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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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Related in: MedlinePlus

Comparison of ss (left) and rs (right) elongation experiments with HIV-1 RT, reaction time 1 h Pss: primer ss, Prs: primer rs, Tm: abasic RNA template (X = rAS), Tn: non-damaged RNA template (X = U).
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Figure 3: Comparison of ss (left) and rs (right) elongation experiments with HIV-1 RT, reaction time 1 h Pss: primer ss, Prs: primer rs, Tm: abasic RNA template (X = rAS), Tn: non-damaged RNA template (X = U).

Mentions: To study the insertion steps preceding the abasic lesion, we performed a rs experiment (Figure 3). At the higher enzyme concentration, the ss as well as the rs experiments showed efficient trans-lesion synthesis. The rs assay confirmed that the slowest nucleotide insertions occur at the positions opposite X and X + 1. The subsequent incorporations then proceed readily, although typically a higher number of truncated fragments are observed compared with the non-damaged template system (X = U).Figure 3.


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)

Comparison of ss (left) and rs (right) elongation experiments with HIV-1 RT, reaction time 1 h Pss: primer ss, Prs: primer rs, Tm: abasic RNA template (X = rAS), Tn: non-damaged RNA template (X = U).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 3: Comparison of ss (left) and rs (right) elongation experiments with HIV-1 RT, reaction time 1 h Pss: primer ss, Prs: primer rs, Tm: abasic RNA template (X = rAS), Tn: non-damaged RNA template (X = U).
Mentions: To study the insertion steps preceding the abasic lesion, we performed a rs experiment (Figure 3). At the higher enzyme concentration, the ss as well as the rs experiments showed efficient trans-lesion synthesis. The rs assay confirmed that the slowest nucleotide insertions occur at the positions opposite X and X + 1. The subsequent incorporations then proceed readily, although typically a higher number of truncated fragments are observed compared with the non-damaged template system (X = U).Figure 3.

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