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

Küpfer PA, Crey-Desbiolles C, Leumann CJ - Nucleic Acids Res. (2007)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012 Bern, Switzerland.

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.

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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.
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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.

Mentions: Standing start experiments were again performed at two different enzyme concentrations. (Figure 5). At higher enzyme concentrations, faint bands are visible for single insertions in the case of dATP and dGTP. No multiple, consecutive incorporations as in the case of the HIV-1 RT could be found. Also, no primer extension takes place in the case of dTTP and dCTP. Instead some 3′–5′-exonuclease activity is present leading to 1–2 nt shortening of the primer. In the case of higher enzyme concentration and in the presence of all four dNTPs, full-length synthesis occurs to a moderate extent in the case of the abasic RNA template. This contrasts to the efficient full-length primer extension in the case of the non-damaged template (X = U) at both enzyme concentrations.Figure 5.


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

Küpfer PA, Crey-Desbiolles C, Leumann CJ - Nucleic Acids Res. (2007)

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175328&req=5

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.
Mentions: Standing start experiments were again performed at two different enzyme concentrations. (Figure 5). At higher enzyme concentrations, faint bands are visible for single insertions in the case of dATP and dGTP. No multiple, consecutive incorporations as in the case of the HIV-1 RT could be found. Also, no primer extension takes place in the case of dTTP and dCTP. Instead some 3′–5′-exonuclease activity is present leading to 1–2 nt shortening of the primer. In the case of higher enzyme concentration and in the presence of all four dNTPs, full-length synthesis occurs to a moderate extent in the case of the abasic RNA template. This contrasts to the efficient full-length primer extension in the case of the non-damaged template (X = U) at both enzyme concentrations.Figure 5.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012 Bern, Switzerland.

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.

Show MeSH
Related in: MedlinePlus