Abstract
Human immunodeficiency virus (HIV-1) is able to recombine by transfer of the growing DNA strand from internal regions of one genome to another. The strand transfer reaction, catalyzed by HIV-1 reverse transcriptase (RT), was conducted in vitro between donor and acceptor RNA templates that were derived from natural HIV-1 nef genes. The donor and acceptor templates shared a nearly homologous region where strand transfer could occur, differing only in that the acceptor had a 36-nucleotide insertion and 6 widely spaced base substitutions compared with the donor. We sequenced elongated primers that underwent transfer. The position of transfer was revealed by the change of sequence from that of the donor to that of the acceptor. Results showed a positive correlation between positions where the RT paused during synthesis and enhancement of strand transfer. Elimination of a pause site, with a minimal change in sequence, decreased the frequency of strand transfer in the immediate area. Analysis of the sequence of DNA products resulting from transfer at a frequently used site showed that mutations had been introduced into the DNA at about the point of transfer. Remarkably, approximately 30% of the products contained mutations. Base substitutions, short additions and deletions were observed. Mutations did not appear in DNA products extended on the donor template without transfer. The identity of the mutations suggests that they were caused by a combination of slippage and non-template-directed nucleotide addition. These results indicated that the detected mutations were related to the process of strand transfer.
Highlights
Analysis of the sequence of DNA products resulting from transfer at a frequently used site showed that mutations had been introduced into the DNA at about the point of transfer
Construction of Templates Used to Measure the Positions of Strand Transfer-NefRNA donor and acceptor templates were used in our strand transfer system in vitro, to simulate internal homologous recombination that could occur during minus strand DNA synthesis in vivo
Since the homologous region in acceptor A contained a 36 nucleotide insertion absent in donor A and 6 base substitutions that were different from donor A (Fig. lB), these sequence differences served as allelic markers allowing us to determine where specific strand transfers occurred
Summary
Analysis of the sequence of DNA products resulting from transfer at a frequently used site showed that mutations had been introduced into the DNA at about the point of transfer. The nascent DNA strand can transfer to a similar or identical sequence on another template to continue primer extension When reverse transcriptase advances along the RNA template, it encounters some sequences or structures at which continued synthesis is difficult To investigate the role of pausing in the process of strand transfer, we measured recombination catalyzed by HIV-RT in vitro between two natural nef gene RNA templates. In this system, a primer initiated on one template, designated the donor, can transfer to the homologous region of the other template, designated the acceptor. At the other recombination junction, we found that over 30% of the recombinant molecules examined had incorrectly
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