Dynamics of phosphodiester synthesis by DNA ligase

作者：Crut Aurelien; Nair Pravin A; Koster Daniel A; Shuman Stewart; Dekker Nynke H^*

来源：Proceedings of the National Academy of Sciences of the United States of America, 2008, 105(19): 6894-6899.

DOI：10.1073/pnas.0800113105

• DNA ligation
• DNA relaxation
• magnetic tweezers

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 approximate 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.

• 出版日期2008-5-13