The Inhibitory Effect of Non-Substrate and Substrate DNA on the Ligation and Self-Adenylylation Reactions Catalyzed by T4 DNA Ligase

PLoS One. 2016 Mar 8;11(3):e0150802. doi: 10.1371/journal.pone.0150802. eCollection 2016.

Abstract

DNA ligases are essential both to in vivo replication, repair and recombination processes, and in vitro molecular biology protocols. Prior characterization of DNA ligases through gel shift assays has shown the presence of a nick site to be essential for tight binding between the enzyme and its dsDNA substrate, with no interaction evident on dsDNA lacking a nick. In the current study, we observed a significant substrate inhibition effect, as well as the inhibition of both the self-adenylylation and nick-sealing steps of T4 DNA ligase by non-nicked, non-substrate dsDNA. Inhibition by non-substrate DNA was dependent only on the total DNA concentration rather than the structure; with 1 μg/mL of 40-mers, 75-mers, or circular plasmid DNA all inhibiting ligation equally. A >15-fold reduction in T4 DNA ligase self-adenylylation rate when in the presence of high non-nicked dsDNA concentrations was observed. Finally, EMSAs were utilized to demonstrate that non-substrate dsDNA can compete with nicked dsDNA substrates for enzyme binding. Based upon these data, we hypothesize the inhibition of T4 DNA ligase by non-nicked dsDNA is direct evidence for a two-step nick-binding mechanism, with an initial, nick-independent, transient dsDNA-binding event preceding a transition to a stable binding complex in the presence of a nick site.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Catalysis
  • DNA / chemistry*
  • DNA Ligases / chemistry*
  • DNA Replication
  • In Vitro Techniques
  • Kinetics

Substances

  • DNA
  • DNA Ligases

Grants and funding

This work was funded internally by New England Biolabs, Ipswich, MA, of which all authors are employees. The specific roles of the authors are articulated in the ‘author contributions’ section. Employees and owners of New England Biolabs not on the author list did not contribute to the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Critical feedback and proofreading of the manuscript was provided by additional members of the NEB Research Department as indicated in the Acknowledgments.