Hydrolysis of RNA/DNA hybrids containing nonpolar pyrimidine isosteres defines regions essential for HIV type 1 polypurine tract selection.
Both x-ray crystallography and chemical footprinting indicate that bases of the HIV type 1 (HIV-1) polypurine tract (PPT)-containing RNA/DNA hybrid deviate from standard Watson-Crick base pairing. However, the contribution of these structural anomalies to the accuracy of plus-strand primer selection by HIV-1 reverse transcriptase is not immediately clear. To address this issue, DNA templates harboring single and pairwise non-hydrogen-bonding isosteres of cytosine (2-fluoro-4-methylbenzene deoxyribonucleoside) and thymine (2,4-difluoro-5-methylbenzene deoxyribonucleoside) were synthesized and hybridized to PPT-containing RNA primers as a means of locally removing hydrogen bonding and destabilizing paired structure. Cleavage of these hybrids was examined with p66/p51 HIV-1 reverse transcriptase and a mutant carrying an alteration in the p66 RNase H primer shown to specifically impair PPT processing. Analog insertion within the PPT (rG):(dC) and central (rA):(dT) tracts repositioned the RNase H domain such that the RNA/DNA hybrid was cleaved 3-4 bp from the site of insertion, a distance corresponding closely to the spatial separation between the catalytic center and RNase H primer grip. However, PPT processing was significantly impaired when the junction between these tracts was substituted. Substitutions within the upstream (rA):(dT) tract, where maximum distortion had previously been observed, destroyed PPT processing. Collectively, our scanning mutagenesis approach implicates multiple regions of the PPT in the accuracy with which it is excised from (+) U3 RNA and DNA, and also provides evidence for close cooperation between the RNase H primer grip and catalytic center in achieving this cleavage.
Rausch
Jason W
JW
HIV Drug Resistance Program, National Cancer Institute, Frederick, MD 21702, USA.
Qu
Jin
J
Yi-Brunozzi
Hye Young
HY
Kool
Eric T
ET
Le Grice
Stuart F J
SF
D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D017931:DNA Primers; D054303:HIV Reverse Transcriptase / Q000378:metabolism; D015497:HIV-1 / Q000201:enzymology / Q000378:metabolism*; D006868:Hydrolysis; D009693:Nucleic Acid Hybridization; D011687:Purines / Q000378:metabolism*; D011743:Pyrimidines / Q000378:metabolism*; D012313:RNA / Q000378:metabolism*
D017931:DNA Primers; D011687:Purines; D011743:Pyrimidines; D012313:RNA; D004247:DNA; D054303:HIV Reverse Transcriptase