title: Dynamics of phosphodiester synthesis by DNA ligase.
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 approximately 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.
authors.affiliation: Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands.
authors.forename: Aurélien
authors.initials: A
authors.lastname: Crut
authors.forename: Pravin A
authors.initials: PA
authors.lastname: Nair
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Koster
authors.forename: Stewart
authors.initials: S
authors.lastname: Shuman
authors.forename: Nynke H
authors.initials: NH
authors.lastname: Dekker
mesh_terms: D000249:Adenosine Monophosphate / Q000378:metabolism; D011088:DNA Ligases / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D008958:Models, Molecular; D050505:Mutant Proteins / Q000378:metabolism; D010755:Organophosphates / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism*
chemical_list: D004278:DNA, Superhelical; D050505:Mutant Proteins; D010755:Organophosphates; D014764:Viral Proteins; D000249:Adenosine Monophosphate; D004259:DNA-Directed DNA Polymerase; D004264:DNA Topoisomerases, Type I; C110456:Chlorella virus DNA ligase; D011088:DNA Ligases