Dynamics of phosphodiester synthesis by DNA ligase.
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.
Crut
Aurélien
A
Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands.
Nair
Pravin A
PA
Koster
Daniel A
DA
Shuman
Stewart
S
Dekker
Nynke H
NH
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*
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