Kinetic mechanism of nick sealing by T4 RNA ligase 2 and effects of 3′-OH base mispairs and damaged base lesions Journal Article
| Authors: | Chauleau, M.; Shuman, S. |
| Article Title: | Kinetic mechanism of nick sealing by T4 RNA ligase 2 and effects of 3′-OH base mispairs and damaged base lesions |
| Abstract: | 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. © 2013 Chauleau and Shuman. |
| Keywords: | controlled study; unclassified drug; nonhuman; dna damage; base pairing; catalysis; adenosine triphosphate; rna sequence; 8 hydroxyguanine; adenosine phosphate; adenylation; ester derivative; rna repair; base mispairing; rna analysis; covalent bond; chemical reaction kinetics; rna ligase; ribonucleoside; bacteriophage t4; adenine derivative; priority journal; article; 8-oxoguanine; abasic lesions; transient state kinetics; t4 rna ligase 2; single turnover nick sealing |
| Journal Title: | RNA |
| Volume: | 19 |
| Issue: | 12 |
| ISSN: | 1355-8382 |
| Publisher: | Cold Spring Harbor Laboratory Press |
| Date Published: | 2013-12-01 |
| Start Page: | 1840 |
| End Page: | 1847 |
| Language: | English |
| DOI: | 10.1261/rna.041731.113 |
| PROVIDER: | scopus |
| PMCID: | PMC3884662 |
| PUBMED: | 24158792 |
| DOI/URL: | |
| Notes: | Export Date: 2 January 2014 -- CODEN: RNARF -- Source: Scopus |
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