- 作者: Belinda B. Oude Essink Ben Berkhout
- 作者服務機構: Department of Human Retrovirology, Academic Medical Center, University of Amsterdam, The Netherlands
- 中文摘要: --
- 英文摘要: Reverse transcriptase enzymes(RT) convert single-stranded retroviral RNA genomes into double-strandedDNA. The RT enzyme can use both RNA and DNA prim-ers, the former being used exclusively during initiation ofminus- and plus-strand synthesis. Initiation of minus-strand DNA synthesis occurs by extension of a tRNAprimer that is associated with the viral genome, andplus-strand DNA synthesis is initiated from an RNase H-resistant polypurine tract of the genomic RNA that re-mains bound to the newly synthesized minus-strandDNA. All other phases of reverse transcription representelongation of a DNA primer. We demonstrate that thepolymerase fidelity of RT enzymes is significantly higherin tRNA-primed reverse transcription compared withDNA-primed reactions. Two mechanistic explanationscan be proposed. First, the type of template-primer (T- P)duplex(RNA-RNA versus RNA-DNA) may affect the RTenzyme conformation such that the discriminationagainst incorrect nucleotides is affected. Second, thetRNA primer may act as a fidelity co-factor through spe-cific association with the RT enzyme. According to thelatter hypothesis, the increased fidelity observed for anRNA-RNA T-P should persist at a distance from the initia-tion site, where the enzyme-bound nucleic acid duplexwill consist of RNA-cDNA. However, we measured thatthe effect of tRNA on the fidelity is detectable only at ashort distance from the initiation site. These results indi-cate that the type of T-P duplex influences the fidelity ofreverse transcription, suggesting that two small seg-ments of the viral genome downstream of the initiationsites for minus- and plus-strand DNA synthesis are cop-ied with a fidelity that is greater than average.
- 中文關鍵字: --
- 英文關鍵字: Reverse transcriptase. Nucleotide misincorporation. Reverse transcription fidelity. RNA-DNA and DNA-DNA duplexes. Retroviral replication