Single unpaired nucleotides facilitate HIV-1 reverse transcriptase displacement synthesis through duplex RNA.
During reverse transcription of viral RNA, HIV-1 reverse transcriptase (RT) encounters RNA stem-loop structures that require displacement synthesis activity in which RT disrupts the RNA helix to access the template strand. A primer extension assay was developed to assess HIV-1 RT RNA displacement synthesis activity in vitro. Initial results revealed that HIV-1 RT performs only limited amounts of RNA displacement through long stretches of RNA duplex, with the majority of synthesis stalling at sequence-dependent pause positions. DNA displacement synthesis through the same sequence, however, proceeded rapidly to the end of the template. The RNA folding algorithm mfold indicated that the presence of an unpaired nucleotide, or "bulge," along the RNA duplex would promote helix melting ahead of the DNA primer terminus to create a small gap of nondisplacement synthesis. Primer extension assays using substrates possessing single-nucleotide bulges in the nontemplate strand near pause sites resulted in diminished pausing at positions within the predicted melted region. Surprisingly, the bulges also reduced pausing distal to the bulge at positions that are expected to remain base-paired. Further analysis revealed that stalling during RNA displacement synthesis results from the displaced RNA re-annealing to the template strand thus forcing the primer terminus to become unpaired and, therefore, not extendable. Introduction of a bulge facilitates displacement synthesis through distal regions by increasing RT processivity in the vicinity of a bulge and reducing the impact of branch migration on primer extension.
Lanciault
Christian
C
Department of Microbiology, University of Washington School of Medicine, Seattle, 98195, USA.
Champoux
James J
JJ
D001483:Base Sequence; D004247:DNA / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D004279:DNA, Viral; D004591:Electrophoresis, Polyacrylamide Gel; D016325:HIV Long Terminal Repeat; D054303:HIV Reverse Transcriptase / Q000737:chemistry*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D012313:RNA / Q000737:chemistry; D012330:RNA, Double-Stranded / Q000737:chemistry*; D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase; D011995:Recombination, Genetic; D012190:Retroviridae / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D013698:Templates, Genetic; D013997:Time Factors
D017931:DNA Primers; D004279:DNA, Viral; D009711:Nucleotides; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D012313:RNA; D004247:DNA; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D015719:Single-Strand Specific DNA and RNA Endonucleases