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Dynamics of phosphodiester synthesis by DNA ligase
- Crut, Aurélien, Nair, Pravin A., Koster, Daniel A., Shuman, Stewart, Dekker, Nynke H.
- Proceedings of the National Academy of Sciences of the United States of America 2008 v.105 no.19 pp. 6894-6899
- DNA replication, dissociation, ligases, probability, supercoiled DNA, toxicity
- 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 [almost equal to]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.