of phosphodiester synthesis by replicative DNA polymerases.

DNA ligation | DNA relaxation | magnetic tweezers

Footnotes

Author contributions: A.C., S.S., and N.H.D. designed research; A.C. and P.A.N. performed research; A.C., D.A.K., S.S., and N.H.D. analyzed data; and A.C., S.S., and N.H.D. wrote the paper.

{dagger}Present address: Laboratoire de Spectrométrie Ionique et Moléculaire, Université Claude Bernard Lyon 1, 43 Boulevard du 11 Novembre 1918, 69622 Villeurbanne Cedex, France.

§Present address: Departments of Physics of Complex Systems and Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

To whom correspondence should be addressed. E-mail: n.h.dekker{at}tudelft.nl

© 2008 by The National Academy of Sciences of the USA


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