Fidelity of human immunodeficiency virus type I reverse transcriptase in copying natural DNA

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Abstract
Reverse transcriptase from the human immunodeflciency 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 singlyprimed ΦX174 (+) DNA info 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 ΦX174am16 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:Ttemplate mismatch, 1/35,000 for the dGMP:Atemplate mismatch, 1/45,000 for the dAMP:Gtemplate mismatch, 1/73,000 for the dCMP:Ttemp mispair, 1/140,000 for the dCMP:Atemplote mispair, and 1/180,000 for the dGMP:Gtemplate mismatch. The dTMP:Ttemplate mispair was below the detection limit of the assay indicating a reversion rate of less than 1/300,000 for this particular mispair.