STRAND TRANSFER MEDIATED BY HUMAN-IMMUNODEFICIENCY-VIRUS REVERSE-TRANSCRIPTASE IN-VITRO IS PROMOTED BY PAUSING AND RESULTS IN MISINCORPORATION

Human immunodeficiency virus (HIV-I) is able to recombine by transfer of the growing DNA strand from internal regions of one genome to anoth er. The strand transfer reaction, catalyzed by HIV-1 reverse transcrip tase (RT), was conducted in vitro between donor and acceptor RNA templ ates that were derived from natural HIV-1 nef genes. The donor and acc eptor templates shared a nearly homologous region where strand transfe r could occur, differing only in that the acceptor had a 36-nucleotide insertion and 6 widely spaced base substitutions compared with the do nor. We sequenced elongated primers that underwent transfer, The posit ion of transfer was revealed by the change of sequence from that of th e donor to that of the acceptor. Results showed a positive correlation between positions where the RT paused during synthesis and enhancemen t of strand transfer. Elimination of a pause site, with a minimal chan ge in sequence, decreased the frequency of strand transfer in the imme diate area. Analysis of the sequence of DNA products resulting from tr ansfer at a frequently used site showed that mutations had been introd uced into the DNA at about the point of transfer. Remarkably, approxim ately 30% of the products contained mutations. Base substitutions, sho rt additions and deletions were observed. Mutations did not appear in DNA products extended on the donor template without transfer. The iden tity of the mutations suggests that they were caused by a combination of slippage and non-template-directed nucleotide addition. These resul ts indicated that the detected mutations were related to the process o f strand transfer.