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.

Two common features are evident for RT slippage directionality by all RT polymerases tested. The 'dNTP rule' is that a higher concentration of the cognate substrate specified by the template base 5 adjacent to the slippage motif, than of the substrate specified by the motif, favors slippage-mediated base omission. When the ratio is reversed, slippage-mediated base addition is favored. The concentration of each dNTP used in our RT reaction with retroviral RT polymerase is in the M range whereas with the Retrotransposon-derived TGIRT polymerase, the highest ratio is in the mM range. The equimolar dNTP conditions used in the present in vitro work are similar to those used in cDNA preparation for NGS analysis. The dNTP imbalance conditions used in the present work were relatively high, 10-fold. Even with ca. 3-fold dNTP imbalance substantial effects on DNA polymerase fidelity have been detected in vivo. Notably, the phenotype associated with an analogue of a colorectal cancer causing DNA polymerase mutator mutant is due to its causing an S-phase checkpoint-dependent elevation of dNTP pools (52) . Other results also point to important correlations of dNTP pool levels with DNA polymerase mutator activities (53) , and mutants of a deoxyribonucleoside triphosphate triphosphohydrolase that influence the level and balance of dNTP pools, are frequent in colon cancer cells (54) .