Nucleic Acids Research, 2001, Vol. 29, No. 24 4963-4972
© 2001 Oxford University Press
A mutation in the primer grip region of HIV-1 reverse transcriptase that confers reduced fidelity of DNA synthesis
Centro de Biología Molecular ‘Severo Ochoa’, Consejo Superior de Investigaciones Científicas–Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain
A compensatory mutation (M230I) in the primer grip of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) restores the replication capacity of virus having a Y115W mutation in their RT coding region. The Y115W substitution impairs DNA polymerase activity and produces an enzyme with a lower fidelity of DNA synthesis. Gel-based fidelity assays with the double mutant Y115W/M230I revealed that the M230I substitution increased the accuracy of mutant Y115W. Y115W/M230I showed wild-type misinsertion fidelity in assays performed with DNA/DNA templates. However, when present alone, M230I conferred reduced fidelity as determined in misinsertion and mispair extension fidelity assays, as well as in primer extension assays carried out with three dNTPs. The mutant M230I showed a 3.3–16-fold increase in misinsertion efficiency for G, C and T opposite T, compared with the wild-type enzyme. Its fidelity was not influenced by nucleotide substitutions in the template/primer around the incorporation site. However, its accuracy was apparently affected by the structure of the 5'-overhang of the template strand. Unlike wild-type HIV-1 RT, nucleotide selectivity of mutant M230I at dT:dG, dT:dC and dT:dT mispairs was almost exclusively dependent on the Km values for correct and incorrect dNTPs, a characteristic that has not been described for other low fidelity mutants of HIV-1 RT.
* To whom correspondence should be addressed. Tel: +34 9139 78477; Fax: +34 9139 74799; Email: lmenendez{at}cbm.uam.esPresent address:Mónica Gutiérrez-Rivas, Centro Nacional de Biología Fundamental, Instituto de Salud Carlos III, Majadahonda (Madrid), Spain
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  A. Sobeck, S. Stone, and M. E. Hoatlin DNA Structure-Induced Recruitment and Activation of the Fanconi Anemia Pathway Protein FANCD2 Mol. Cell. Biol., June 15, 2007; 27(12): 4283 - 4292. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Furio, A. Moya, and R. Sanjuan The cost of replication fidelity in human immunodeficiency virus type 1 Proc R Soc B, January 22, 2007; 274(1607): 225 - 230. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Dash, T. S. Fisher, V. R. Prasad, and S. F. J. Le Grice Examining Interactions of HIV-1 Reverse Transcriptase with Single-stranded Template Nucleotides by Nucleoside Analog Interference J. Biol. Chem., September 22, 2006; 281(38): 27873 - 27881. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. F. Elena and R. Sanjuan Adaptive Value of High Mutation Rates of RNA Viruses: Separating Causes from Consequences J. Virol., September 15, 2005; 79(18): 11555 - 11558. [Full Text] [PDF]
|
|
|
|
|
|
C. E. Cases-Gonzalez and L. Menendez-Arias Increased G->A Transition Frequencies Displayed by Primer Grip Mutants of Human Immunodeficiency Virus Type 1 Reverse Transcriptase J. Virol., January 15, 2004; 78(2): 1012 - 1019. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Xu, Y. Liu, S. Weiss, E. Arnold, S. G. Sarafianos, and J. Ding Molecular model of SARS coronavirus polymerase: implications for biochemical functions and drug design Nucleic Acids Res., December 15, 2003; 31(24): 7117 - 7130. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. M. Mansky, E. Le Rouzic, S. Benichou, and L. C. Gajary Influence of Reverse Transcriptase Variants, Drugs, and Vpr on Human Immunodeficiency Virus Type 1 Mutant Frequencies J. Virol., February 1, 2003; 77(3): 2071 - 2080. [Abstract] [Full Text] [PDF]
|
|