Trans-lesion synthesis and RNaseH activity by reverse transcriptases on a true abasic RNA template

Abstract

While much is known about abasic DNA, the biological impact of abasic RNA is largely unexplored. To test the mutagenic potential of this RNA lesion in the context of retroviruses, we synthesized a 31-mer oligoribonucleotide containing an abasic (rAS) site and used it as a template for studying DNA primer extension by HIV-1, avian myeloblastosis virus (AMV) and moloney murine leukemia virus (MMLV) reversed transcriptases (RT). We found that trans-lesion synthesis readily takes place with HIV-1 RT and to a lesser extent with AMV RT while MMLV RT aborts DNA synthesis. The preference of dNTP incorporation follows the order A∼G > C∼T and thus obeys to the ‘A-rule’. In the case of HIV-1 RT, we measured the kinetic data of dNTP incorporation and compared it to abasic DNA. We found that A-incorporation is only 2-fold slower relative to a matched (undamaged) RNA template while it is 7-fold slower in the case of DNA. Furthermore, there is less discrimination in incorporation between the four dNTPs in the case of abasic RNA compared to abasic DNA. These experiments clearly point to a higher promiscuity of lesion bypass on abasic RNA. Given their known higher chemical stability, such rAS sites can clearly contribute to (retro)viral evolution.

Quantitative profiling of the in vivo enzymatic activity of ricin reveals disparate depurination of different pulmonary cell types

“…Depurinated RNA species are detected indirectly by measuring the cDNA reverse transcription products which have incorporated deoxyadenosine at the expected site. In our experimental setup, deoxyadenosine-containing cDNA species spanning the abasic site were not generated since we consistently used mMLV reverse transcriptase which does not promote nucleotide insertion opposite the abasic site in the RNA template (Kupfer et al, 2007), while in the studies mentioned above, recombinant MuLV reverse transcriptase was utilized. It should be noted in this context that the incorporation efficiency of dNTPs in case of an abasic site where RNA serves as template, is considerably low.…”

RNA duplexes with abasic substitutions are potent and allele-selective inhibitors of huntingtin and ataxin-3 expression

“…RNA that contains abasic sites is more stable than abasic site-containing DNA (3,4). Abasic RNA can interact with enzymes, including HIV reverse transcriptase (4,5), APE1 endonuclease (6,7) and mutated Thermococcus DNA polymerase (8). Abasic RNA has been used to probe RNA structure (9) and can be compatible with RNA interference (RNAi) (10).…”

An assay for RNA oxidation induced abasic sites using the Aldehyde Reactive Probe

“…To further examine the ARP reactivity to RNA abasic sites, an oligo ribonucleotide, which contains abasic sites, was prepared by chemical synthesis according to a previous report [28]. Abasic sites in the oligo ribonucleotide were deprotected from a photocleavable 1-(2-nitrophenyl)ethyl group by visible light illumination.…”