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 approximately G > C approximately 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.

Figure 1.

Primers and templates (X = dAS, rAS, T or U) used for ss primer and rs primer reverse transcription assays.

Figure 2.

Standing start HIV-1 RT assay with abasic RNA template (X = rAS), enzyme concentrations 0.5 and 2.0 U, reaction time 1 h. Ref: without enzyme and dNTPs. A, T, G, C: reactions in presence of the according dNTP; N: reactions in presence of all four dNTPs; Nat: unmodified RNA template (X = U) and all four dNTPs.

Figure 3.

Comparison of ss (left) and rs (right) elongation experiments with HIV-1 RT, reaction time 1 h P_ss: primer ss, P_rs: primer rs, T_m: abasic RNA template (X = rAS), T_n: non-damaged RNA template (X = U).

Figure 4.

Comparison of the RNaseH activity of HIV-1 RT with (lanes 1–4) and without (lanes 5–8) dNTPs at different enzyme concentrations: 0.5 U (lanes 1, 3, 5, 7) and 2.0 U (lanes 2, 4, 6, 8). Nat = unmodified RNA template (X = U), Mod = abasic RNA template (X = rAS).

Figure 5.

Standing start AMV RT assay with abasic RNA template (X = rAS). Enzyme concentrations 2.0 and 8.0 U, reaction time 1 h. Ref: without enzyme and dNTPs. A, T, G, C: reactions in presence of the respective dNTP; N: reactions in presence of all four dNTPs; Nat: unmodified RNA template (X = U) and all four dNTPs.

Figure 6.

Comparison of ss (left) and rs (right) primer elongation experiments with AMV RT, reaction time 1 h. P_ss: primer ss, P_rs: primer rs, T_m: abasic RNA template (X = rAS), T_n: natural RNA template (X = U).

Figure 7.

Comparison of the RNaseH activity of AMV reverse transcriptase with (lanes 1–4) and without (lanes 5–8) dNTPs for different enzyme concentrations: 2.0 U (lanes 1, 3, 5, 7) and 8.0 U (lanes 2, 4, 6, 8). Nat = unmodified RNA template (X = U); Mod = abasic RNA template (X = rAS).

Figure 8.

Standing start MMLV RT (H−) assay with abasic RNA template (X = rAS), enzyme concentrations 4.0, 16 and 32 U, reaction time 1 h. Ref: without enzyme and dNTPs. A, T, G, C: reactions in presence of the according dNTP; N: reactions in presence of all four dNTPs; Nat: non-damaged template (X = U) and all four dNTPs.