DNTP BINDING TO HIV-1 REVERSE-TRANSCRIPTASE AND MAMMALIAN DNA-POLYMERASE-BETA AS REVEALED BY AFFINITY LABELING WITH A PHOTOREACTIVE DNTP ANALOG

The dNTP binding pocket of human immunodeficiency virus type 1 reverse transcriptase (RT) and DNA polymerase beta (beta-pol) were labeled us ing a photoreactive analog of dCTP, uorobenzamido)ethyl]-deoxycytidine -5'-triphosphate (FABdCTP), Two approaches of photolabeling were utili zed, In one approach, photoreactive FABdCTP and radiolabeled primer-te mplate were UV-irradiated in the presence of each enzyme and resulted in polymerase radiolabeling. In an alternate approach, FABdCTP was fir st UV-cross linked to enzyme; subsequently, radiolabeled primer-templa te was added, and the enzyme-linked dCTP analog was incorporated onto the 3'-end of the radiolabeled primer. The results showed strong label ing of the p66 subunit of RT, with only minor labeling of p51. No diff erence in the intensity of cross linking was observed with either appr oach, FABdCTP crosslinking was increased in the presence of a dideoxyt erminated primer-template with RT, but not with beta-pol, suggesting a significant influence of prior primer-template binding on dNTP bindin g for RT. Mutagenesis of beta-pol residues observed to interact with t he incoming dNTP in the crystal structure of the ternary complex resul ted in labeling consistent with kinetic characterization of these muta nts and indicated specific labeling of the dNTP binding pocket.