Affinity modification of human immunodeficiency virus reverse transcriptase and DNA template by photoreactive dCTP analogs.
New base-substituted analogs of dCTP containing an azido group have been synthesized and applied to a selective photoaffinity modification of HIV-RT (p66/p51 heterodimer). The labeling of only the 66 kDa subunit of HIV-RT was detected when the enzyme was first irradiated with the analogs and then template (5'-(d)GGTTAAATAAAATAGTAAGAATGTATAGCCCCTACCA-3') and 5' 32P end-labeled 3'-(d)TTACATATCGGGGATGGT-5' primer were added. The 5' 32P end-labeled primer elongated by dCTP analogs in the presence of both HIV-RT and DNA template is able to modify both subunits of HIV-RT and DNA template. This way of specific cross-linking to both DNA (RNA) template and HIV-RT opens up new possibilities to study the HIV-RT active site.
Doronin
S V
SV
Institute of Bioorganic Chemistry, Siberian Division of the Russian Academy of Sciences, Novosibirsk.
Dobrikov
M I
MI
Buckle
M
M
Roux
P
P
Buc
H
H
Lavrik
O I
OI
D000345:Affinity Labels; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D003845:Deoxycytosine Nucleotides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D006678:HIV / Q000201:enzymology*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010777:Photochemistry; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism; D013698:Templates, Genetic; D014466:Ultraviolet Rays
C090362:5-(3-(4-azidotetrafluorobenzamido)allyl)-2'-deoxycytidine-5'-triphosphate; D000345:Affinity Labels; D003845:Deoxycytosine Nucleotides; C078023:exo-N-(2-(4-azidotetrafluorobenzamido)ethyl)-deoxycytidine-5'-triphosphate; C024107:2'-deoxycytidine 5'-triphosphate; D004247:DNA; D012194:RNA-Directed DNA Polymerase