Mispair extension fidelity of human immunodeficiency virus type 1 reverse transcriptases with amino acid substitutions affecting Tyr115.
The role of Tyr115 of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) in the mispair extension fidelity of DNA dependent DNA synthesis was analysed by using a series of 15 mutant enzymes with substitutions at Tyr115. Their kinetic parameters for elongation using homopolymeric RNA-DNA and heteropolymeric DNA-DNA complexes showed major effects of the amino acid substitutions on the Km value for dNTP. Enzymes with large hydrophobic residues at position 115 displayed lower Km values than enzymes with small and charged amino acids at this position. The influence of all these amino acid replacements in mispair extension fidelity assays was analyzed using three different mismatches (A:C, A:G and A:A) at the 3'-terminal position of the primer DNA. For the A:C mispair, a 2. 6-33.4-fold increase in mispair extension efficiency (fext) was observed as compared with the wild-type enzyme. Unexpectedly, all the mutants tested as well as the wild-type RT were very efficient in extending the A:G and A:A transversion mispairs. This effect was due to the template-primer sequence context and not to the buffer conditions of the assay. The data support a role of Tyr115 in accommodating the complementary nucleotide into the nascent DNA while polymerization takes place.
Martín-Hernández
A M
AM
Centro de Biología Molecular 'Severo Ochoa', Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, 28049 Cantoblanco, Madrid, Spain.
Gutiérrez-Rivas
M
M
Domingo
E
E
Menéndez-Arias
L
L
D004260:DNA Repair; D004261:DNA Replication; D054303:HIV Reverse Transcriptase / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009692:Nucleic Acid Heteroduplexes / Q000378:metabolism*; D016914:Ribonuclease H / Q000378:metabolism; D013329:Structure-Activity Relationship; D013942:Thymine Nucleotides / Q000378:metabolism; D014443:Tyrosine / Q000235:genetics*
D009692:Nucleic Acid Heteroduplexes; D013942:Thymine Nucleotides; D014443:Tyrosine; D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H; C024157:thymidine 5'-triphosphate