Human immunodeficiency virus type 1 reverse transcriptase Affinity labeling of the primer binding site

AbstractAffinity modification of the primer site of HIV1-RT was performed with an oligonucleotide derivative containing a photoreactive azido group at the 5′ end of d(pT)10. The affinity of HIV1-RT for d(pT)10 and for its derivative was first estimated by measuring the Michaelis constants of these two oligonucleotides acting as primers in the retrotranscription of poly(rA). The enzyme was then inactivated under UV-irradiation at 303–365 nm in the presence of ArN3-d(U*T9); the dependence of the rate of inactivation on primer concentration was found to be consistent with the Km value. Last, selectivity of affinity modification was demonstrated through elongation of the covalently bound primer and selective protection of inactivation by d(pT)10 or tRNALy5

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

AbstractNew 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