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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.