Studies on the effects of truncating alpha-helix E' of p66 human immunodeficiency virus type 1 reverse transcriptase on template-primer binding and fidelity of DNA synthesis.
The role of alpha-helix E' of the RNase H domain of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) in template-primer binding and fidelity of DNA synthesis was investigated by using a series of mutant enzymes with deletions of 4, 8, 12, 16, and 20 amino acids at the C-terminal end of the 66 kDa subunit. The dissociation equilibrium constants (Kd) of wild-type HIV-1 RT and 38/16mer and 47/25mer DNA/DNA template-primer complexes were 2.2 +/- 0.7 and 0.69 +/- 0.35 nM, respectively. Deletions involving partial or total removal of alpha-helix E' rendered enzymes with a 2-5-fold decrease in binding affinity. Misinsertion and mispair extension fidelity of DNA synthesis of the wild-type enzyme and truncated mutants were determined by using both DNA/DNA template-primers and a 47/25mer RNA/DNA complex. In all cases, incorporation assays were done in the same sequence context, which was taken from the viral gag gene. The removal of alpha-helix E' had little effect on fidelity as determined with the three template-primers. Misinsertion fidelity assays showed that the specificity of mismatch formation was A:C approximately A:G > A:A for the DNA template and A:C > A:G approximately A:A for the RNA template, in 47/25mers. The specificity of extending mispaired 3'-termini was similar with both 47/25mers: A:C > A:A approximately A:G. However, the efficiency of transversion mispair extension was higher with RNA templates. The results reported in this paper suggest that alpha-helix E' may stabilize the RT/template-primer interaction, but does not have a strong influence in the correct positioning of the template-primer at the polymerase active site.
Menéndez-Arias
L
L
Centro de Biología Molecular "Severo Ochoa", CSIC-Universidad Autónoma de Madrid, Spain. lmenedez@cbm.uam.es
D020137:Base Pair Mismatch / Q000235:genetics; D018360:Crystallography, X-Ray; D017931:DNA Primers / Q000235:genetics / Q000378:metabolism*; D004279:DNA, Viral / Q000096:biosynthesis* / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D054303:HIV Reverse Transcriptase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D012367:RNA, Viral / Q000378:metabolism; D013698:Templates, Genetic
D017931:DNA Primers; D004279:DNA, Viral; D003854:Deoxyribonucleotides; D010446:Peptide Fragments; D012367:RNA, Viral; D054303:HIV Reverse Transcriptase; D004259:DNA-Directed DNA Polymerase