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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 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).
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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).

Mentions: RNaseH activity was again assayed in the presence and absence of dNTPs and at two different enzyme concentrations. From the gels, it becomes clear that RNaseH activity is generally slightly higher on the damaged template as compared to the non-damaged template irrespective of the enzyme concentration and the presence or absence of dNTPs (Figure 7). In the presence of the dNTPs, there is a clear difference in the preferred sites of cleavage. In the non-damaged template, the preferred site of cleavage is near to the 3′-end of the primer (positions X − 5 and X − 6, direction of DNA synthesis) while on the abasic template, the preferred site of cleavage is deep into the non-extended primer/template region. In the absence of dNTPs, the cleavage pattern is essentially the same in both cases with the preferred cleavage site deep in the non-extended primer/template part. The similar behavior in the presence or absence of dNTPs in the case of the damaged template can easily be explained by the very inefficient primer extension, excluding cleavage near to the 3′-end of the primer due to lack of elongation. The slightly higher cleavage at the abasic site in the damaged template is most likely not due to enzymatic activity but of β-elimination chemistry as a consequence of the denaturation of the sample after reaction and before application onto the gel.Figure 7.


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

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

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).
Mentions: RNaseH activity was again assayed in the presence and absence of dNTPs and at two different enzyme concentrations. From the gels, it becomes clear that RNaseH activity is generally slightly higher on the damaged template as compared to the non-damaged template irrespective of the enzyme concentration and the presence or absence of dNTPs (Figure 7). In the presence of the dNTPs, there is a clear difference in the preferred sites of cleavage. In the non-damaged template, the preferred site of cleavage is near to the 3′-end of the primer (positions X − 5 and X − 6, direction of DNA synthesis) while on the abasic template, the preferred site of cleavage is deep into the non-extended primer/template region. In the absence of dNTPs, the cleavage pattern is essentially the same in both cases with the preferred cleavage site deep in the non-extended primer/template part. The similar behavior in the presence or absence of dNTPs in the case of the damaged template can easily be explained by the very inefficient primer extension, excluding cleavage near to the 3′-end of the primer due to lack of elongation. The slightly higher cleavage at the abasic site in the damaged template is most likely not due to enzymatic activity but of β-elimination chemistry as a consequence of the denaturation of the sample after reaction and before application onto the gel.Figure 7.

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