Examining Ty3 polypurine tract structure and function by nucleoside analog interference.
We have combined nucleoside analog interference with chemical footprinting, thermal denaturation, NMR spectroscopy, and biochemical studies to understand recognition of the polypurine tract (PPT) primer of the Saccharomyces cerevisiae long terminal repeat-containing retrotransposon Ty3 by its cognate reverse transcriptase. Locked nucleic acid analogs, which constrain sugar ring geometry, were introduced pairwise throughout the PPT (-)-DNA template, whereas abasic tetrahydrofuran linkages, which lack the nucleobase but preserve the sugar phosphate backbone, were introduced throughout the (-)-strand DNA template and (+)-strand RNA primer. Collectively, our data suggest that both the 5'- and 3'-portions of the PPT-containing RNA/DNA hybrid are sensitive to nucleoside analog substitution, whereas the intervening region can be modified without altering cleavage specificity. These two regions most likely correspond to portions of the PPT that make close contact with the Ty3 reverse transcriptase thumb subdomain and RNase H catalytic center, respectively. Achieving a similar phenotype with nucleoside analogs that have different effects on duplex geometry reveals structural features that are important mediators of Ty3 PPT recognition. Finally, the results from introducing tetrahydrofuran lesions around the scissile PPT/unique 3'-sequence junction indicate that template nucleobase -1 is dispensable for catalysis, whereas a primer nucleobase on either side of the junction is necessary.
Dash
Chandravanu
C
Resistance Mechanisms Laboratory, HIV Drug Resistance Program, NCI-Frederick, National Institutes of Health, Frederick, Maryland 21702, USA.
Marino
John P
JP
Le Grice
Stuart F J
SF
D001483:Base Sequence; D002384:Catalysis; D005663:Furans; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D009705:Nucleosides / Q000737:chemistry; D034741:RNA, Small Interfering / Q000494:pharmacology; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000502:physiology*; D016914:Ribonuclease H; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000502:physiology*
D005663:Furans; D009705:Nucleosides; D034741:RNA, Small Interfering; D029701:Saccharomyces cerevisiae Proteins; C018674:tetrahydrofuran; D012194:RNA-Directed DNA Polymerase; C496084:reverse transcriptase Ty3, S cerevisiae; D016914:Ribonuclease H