Kinetic mechanism of nick sealing by T4 RNA ligase 2 and effects of 3'-OH base mispairs and damaged base lesions.

T4 RNA ligase 2 (Rnl2) repairs 3'-OH/5'-PO4 nicks in duplex nucleic acids in which the broken 3'-OH strand is RNA. Ligation entails three chemical steps: reaction of Rnl2 with ATP to form a covalent Rnl2-(lysyl-Nζ)-AMP intermediate (step 1); transfer of AMP to the 5'-PO4 of the nick to form an activated AppN- intermediate (step 2); and attack by the nick 3'-OH on the AppN- strand to form a 3'-5' phosphodiester (step 3). Here we used rapid mix-quench methods to analyze the kinetic mechanism and fidelity of single-turnover nick sealing by Rnl2-AMP. For substrates with correctly base-paired 3'-OH nick termini, kstep2 was fast (9.5 to 17.9 sec(-1)) and similar in magnitude to kstep3 (7.9 to 32 sec(-1)). Rnl2 fidelity was enforced mainly at the level of step 2 catalysis, whereby 3'-OH base mispairs and oxoguanine, oxoadenine, or abasic lesions opposite the nick 3'-OH elicited severe decrements in the rate of 5'-adenylylation and relatively modest slowing of the rate of phosphodiester synthesis. The exception was the noncanonical A:oxoG base pair, which Rnl2 accepted as a correctly paired end for rapid sealing. These results underscore (1) how Rnl2 requires proper positioning of the 3'-terminal ribonucleoside at the nick for optimal 5'-adenylylation and (2) the potential for nick-sealing ligases to embed mutations during the repair of oxidative damage.