Effects of 3′-OH and 5′-PO 4 Base Mispairs and Damaged Base Lesions on the Fidelity of Nick Sealing by Deinococcus radiodurans RNA Ligase

Abstract

Deinococcus radiodurans RNA ligase (DraRnl) is the founding member of a family of end-joining enzymes encoded by diverse microbes and viruses. DraRnl ligates 3'-OH, 5'-PO4 nicks in double-stranded nucleic acids in which the nick 3'-OH end is RNA. Here we gauge the effects of 3'-OH and 5'-PO4 base mispairs and damaged base lesions on the rate of nick sealing. DraRnl is indifferent to the identity of the 3'-OH nucleobase, provided that it is correctly paired. With 3'-OH mispairs, the DraRnl sealing rate varies widely, with G-T and A-C mispairs being the best substrates and G-G, G-A, and A-A mispairs being the worst. DraRnl accepts 3' A-8-oxoguanine (oxoG) to be correctly paired, while it discriminates against U-oxoG and G-oxoG mispairs. DraRnl displays high activity and low fidelity in sealing 3'-OH ends opposite an 8-oxoadenine lesion. It prefers 3'-OH adenosine when sealing opposite an abasic template site. With 5'-PO4 mispairs, DraRnl seals a 5' T-G mispair as well as it does a 5' C-G pair; in most other respects, the ligation fidelity at 5' mispairs is similar to that at 3' mispairs. DraRnl accepts a 5' A-oxoG end to be correctly paired, yet it is more tolerant of 5' T-oxoG and 5' G-oxoG mispairs than the equivalent configurations on the 3' side of the nick. At 5' nucleobase-abasic site nicks, DraRnl prefers to ligate when the nucleobase is a purine. The biochemical properties of DraRnl are compatible with its participation in the templated repair of RNA damage or in the sealing of filled DNA gaps that have a 3' ribopatch.