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

Hypoxanthine (H), the deamination product of adenine, has been implicated i n the high frequency of A to G transitions observed in retroviral and other RNA genomes. Although H.C base pairs are thermodynamically more stable tha n other H.N pairs, polymerase selection may be determined in part by kineti c factors. Therefore, the hypoxanthine induced substitution pattern resulti ng from replication by viral polymerases may be more complex than that pred icted from thermodynamics, We have examined the steady-state kinetics of fo rmation of base pairs opposite template H in RNA by HIV-RT, and for the inc orporation 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 o rder 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 sequen ce, Insertion of dITP opposite all bases occurs 2-20 times more frequently on RNA templates. This bias for higher insertion frequencies on RNA relativ e to DNA templates is also observed for formation of mispairs at template A . This kinetic advantage afforded by RNA templates for mismatches and pairi ng involving H suggests a higher induction of mutations at adenines during first-strand synthesis by HIV-RT.