Kinetic mechanism of human DNA ligase I reveals magnesium-dependent changes in the rate-limiting step that compromise ligation efficiency

J Biol Chem. 2011 Jul 1;286(26):23054-62. doi: 10.1074/jbc.M111.248831. Epub 2011 May 10.

Abstract

DNA ligase I (LIG1) catalyzes the ligation of single-strand breaks to complete DNA replication and repair. The energy of ATP is used to form a new phosphodiester bond in DNA via a reaction mechanism that involves three distinct chemical steps: enzyme adenylylation, adenylyl transfer to DNA, and nick sealing. We used steady state and pre-steady state kinetics to characterize the minimal mechanism for DNA ligation catalyzed by human LIG1. The ATP dependence of the reaction indicates that LIG1 requires multiple Mg(2+) ions for catalysis and that an essential Mg(2+) ion binds more tightly to ATP than to the enzyme. Further dissection of the magnesium ion dependence of individual reaction steps revealed that the affinity for Mg(2+) changes along the reaction coordinate. At saturating concentrations of ATP and Mg(2+) ions, the three chemical steps occur at similar rates, and the efficiency of ligation is high. However, under conditions of limiting Mg(2+), the nick-sealing step becomes rate-limiting, and the adenylylated DNA intermediate is prematurely released into solution. Subsequent adenylylation of enzyme prevents rebinding to the adenylylated DNA intermediate comprising an Achilles' heel of LIG1. These ligase-generated 5'-adenylylated nicks constitute persistent breaks that are a threat to genomic stability if they are not repaired. The kinetic and thermodynamic framework that we have determined for LIG1 provides a starting point for understanding the mechanism and specificity of mammalian DNA ligases.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / chemistry*
  • Adenosine Triphosphate / genetics
  • Adenosine Triphosphate / metabolism
  • Catalysis
  • DNA / chemistry*
  • DNA / metabolism
  • DNA Breaks
  • DNA Ligase ATP
  • DNA Ligases / chemistry*
  • DNA Ligases / metabolism
  • DNA Repair / physiology
  • DNA Replication / physiology
  • Humans
  • Kinetics
  • Magnesium / chemistry*
  • Magnesium / metabolism

Substances

  • LIG1 protein, human
  • Adenosine Triphosphate
  • DNA
  • DNA Ligases
  • DNA Ligase ATP
  • Magnesium