Rapid Time Scale Analysis of T4 DNA Ligase-DNA Binding.
DNA ligases, essential to both in vivo genome integrity and in vitro molecular biology, catalyze phosphodiester bond formation between adjacent 3'-OH and 5'-phosphorylated termini in dsDNA. This reaction requires enzyme self-adenylylation, using ATP or NAD+as a cofactor, and AMP release concomitant with phosphodiester bond formation. In this study, we present the first fast time scale binding kinetics of T4 DNA ligase to both nicked substrate DNA (nDNA) and product-equivalent non-nicked dsDNA, as well as binding and release kinetics of AMP. The described assays utilized a fluorescein-dT labeled DNA substrate as a reporter for ligase·DNA interactions via stopped-flow fluorescence spectroscopy. The analysis revealed that binding to nDNA by the active adenylylated ligase occurs in two steps, an initial rapid association equilibrium followed by a transition to a second bound state prior to catalysis. Furthermore, the ligase binds and dissociates from nicked and nonsubstrate dsDNA rapidly with initial association affinities on the order of 100 nM regardless of enzyme adenylylation state. DNA binding occurs through a two-step mechanism in all cases, confirming prior proposals of transient binding followed by a transition to a productive ligase·nDNA (Lig·nDNA) conformation but suggesting that weaker nonproductive "closed" complexes are formed as well. These observations demonstrate the mechanistic underpinnings of competitive inhibition by rapid binding of nonsubstrate DNA, and of substrate inhibition by blocking of the self-adenylylation reaction through nick binding by deadenylylated ligase. Our analysis further reveals that product release is not the rate-determining step in turnover.
DNA Enzymes Division, New England BioLabs, Inc. , Ipswich, Massachusetts 01938-2723, United States.
Robert J
0000-0001-6317-6933
RJ
Bauer
University of Michigan Medical School , Ann Arbor, Michigan 48109-0600, United States.
Thomas J
TJ
Jurkiw
DNA Enzymes Division, New England BioLabs, Inc. , Ipswich, Massachusetts 01938-2723, United States.
Thomas C
TC
Evans
DNA Enzymes Division, New England BioLabs, Inc. , Ipswich, Massachusetts 01938-2723, United States.
Gregory J S
GJ
Lohman
D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D011088:DNA Ligases / Q000378:metabolism*; D007700:Kinetics; D011485:Protein Binding
D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D004247:DNA; D011088:DNA Ligases