High fidelity of internal strand transfer catalyzed by human immunodeficiency virus reverse transcriptase.
A system to study the fidelity of internal strand transfer events was constructed. A donor RNA, on which reverse transcriptase (RT)-directed DNA synthesis was initiated, shared homology with an acceptor RNA, to which DNAs initiated on the donor could transfer. The homology occurred over a 119-base internal region of the donor which coded for the N-terminal portion of the alpha-lac gene. Polymerase chain reaction (PCR) was used to amplify DNA synthesis products. The PCR products were then digested with PvuII and EcoRI and ligated into a vector which had this same region excised. Transformed Escherichia coli were screened for the ability to produce a functional beta-galactosidase protein by blue-white phenotype analysis with white colonies scored as those with errors in alpha-lac. Products synthesized on the donor were used to assess the error rate of human immunodeficiency virus-RT while products transferring to and subsequently extended on the acceptor (transfer products) were used to monitor transfer fidelity. Human immunodeficiency virus-RT made approximately 1 error per 7500 bases copied in the assay. Nucleocapsid protein (NCp), although stimulating strand transfer 3-fold, had no effect on RT fidelity. Transfer products in the absence of NCp had essentially the same amount of errors as donor-directed products while those produced with NCp showed a slight increase in error frequency. Overall, strand transfer events on this template were highly accurate. Since experiments with other templates have suggested that transfer is error prone, the fidelity of strand transfer may be highly sequence dependent.
DeStefano
J
J
Department of Microbiology, University of Maryland, College Park 20742, USA. jd146@umail.umd.edu
Ghosh
J
J
Prasad
B
B
Raja
A
A
D001483:Base Sequence; D002384:Catalysis; D004252:DNA Mutational Analysis; D004261:DNA Replication; D054303:HIV Reverse Transcriptase / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D019590:Nucleocapsid Proteins / Q000378:metabolism; D010957:Plasmids; D011994:Recombinant Proteins / Q000378:metabolism; D014779:Virus Replication
D019590:Nucleocapsid Proteins; D011994:Recombinant Proteins; D054303:HIV Reverse Transcriptase