Using pyrrolo-deoxycytosine to probe RNA/DNA hybrids containing the human immunodeficiency virus type-1 3' polypurine tract.
Recent structural analyses indicate that localized regions of abnormal base pairing exist within RNA/DNA hybrids containing the HIV-1 polypurine tract (PPT) and that these distortions may play a role in PPT function. To examine this directly, we have introduced pyrrolo-deoxycytosine (pdC), a fluorescent, environmentally sensitive analog of deoxycytosine (dC), into the DNA strand of PPT-containing hybrids. Steady-state fluorescence analysis of these hybrids reveals that the DNA base 11 nt from the PPT-U3 junction is unpaired even in the absence of reverse transcriptase (RT). Unstable base pairing is also observed within the (rG:dC)6 tract in the downstream portion of the duplex, suggesting that HIV-1 RT may recognize multiple pre-existing distortions during PPT selection. HIV-1 RT hydrolyzes pdC-containing hybrids primarily at the PPT-U3 junction, indicating that the analog does not induce a gross structural deformation of the duplex. However, aberrant cleavage is frequently observed 3 bp from the site of pdC substitution, most likely reflecting a specific interaction between the analog and amino acid residues within the RNase H primer grip. pdC substitution within the template strand of a DNA duplex does not appear to significantly affect RT-catalyzed DNA synthesis. Implications of these findings on the use of pdC to examine nucleic acid structure are discussed.
Dash
Chandravanu
C
Resistance Mechanisms Laboratory, HIV Drug Resistance Program, National Cancer Institute-Frederick, Frederick, MD 21702, USA.
Rausch
Jason W
JW
Le Grice
Stuart F J
SF
D000225:Adenine / Q000737:chemistry; D020029:Base Pairing; D001483:Base Sequence; D003596:Cytosine / Q000031:analogs & derivatives / Q000737:chemistry*; D004279:DNA, Viral / Q000096:biosynthesis / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry; D006147:Guanine / Q000737:chemistry; D054303:HIV Reverse Transcriptase / Q000378:metabolism; D015497:HIV-1 / Q000201:enzymology / Q000235:genetics*; D006860:Hydrogen Bonding; D009154:Mutation; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry / Q000378:metabolism; D015341:Nucleic Acid Probes / Q000737:chemistry*; D011687:Purines / Q000737:chemistry*; D012367:RNA, Viral / Q000737:chemistry*; D016914:Ribonuclease H / Q000235:genetics / Q000378:metabolism; D013698:Templates, Genetic
D004279:DNA, Viral; D005456:Fluorescent Dyes; D009692:Nucleic Acid Heteroduplexes; D015341:Nucleic Acid Probes; D011687:Purines; D012367:RNA, Viral; C486438:pyrrolo-deoxycytosine; D006147:Guanine; D003596:Cytosine; D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H; D000225:Adenine