Human immunodeficiency virus reverse transcriptase base misincorporations can promote strand transfer.
A system to determine if HIV-reverse transcriptase (RT) base misincorporations can promote strand transfer was constructed. A donor RNA, on which RT-directed DNA synthesis was initiated, shared homology over a 119 base internal region with an acceptor RNA, to which DNAs initiated on the donor could transfer. Products completed on the donor in the presence or absence of acceptor were isolated and PCR was used to amplify these DNAs. PCR products were ligated into a vector which had this same region (near the N-terminus of the alpha-lac gene) removed. Transformed E. coli were screened in an alpha-complementation assay by blue-white phenotype analysis with white colonies scored as those with errors in plasmid-derived alpha-lac. The frequence of white colonies +/- standard deviations was 0.031 +/- 0.006 and 0.0037 +/- 0.009, for plasmids with inserts derived from donor-directed products synthesized with 100 microM dNTPs in the presence and absence of acceptor template, respectively. Statistical analysis indicated a lower white colony frequency in the presence of acceptor (p = 0.0025). The lower frequency with acceptor implies that a portion of the errors made on the donor are transferred to the acceptor suggesting that base misincorporations can induce strand transfer.
Diaz
L
L
Department of Cell Biology and Molecular Genetics, University of Maryland College Park, 20742, USA.
Cristofaro
J V
JV
DeStefano
J J
JJ
D020137:Base Pair Mismatch; D001483:Base Sequence; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D054303:HIV Reverse Transcriptase / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009693:Nucleic Acid Hybridization; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA; D013698:Templates, Genetic
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D012367:RNA, Viral; D054303:HIV Reverse Transcriptase