Fidelity of mutant HIV-1 reverse transcriptases: interaction with the single-stranded template influences the accuracy of DNA synthesis

Biochemistry
Baek KimL A Loeb

Abstract

We have used random sequence mutagenesis and complementation in a bacterial selection system to establish a large library of immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) mutants with amino acid substitutions in the beta3-beta4 region of the fingers subdomain [Kim, B., Hathaway, T. R., and Loeb, L. A. (1996) J. Biol. Chem. 271, 4872-4878]. We show here that one of these mutants, D76V, exhibits increased accuracy of copying both DNA and RNA templates in a primer extension assay with biased dNTP pools. More detailed analysis of DNA-dependent polymerization showed that the D76V mutation conferred an up to 14-fold increase in fidelity of nucleotide insertion and a 9-fold reduced mutation rate in an M13mp2 lacZalpha forward mutation assay. Substitution at D76 with positively charged (D76R) and nonpolar (D76V and D76I) residues increased replicational accuracy, while substitutions with negatively charged (D76E) and polar residues (D76S and D76C) had little effect on fidelity. We propose that D76 affects replicational accuracy by mediating interaction between the fingers subdomain and the single-stranded template. Our work shows that the Escherichia coli complementation system can yield HIV RT mutants with increased...Continue Reading

References

May 25, 1977·Journal of Molecular Biology·F C BernsteinM Tasumi
Jan 1, 1994·Annual Review of Biochemistry·C M Joyce, T A Steitz
Nov 1, 1994·Acta Crystallographica. Section D, Biological Crystallography·E A Merritt, M E Murphy

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Citations

Jul 23, 1999·Annals of the New York Academy of Sciences·A Gabriel, E H Mules
Jun 5, 2002·Annual Review of Biochemistry·Eric T Kool
Jul 18, 2002·Proceedings of the National Academy of Sciences of the United States of America·Wen-Hui ZhangVinay K Pathak
Mar 21, 2012·Virus Research·Apostolos BeloukasDimitrios Paraskevis
Sep 25, 1999·The Journal of Biological Chemistry·M WisniewskiR A Bambara
Jan 21, 2006·The Journal of Biological Chemistry·Monica E HamburghVinayaka R Prasad
Nov 9, 2000·The Journal of Biological Chemistry·P H PatelL A Loeb

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