The steric flexibility or rigidity of polymerase active sites may play an important role in their fidelity of nucleic acid synthesis. In this regard, reverse transcriptases offer an unusual opportunity to compare two enzymatic activities that proceed in the same active site. For HIV-1 reverse transcriptase, reverse transcription (RNA-templated synthesis) is known to proceed with lower fidelity than DNA-templated synthesis. Here, we describe the use of a set of variably sized nonpolar thymidine and uracil mimics as molecular rulers to probe the active site steric constraints of HIV-1 RT, and for the first time, we directly compare the functional flexibility of these two activities. Steady-state kinetics of incorporation for natural dNTPs opposite unnatural template bases as well as for unnatural dNTPs opposite natural template bases are reported for the DNA-templated DNA synthesis, and comparison is made with recent data for the RNA-templated activity. Kinetics for extension beyond a base pair containing the analogue template bases are also reported both for RNA and DNA templates. Our results show that the DNA-dependent polymerization by HIV-RT is highly sensitive to size, strongly biasing against both too-small and too-large base pairs, while, by contrast, the RNA-dependent polymerization is only biased against analogues that are too small, and is much more accepting of larger base pairs. In addition, base pair extension with HIV-RT is found to be relatively insensitive to varied base pair size, consistent with its high mutagenicity. Overall, the data show greater rigidity with a DNA template as compared with an RNA template, which correlates directly with the higher fidelity of the DNA-templated synthesis. Possible structural explanations for these differences are discussed. We also report kinetics data for two HIV-1 RT mutants reported to have altered fidelity (F61A and K65R) using DNA templates containing nonpolar base analogues, and find that one of these (F61A) is a high-fidelity enzyme that appears to be sensitive to a loss of hydrogen-bonding groups.
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