Kinetic mechanism of human DNA ligase I

Abstract

DNA ligase I (Lig1) is essential for DNA replication and repair. It catalyzes the joining of single‐strand DNA breaks via three chemical reactions. In the first step the enzyme is activated by transferring the adenylyl group from ATP to an active site lysine; in the second step the adenylyl group is transferred to the 5′‐phosphate at the nick; in the third step the new phosphodiester bond is formed and AMP is released. We used transient kinetic approaches to determine the minimal kinetic mechanism for human Lig1 in the presence of Mg2+ and other divalent metal ions. Although all three reactions catalyzed by Lig1 require divalent metal ions, individual steps exhibit different affinity for metal and are susceptible to inhibition by different metal ions. These different patterns of inhibition suggest that there are multiple divalent metal ion binding sites and that the metal sites change throughout the reaction cycle. This work provides deeper insight into the mechanism of DNA ligation and illustrates how divalent metal ions can inhibit a single enzyme via very different mechanisms. The results have implications for abortive ligation, which generates blocked (adenylylated) 5′ends and for the inhibition of DNA ligation by transition metals.