Kinetic characterization of single strand break ligation in duplex DNA by T4 DNA ligase.
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.
New England Biolabs Inc., Ipswich, Massachusetts 01938-2723.
Gregory J S
GJS
Lohman
New England Biolabs Inc., Ipswich, Massachusetts 01938-2723.
Lixin
L
Chen
New England Biolabs Inc., Ipswich, Massachusetts 01938-2723. Electronic address: evanst@neb.com.
Thomas C
TC
Evans
D001483:Base Sequence; D001671:Biochemistry / Q000379:methods*; D004247:DNA / Q000737:chemistry*; D011088:DNA Ligases / Q000737:chemistry*; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000737:chemistry; D019075:Electrophoresis, Capillary; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry
D004268:DNA-Binding Proteins; D011506:Proteins; D004247:DNA; D011088:DNA Ligases