Kinetic characterization of single strand break ligation in duplex DNA by T4 DNA ligase

Abstract

T4 DNA ligase catalyzes phosphodiester bond formation between juxtaposed 5′-phosphate and 3′-hydroxyl termini in duplex DNA in three steps: 1) enzyme-adenylylate formation by reaction with ATP; 2) adenylyl transfer to a 5′-phosphorylated polynucleotide to generate adenylylated DNA; and 3) phosphodiester bond formation with release of AMP. This investigation used synthetic, nicked DNA substrates possessing either a 5′-phosphate or a 5′-adenylyl phosphate. Steady state experiments with a nicked substrate containing juxtaposed dC and 5′-phosphorylated dT deoxynucleotides (substrate 1) yielded kcat and kcat/Km values of 0.4 ± 0.1 s-1 and 150 ± 50 μM-1 s -1, respectively. Under identical reaction conditions, turnover of an adenylylated version of this substrate (substrate 1A) yielded k catand kcat/Km values of 0.64 ± 0.08 s-1 and 240 ± 40 μM-1 s-1. Single turnover experiments utilizing substrate 1 gave fits for the forward rates of Step 2 (k2) and Step 3 (k3) of 5.3 and 38 s-1, respectively, with the slowest step ∼10-fold faster than the rate of turnover seen under steady state conditions. Single turnover experiments with substrate 1A produced a Step 3 forward rate constant of 4.3 s-1, also faster than the turnover rate of 1A. Enzyme self-adenylylation was confirmed to also occur on a fast time scale (∼6 s-1), indicating that the rate-limiting step for T4 DNA ligase nick sealing is not a chemical step but rather is most likely product release. Pre-steady state reactions displayed a clear burst phase, consistent with this conclusion. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.