High-resolution NMR analysis of the conformations of native and base analog substituted retroviral and LTR-retrotransposon PPT primers.
A purine-rich region of the plus-strand RNA genome of retroviruses and long terminal repeat (LTR)-containing retrotransposons, known as the polypurine tract (PPT), is resistant to hydrolysis by the RNase H domain of reverse transcriptase (RT) and ultimately serves as a primer for plus-strand DNA synthesis. The mechanisms underlying PPT resistance and selective processing remain largely unknown. Here, two RNA/DNA hybrids derived from the PPTs of HIV-1 and Ty3 were probed using high-resolution NMR for preexisting structural distortions in the absence of RT. The PPTs were selectively modified through base-pair changes or by incorporation of the thymine isostere, 2,4-difluoro-5-methylbenzene (dF), into the DNA strand. Although both wild-type (WT) and mutated hybrids adopted global A-form-like helical geometries, observed structural perturbations in the base-pair and dF-modified hybrids suggested that the PPT hybrids may function as structurally coupled domains.
Yi-Brunozzi
Hye Young
HY
HIV Drug Resistance Program, NCI-Frederick National Institutes of Health, Frederick, MD 21702, USA.
Brinson
Robert G
RG
Brabazon
Danielle M
DM
Lener
Daniela
D
Le Grice
Stuart F J
SF
Marino
John P
JP
D001482:Base Composition; D020029:Base Pairing; D001483:Base Sequence; D001554:Benzene / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D011685:Purine Nucleotides / Q000235:genetics*; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry; D018626:Retroelements / Q000235:genetics*; D020079:Terminal Repeat Sequences / Q000235:genetics*; D044366:Transition Temperature
D017931:DNA Primers; D011685:Purine Nucleotides; D018626:Retroelements; D012194:RNA-Directed DNA Polymerase; D001554:Benzene