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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

Standing start HIV-1 RT assay with abasic RNA template (X = rAS), enzyme concentrations 0.5 and 2.0 U, reaction time 1 h. Ref: without enzyme and dNTPs. A, T, G, C: reactions in presence of the according dNTP; N: reactions in presence of all four dNTPs; Nat: unmodified RNA template (X = U) and all four dNTPs.
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Figure 2: Standing start HIV-1 RT assay with abasic RNA template (X = rAS), enzyme concentrations 0.5 and 2.0 U, reaction time 1 h. Ref: without enzyme and dNTPs. A, T, G, C: reactions in presence of the according dNTP; N: reactions in presence of all four dNTPs; Nat: unmodified RNA template (X = U) and all four dNTPs.

Mentions: Standing start experiments with two different enzyme concentrations showed that HIV-1 reverse transcriptase incorporates all four natural dNTPs opposite an abasic RNA lesion with varying preferences (Figure 2). The efficiency of incorporation of the four dNTPs followed the order A∼G > C∼T. A relative quantification of the incorporation of the different dNTPs at the higher (2 U) enzyme concentration is given in Table 1. The amount of residual non-extended primer ranged from 30% to 60% depending on the nature of the dNTP. While insertion stopped after bypassing the abasic site in the case of the pyrimidine dTPs, additional insertions were observed in the case of the purine dTPs. For dATP, a second adenosine was misincorporated after that opposite to the abasic site. In the case of dGTP, up to five guanosines were inserted before primer extension came to an end. While positions X + 1 and X + 3 correspond to a correct G–C pair, the positions X + 2 and X + 4 correspond to G–A and G–G mispairs. In the case of dCTP and dTTP insertion, substantial residual 3′–5′-exonucleolytic activity on the primer was observed. With all four dNTPs available, full-length elongation took place readily. The size of the band at the position X + 1 thereby indicates that extension of the first nucleotide after the abasic site bypass is relatively slow in comparison with a non-damaged template (X = U).Figure 2.


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 HIV-1 RT assay with abasic RNA template (X = rAS), enzyme concentrations 0.5 and 2.0 U, reaction time 1 h. Ref: without enzyme and dNTPs. A, T, G, C: reactions in presence of the according 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 2: Standing start HIV-1 RT assay with abasic RNA template (X = rAS), enzyme concentrations 0.5 and 2.0 U, reaction time 1 h. Ref: without enzyme and dNTPs. A, T, G, C: reactions in presence of the according dNTP; N: reactions in presence of all four dNTPs; Nat: unmodified RNA template (X = U) and all four dNTPs.
Mentions: Standing start experiments with two different enzyme concentrations showed that HIV-1 reverse transcriptase incorporates all four natural dNTPs opposite an abasic RNA lesion with varying preferences (Figure 2). The efficiency of incorporation of the four dNTPs followed the order A∼G > C∼T. A relative quantification of the incorporation of the different dNTPs at the higher (2 U) enzyme concentration is given in Table 1. The amount of residual non-extended primer ranged from 30% to 60% depending on the nature of the dNTP. While insertion stopped after bypassing the abasic site in the case of the pyrimidine dTPs, additional insertions were observed in the case of the purine dTPs. For dATP, a second adenosine was misincorporated after that opposite to the abasic site. In the case of dGTP, up to five guanosines were inserted before primer extension came to an end. While positions X + 1 and X + 3 correspond to a correct G–C pair, the positions X + 2 and X + 4 correspond to G–A and G–G mispairs. In the case of dCTP and dTTP insertion, substantial residual 3′–5′-exonucleolytic activity on the primer was observed. With all four dNTPs available, full-length elongation took place readily. The size of the band at the position X + 1 thereby indicates that extension of the first nucleotide after the abasic site bypass is relatively slow in comparison with a non-damaged template (X = U).Figure 2.

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