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

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

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

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).

Mentions: We also determined the RNaseH activity of HIV-1 RT (23) on the abasic RNA template in the presence and absence of dNTPs. For this, 5′-32P-labeled template RNA and unlabeled DNA ss primer were used. At low enzyme concentration, only weak RNaseH activity in the case of both damaged and non-damaged RNA template, independent on the presence or absence of dNTPs, was observed (lanes 1, 3, 5 and 7 in Figure 4). Increasing the enzyme concentration resulted in complete disappearance of the full-length RNA template in all cases (lanes 2, 4, 6 and 8). Interestingly, in the presence of dNTPs (where primer extension is possible) there are only small differences in the RNA degradation pattern as a function of the presence or absence of the RNA abasic site. In the unmodified RNA template, cleavage occurs predominantly at the positions X and X − 4 (direction of DNA synthesis) whereas in the abasic template, cleavage is effected predominantly at positions X − 3 and X − 4. The same experiment performed in the absence of dNTPs (no primer extension possible) shows again similar degradation characteristics for the natural and the abasic template. In these cases, however, the major cleavage sites are deep in the double helical primer/template region and not near to the X site. This differential behavior is an indication that primer extension in the presence of dNTPs is faster than RNA degradation also in the case of the damaged template.Figure 4.


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

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

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).
Mentions: We also determined the RNaseH activity of HIV-1 RT (23) on the abasic RNA template in the presence and absence of dNTPs. For this, 5′-32P-labeled template RNA and unlabeled DNA ss primer were used. At low enzyme concentration, only weak RNaseH activity in the case of both damaged and non-damaged RNA template, independent on the presence or absence of dNTPs, was observed (lanes 1, 3, 5 and 7 in Figure 4). Increasing the enzyme concentration resulted in complete disappearance of the full-length RNA template in all cases (lanes 2, 4, 6 and 8). Interestingly, in the presence of dNTPs (where primer extension is possible) there are only small differences in the RNA degradation pattern as a function of the presence or absence of the RNA abasic site. In the unmodified RNA template, cleavage occurs predominantly at the positions X and X − 4 (direction of DNA synthesis) whereas in the abasic template, cleavage is effected predominantly at positions X − 3 and X − 4. The same experiment performed in the absence of dNTPs (no primer extension possible) shows again similar degradation characteristics for the natural and the abasic template. In these cases, however, the major cleavage sites are deep in the double helical primer/template region and not near to the X site. This differential behavior is an indication that primer extension in the presence of dNTPs is faster than RNA degradation also in the case of the damaged template.Figure 4.

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