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 occurr ed 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 th en digested with PvuII and EcoRI and ligated into a vector which had t his same region excised. Transformed Escherichia coli were screened fo r the ability to produce a functional beta-galactosidase protein by bl ue-white phenotype analysis with white colonies scored as those with e rrors in alpha-lac, Products synthesized on the donor were used to ass ess the error rate of human immunodeficiency virus-RT while products t ransferring to and subsequently extended on the acceptor (transfer pro ducts) 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-f old, had no effect on RT fidelity. Transfer products in the absence of NCp had essentially the same amount of errors as donor-directed produ cts while those produced with NCp showed a slight increase in error fr equency. Overall, strand transfer events on this template were highly accurate, Since experiments with other templates have suggested that t ransfer is error prone, the fidelity of strand transfer may be highly sequence dependent.