Affinity modification of human immunodeficiency virus reverse transcriptase and DNA template by photoreactive dCTP analogs

Abstract

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.