Kinetics of formation of hypoxanthine containing base pairs by HIV-RT: RNA template effects on the base substitution frequencies.

Hypoxanthine (H), the deamination product of adenine, has been implicated in the high frequency of A to G transitions observed in retroviral and other RNA genomes. Although H.C base pairs are thermodynamically more stable than other H.N pairs, polymerase selection may be determined in part by kinetic factors. Therefore, the hypoxanthine induced substitution pattern resulting from replication by viral polymerases may be more complex than that predicted from thermodynamics. We have examined the steady-state kinetics of formation of base pairs opposite template H in RNA by HIV-RT, and for the incorporation of dITP during first- and second-strand synthesis. Hypoxanthine in an RNA template enhances the k(2app) for pairing with standard dNTPs by factors of 10-1000 relative to adenine at the same sequence position. The order of base pairing preferences for H in RNA was observed to be H.C >> H.T > H.A > H.G. Steady-state kinetics of insertion for all possible mispairs formed with dITP were examined on RNA and DNA templates of identical sequence. Insertion of dITP opposite all bases occurs 2-20 times more frequently on RNA templates. This bias for higher insertion frequencies on RNA relative to DNA templates is also observed for formation of mispairs at template A. This kinetic advantage afforded by RNA templates for mismatches and pairing involving H suggests a higher induction of mutations at adenines during first-strand synthesis by HIV-RT.