Fidelity of human immunodeficiency virus type I reverse transcriptase in copying natural DNA.
Reverse transcriptase from the human immunodeficiency virus type I (HIV-1) was expressed in E. coli and purified to near homogeneity. The enzyme was shown to contain reverse transcriptase, DNA polymerase and ribonuclease H activities. The DNA polymerase activity converted singly-primed phi X174 (+) DNA into the double-stranded form. Two third of the replication product is ligatable to covalently closed circular DNA (RFIV-form DNA) indicating that DNA synthesis by HIV reverse transcriptase can proceed until the enzyme matches the 5'-end of a pre-existing primer molecule. The in vitro accuracy of HIV reverse transcriptase was measured with the phi X174am16 reversion assay to be 1/7,400. Reversion rates for the individual mispairs were determined from pool bias studies to be 1/8,000 for the dGMP:T template mismatch, 1/35,000 for the dGMP:A template mismatch, 1/45,000 for the dAMP:G template mismatch, 1/73,000 for the dCMP:T template mispair, 1/140,000 for the dCMP:A template mispair, and 1/180,000 for the dGMP:G template mismatch. The dTMP:T template mispair was below the detection limit of the assay indicating a reversion rate of less than 1/300,000 for this particular mispair.
Weber
J
J
Department of Chemistry, Max-Planck-Institute for Experimental Medicine, Göttingen, FRG.
Grosse
F
F
D003001:Cloning, Molecular; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004722:Endoribonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D016914:Ribonuclease H
D004279:DNA, Viral; D011994:Recombinant Proteins; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase; D004722:Endoribonucleases; D016914:Ribonuclease H