Kinetic Characterization of Single Strand Break Ligation in Duplex DNA by T4 DNA Ligase

• Published in: The Journal of biological chemistry 2011-12, Vol.286 (51), p.44187-44196
• Main Authors: Lohman, Gregory J.S., Chen, Lixin, Evans, Thomas C.
• Format: Article
• Language: English
• Subjects: Base Sequence; Biochemistry - methods; DNA - chemistry; DNA Enzymes; DNA Ligases - chemistry; DNA Repair; DNA-Binding Proteins - chemistry; DNA-Protein Interaction; Electrophoresis, Capillary; Enzyme Kinetics; Enzyme Mechanisms; Enzymology; Kinetics; Models, Chemical; Molecular Sequence Data; Pre-steady State Kinetics; Protein Binding; Proteins - chemistry; Rapid Quench-Flow; T4 DNA Ligase
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M111.284992

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 kcat and 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. Background: T4 DNA ligase catalyzes the formation of phosphodiester bonds in dsDNA. Results: Single turnover rates of all chemical reaction steps exceed steady state turnover rates by 10-fold. Conclusion: Product release or a postligation conformational change is rate-limiting during turnover. Significance: This study represents the first detailed analysis of the kinetic mechanism of nick ligation by T4 DNA ligase.