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

RNA. 2013 Dec;19(12):1840-7. doi: 10.1261/rna.041731.113. Epub 2013 Oct 24.


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.

Keywords: 8-oxoguanine; RNA repair; abasic lesions; transient state kinetics.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenine / analogs & derivatives
  • Adenine / chemistry
  • Apurinic Acid / chemistry
  • Base Pairing
  • Base Sequence
  • Escherichia coli
  • Guanine / analogs & derivatives
  • Guanine / chemistry
  • Kinetics
  • RNA Ligase (ATP) / chemistry*
  • RNA, Double-Stranded / chemistry*
  • RNA, Double-Stranded / genetics
  • Viral Proteins / chemistry*


  • RNA, Double-Stranded
  • Viral Proteins
  • Apurinic Acid
  • 8-hydroxyadenine
  • 8-hydroxyguanine
  • Guanine
  • RNA Ligase (ATP)
  • bacteriophage T4 RNA ligase 2
  • Adenine