Stimulation of reverse transcriptase generated cDNAs with specific indels by template RNA structure: retrotransposon, dNTP balance, RT-reagent usage

Penno, Christophe; Kumari, Romika; Baranov, Pavel V.; van Sinderen, Douwe; Atkins, John F.

Our 'roadblock stimulatory RT slippage' model highlights the dependence of productive slippage on RT polymerase translocation state and cognate substrate incorporation. Though this model involves realignment of the 3 end of the cDNA in either direction with respect to the template, it does not give any explanation about the trigger for the realignment process. A previous model for HIV-1 RT slippage-mediated base omission involves 'template-strand slippage' with a separate model for HIV-1 RT base addition involving 'primer-strand slippage' (35) . These models feature either potential formation of an extrahelical base (a bulge) or base sharing. WT HIV-1 RT enzyme has a fivefold greater efficiency for base addition than base omission (35) . Substitution of HIV-1RT Glu89 by other residues leads not only to a decrease of slippage-mediated base omission in favor of an increase of base addition, but it also leads to a decrease of base substitution errors. Glu89 is in close proximity to the sugar phosphate backbone of the template strand near the penultimate base pair. Its importance for fidelity and slippage directionality has been suggested to be due to increased dNTP binding pocket stability (35) . An alternative explanation linked to our model, is that WT and position 89 variants differentially favor the pre-and posttranslocation states. Knowledge of a possible correlation between WT and position 89 variants and both HIV-1 RT translocation state and slippage directionality, would be informative.