Abasic sites are well-known DNA lesions that occur spontaneously via depurination at a frequency of ca. 10 000 per genome per day in mammalian cells (1
). Acidic or alkylating conditions as well as oxidative stress greatly increase this number. Abasic DNA is unstable and undergoes strand cleavage 3′ to the abasic site with an average lifetime of 8 days at 37°C, pH 7.4 and physiological ionic strength (2
). Due to the degradation of the genetic material and to missing coding information, such sites are highly mutagenic and are a major threat to living cells (3
). Living organisms have therefore evolved a highly efficient and complex DNA repair machinery that maintains genome integrity (5
). An important component of this machinery is the base excision repair (BER) pathway in which abasic sites are produced via removal of damaged bases by specific glycosylases, followed by endonucleolytic cleavage 5′ to the abasic site by the endonuclease APE1 to generate a nick. Polymerase β (Polβ) then removes the abasic moiety with its AP lyase activity and reinserts a correct nucleotide. In the final step, the ends of the repaired strand are ligated by DNA ligase III.
Abasic lesions are a priori
not restricted to DNA but can also occur in RNA. Given the fact that not only short-lived mRNA but long-lived tRNAs and ribosomal RNAs are present at all times in a cell in much higher concentrations than DNA, and given also the fact that RNA viruses as well as retroviruses store their genetic information in form of RNA, it is not excluded that abasic RNA is of biological relevance. Literature about the chemical biology of RNA abasic sites is, however, rare and their biological impact is largely unexplored with a few exceptions. For example, RNA abasic sites are known to be the result of the action of RNA N
) that deactivate eukaryotic ribosomes by depurination of a specific adenosine residue on 28S rRNA. Such ribosome-inactivating proteins (RIPs) are long known, but only recently it was found that class-I RIPs as e.g. the pokeweed antiviral protein (PAP) cannot only depurinate the sarcin/ricin loop of the large rRNA but also specific adenine and guanine residues throughout the sequence of capped mRNAs (7
) or other viral RNAs (8
). Besides this there is also evidence for a class of RNA-specific lyases that act on ribosomal RNA abasic sites, leading to complete inactivation of ribosomes (9
). Taken together, this proves that there exist not only spontaneous but also enzymatic pathways to abasic RNA for which further metabolic processing may be required.
Given the lack of knowledge on the chemical biology of RNA abasic sites, we set out to investigate it in more detail. We (11
) and others (12
) developed a chemical synthesis of abasic RNA based on a precursor phosphoramidite carrying a photocleavable protecting group at the anomeric center. Using this method, we recently investigated the chemical reactivity of RNA abasic sites toward 3′-β-elimination chemistry. We found that, compared to abasic DNA, abasic RNA is ca. 150-fold more stable under basic conditions (0.1 M NaOH) and ca. 15-fold more stable under slightly acidic conditions in the presence of an aromatic amine (11
). This has of course to be put into perspective with the ca. 100- to 1000-fold higher stability of the glycosidic bond in a ribonucleoside compared to a 2′-deoxyribonucleoside (13
), which makes the spontaneous formation of abasic RNA a much rarer event compared to DNA. Anyway, once formed the relative longevity of abasic RNA can be expected to have a non-negligible impact on mutation in RNA viruses and retroviruses.
Reverse transcriptases (RT) are the key enzymes in retroviral replication. They synthesize in a first step a complementary DNA strand on the retroviral RNA template followed by degradation of the RNA strand by the RNaseH subunit and synthesis of the complementary DNA strand by the polymerase subunit. An abasic site on the RNA template can affect both the polymerase and the RNaseH activity of an RT. In preliminary work, we reported on the trans-lesion synthesis of HIV-1 reverse transcriptase on a RNA-template/DNA-primer system with an abasic site in the RNA template (14
). Here we present a detailed investigation of the impact of abasic RNA on reverse transcription by HIV-1 RT, avian myeloblastosis virus (AMV) RT and moloney murine leukemia virus (MMLV) (H−) RT. More specifically, we investigated the insertion preferences for any of the natural dNTPs opposite the lesion in standing start (ss) and running start (rs) experiments with DNA primers. We also investigated the RNaseH activity of HIV-1 RT and AMV RT on the abasic template. In the case of HIV-1 RT, we determined the relative kinetic efficiencies of dNTP incorporation and compared it to those of a true abasic DNA template/DNA primer system of the same sequence.