Limits...
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.

Show MeSH

Related in: MedlinePlus

Primers and templates (X = dAS, rAS, T or U) used for ss primer and rs primer reverse transcription assays.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2175328&req=5

Figure 1: Primers and templates (X = dAS, rAS, T or U) used for ss primer and rs primer reverse transcription assays.

Mentions: For the polymerase and RNaseH assays, we chose the primer/template systems depicted in Figure 1. We used two formats, namely a ss system consisting of a 31-mer abasic RNA template (X = rAS) and a 20-mer DNA primer with its 3′-end directly ending before the rAS site, and a rs system with the same RNA template and a DNA primer which was 4 nt shorter. For reference, we used a non-damaged RNA template (X = U). The nucleotide sequence of the template was chosen randomly with the annealing region to have a GC content of 60% and every base to occur at least twice in the single strand overhang to be transcribed. For determining the insertion kinetics in the case of HIV-1 RT, the ss system with both the abasic RNA and the DNA template (X = rAS and dAS, respectively) was used. Also here, the templates with X = U or T served as undamaged references. The template strands were chemically synthesized using the corresponding caged abasic site precursor phosphoramidites as described previously (11,14) and the abasic sites rAS and dAS, respectively, were revealed by UV-irradiation after primer annealing. RNaseH activity was tested in the same way on the ss primer/template system using labeled template instead of labeled primer.Figure 1.


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)

Primers and templates (X = dAS, rAS, T or U) used for ss primer and rs primer reverse transcription assays.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: Primers and templates (X = dAS, rAS, T or U) used for ss primer and rs primer reverse transcription assays.
Mentions: For the polymerase and RNaseH assays, we chose the primer/template systems depicted in Figure 1. We used two formats, namely a ss system consisting of a 31-mer abasic RNA template (X = rAS) and a 20-mer DNA primer with its 3′-end directly ending before the rAS site, and a rs system with the same RNA template and a DNA primer which was 4 nt shorter. For reference, we used a non-damaged RNA template (X = U). The nucleotide sequence of the template was chosen randomly with the annealing region to have a GC content of 60% and every base to occur at least twice in the single strand overhang to be transcribed. For determining the insertion kinetics in the case of HIV-1 RT, the ss system with both the abasic RNA and the DNA template (X = rAS and dAS, respectively) was used. Also here, the templates with X = U or T served as undamaged references. The template strands were chemically synthesized using the corresponding caged abasic site precursor phosphoramidites as described previously (11,14) and the abasic sites rAS and dAS, respectively, were revealed by UV-irradiation after primer annealing. RNaseH activity was tested in the same way on the ss primer/template system using labeled template instead of labeled primer.Figure 1.

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