Increased G-->A transition frequencies displayed by primer grip mutants of human immunodeficiency virus type 1 reverse transcriptase.
A genetic screen based on the blue-white beta-galactosidase complementation assay designed to detect G-->A mutations arising during RNA-dependent DNA synthesis was used to compare the fidelity of mutant human immunodeficiency virus type 1 reverse transcriptases (RTs) with the mutations M230L and M230I with the wild-type enzyme, in the presence of biased deoxynucleoside triphosphate (dNTP) pools. The mutant RTs with the M230L and M230I changes were found to be 20 to 70 times less faithful than the wild-type RT in the presence of low [dCTP]/[dTTP] ratios but showed similar fidelity in assays carried out with equimolar concentrations of each nucleotide. Biased dNTP pools led to short tandem repeat deletions in the target sequence, which were also detectable with the assay. However, deletion frequencies were similar for all of the RTs tested. The reported data suggest that RT pausing due to the low dNTP levels available in the RT reaction mixture facilitates strand transfer, in a process that is not necessarily mediated by nucleotide misinsertion.
Cases-González
Clara E
CE
Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain.
Menéndez-Arias
Luis
L
D001483:Base Sequence; D017931:DNA Primers; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis; D003845:Deoxycytosine Nucleotides; D054303:HIV Reverse Transcriptase / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D013698:Templates, Genetic; D013942:Thymine Nucleotides; D014158:Transcription, Genetic
D017931:DNA Primers; D004279:DNA, Viral; D003845:Deoxycytosine Nucleotides; D013942:Thymine Nucleotides; C024107:2'-deoxycytidine 5'-triphosphate; D054303:HIV Reverse Transcriptase; C024157:thymidine 5'-triphosphate