title: Kinetic mechanism of human DNA ligase I reveals magnesium-dependent changes in the rate-limiting step that compromise ligation efficiency.
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.
authors.affiliation: Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109-0600, USA.
authors.forename: Mark R
authors.initials: MR
authors.lastname: Taylor
authors.forename: John A
authors.initials: JA
authors.lastname: Conrad
authors.forename: Daniel
authors.initials: D
authors.lastname: Wahl
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: O'Brien
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D053960:DNA Breaks; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000737:chemistry* / Q000378:metabolism; D004260:DNA Repair / Q000502:physiology; D004261:DNA Replication / Q000502:physiology; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry* / Q000378:metabolism
chemical_list: C000607892:LIG1 protein, human; D000255:Adenosine Triphosphate; D004247:DNA; D011088:DNA Ligases; D000072481:DNA Ligase ATP; D008274:Magnesium