Preparation of photoreactive oligonucleotide duplexes and their application to photoaffinity modification of DNA-binding proteins

To introduce photoreactive dNMP residues to the 3'-end of a mononucleotide gap, base-substituted photoreactive deoxynucleoside triphosphate derivative s, (5-[N-(2,3,5,6-tetrafluoro-4-azidobenzoyl)-trans-3-aminopropenyl-1]- and 5-{N-[N-(4-azido-2,5-difluoro-3-chloropyridine-6-yl)-3-aminopropionyl]-tra ns-3-aminopropenyl-1}-2'-deoxyuridine 5'-triphosphates, were used as substr ates in the DNA polymerase beta -catalyzed reaction. The resulting nick, co ntaining a modified base at the 3'-end, was sealed by T4 phage DNA ligase. This approach enables the preparation of DNA duplexes bearing photoreactive groups at a predetermined position of the nucleotide chain. Using the gene rated photoreactive DNA duplexes, the photoaffinity modifications of DNA po lymerase beta and human replication protein A (hRPA) were carried out. It w as shown that DNA polymerase beta and hRPA subunits were modified with the photoreactive double-stranded DNA considerably less effectively than by the nicked DNA. In the case of double-stranded DNA, the hRPA p70 subunit was p referentially labeled, implying a crucial role of this subunit in the prote in-DNA interaction.