A binary system of photoreagents for high-efficiency labeling of DNA polymerases

To increase the efficiency of photoaffinity labeling of DNA polymerases, a binary system of photoaffinity reagents was applied. Photoreactive radioact ive primers were synthesized by DNA polymerases beta (pol beta) or DNA poly merase from Thermus thermophilus (pol Tte) using a template-primer duplex i n the presence of a dTTP analogue containing 4-azidotetrafluorobenzoyl grou p linked via spacers of varying length to 5-position of uridine ring-5-[N-( 2,3,5,6-tetrafluoro-4-azidobenzoyl)-amino-trans-propenyl-1]-2 ' -deoxyuridi ne-5 ' -triphosphate (FAB-4-dUTP) or 5-[N-[[(2,3,5,6-tetrafluoro-4-azidoben zoyl)butanoyl]-amino]-trans-3-aminopropenyl-1]-2 ' -deoxyuridine-5 ' -triph osphate (FAB-9-dUTP). The reaction mixtures were UV irradiated (lambda = 36 5-450 nm) in the absence or presence of a dTTP analog, containing a pyrene moiety-5-[N-(4-(1-pyrenyl)-butylcarbonyl)-amino-trans-propenyl-1]-2 ' -deox yuridine-5 ' -triphosphate (Pyr-8-dUTP) or 5-[N(4-(1-pyrenyl)-ethylcarbonyl )-amino-trans-propenyl-1]-2 ' -deoxyuridine-5 ' -triphosphate (Pyr-6-dUTP). The most efficient crosslinking of both DNA polymerases was observed in th e case of photoreactive DNA primer, carrying the FAB-4-dUMP moiety at the 3 ' -end, and Pyr-6-dUTP as a sensitizer. The binary system of photoaffinity reagents allows increasing photoaffinity labeling of the both DNA polymera ses in comparison to the primer crosslinking without photosensitizer. (C) 2 001 Academic Press.