RNA dependent DNA replication fidelity of HIV-1 reverse transcriptase: evidence of discrimination between DNA and RNA substrates.

Important note: Information in this article was accurate in 1998. The state of the art may have changed since the publication date.

RNA dependent DNA replication fidelity of HIV-1 reverse transcriptase: evidence of discrimination between DNA and RNA substrates.

Biochemistry. 1997 Nov 18;36(46):14056-63. Unique Identifier : AIDSLINE MED/98039136
Kerr SG; Anderson KS; Department of Pharmacology, 333 Cedar Street, Yale University; School of Medicine, New Haven, Connecticut 06520-8066, USA.

Abstract: The RNA dependent DNA replication fidelity of HIV-1 reverse transcriptase has been investigated using pre-steady-state kinetics under single turnover conditions. In contrast to previous estimates of low replication fidelity of HIV-1 reverse transcriptase, the present study finds the enzyme to be more highly discriminating when an RNA/DNA template-primer is employed as compared with the corresponding DNA/DNA template-primer. The basis of this selectivity is due to extremely slow polymerization kinetics for incorporation of an incorrect deoxynucleotide. The maximum rates for misincorporation (kpol) of dGTP, dCTP, and dTTP opposite a template uridine were 0.2, 0.03, and 0.003 s-1, respectively. The equilibrium dissociation constants (Kd) for the incorrect nucleotide opposite a template uridine were 1.0, 1.1, and 0.7 mM for dGTP, dCTP, and dTTP, respectively. These kinetic values provide fidelity estimates of 26 000 for discrimination against dGTP, 176 000 for dCTP, and 1 x 10(6) for dTTP misincorporation at this position. Similar observations were obtained when incorrect nucleotide misincorporation was examined opposite a template adenine. Thus in a direct comparison of RNA/DNA and DNA/DNA template-primer substrates, HIV-1 RT exhibits approximately a 10-60-fold increase in fidelity. This study augments our current understanding of the similarities and differences of catalytic activity of HIV-1 reverse transcriptase using RNA and DNA substrates. Moreover, these studies lend further support for a model for nucleotide incorporation by HIV-1 reverse transcriptase involving a two-step binding mechanism governed by a rate-limiting conformational change for correct incorporation.

Keywords: Comparative Study Deoxycytosine Nucleotides/METABOLISM Deoxyguanine Nucleotides/METABOLISM DNA Primers *DNA Replication DNA, Viral/*BIOSYNTHESIS Escherichia coli/GENETICS HIV-1/*ENZYMOLOGY HIV-1 Reverse Transcriptase/GENETICS/*METABOLISM Kinetics Recombinant Proteins/METABOLISM Substrate Specificity Support, U.S. Gov't, P.H.S. Thymine Nucleotides/METABOLISM JOURNAL ARTICLE
980228
M9820704

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