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.