We continued biochemical studies of mammalian DNA replication proteins. Steady-state kinetic analysis of the HIV reverse transcriptase revealed an over-all kinetic scheme for reverse transcriptase mechanism. A DNA segment containing the coding region for this enzyme was subcloned in an expression vector and the enzyme was overproduced in E. coli. This recombinant enzyme is under study. The model DNA polymerase, Pol I l.f., forms DNA in a processive fashion, remaining associated with the template for many dNMP additions. However, after any given dNMP addition along a template some enzyme molecules terminate synthesis and fall off the template. This termination is quantified by analysis of reaction product size using a sequencing gel. We found that with a natural DNA (0X174) template primer system, certain points along the single-stranded DNA template are strong termination sites for Pol I l.f. The mechanism of this termination was studied. Observations were extended with a second defined template, containing one inosine reside in an otherwise d(T)n homopolymer. Termination at the I residue is modulated by dCTP and by other modifications in the reaction conditions. The results are consistent with a model in which termination occurs with several enzyme forms that are in equilibrium in an ordered catalytic mechanism.
