Chemical cross-linking agents having a photoactivable azido group are promising for the study of the spatial organization of biopolymers. We describe here a variety of (d)NTPs derivatives (6a, 6b, 7, 11, 12, 14, and 16) bearing the residues of three different photoreagents containing an aromatic azido group (1a, 2a, and 3a). These conjugates provide a wide choice of instruments to investigate nucleic acid-nucleic acid and nucleic acid-protein interaction. The synthesis of new photoreagent 2a has been also fulfilled. This compound is the most attractive for affinity modification of the nucleic acids.

Two simple methods for the synthesis of oligonucleotides bearing a N-(2-hydroxyethyl)phenazinium (Phn) residue at the 5'- and/or 3'-terminal phosphate groups are proposed. By forming complexes between a dodecanucleotide d(pApApCpCpTpGpTpTpTpGpGpC), a heptanucleotide d(pCpCpApApApCpA), and Phn derivatives of the latter, it is shown that the introduction of a dye at the end of an oligonucleotide chain strongly stabilizes its complementary complexes. The Tmax and the thermodynamic parameters (delta H, delta S, delta G) of complex formation were determined. According to these data, coupling of a dye with the 5'-terminal phosphate group is the most advantageous: delta G(37 degrees C) is increased by 3.59 +/- 0.04 kcal/mol compared to 2.06 +/- 0.04 kcal/mol for 3'-Phn derivatives. The elongation of the linker, which connects the dye to the oligonucleotide, from a dimethylene up to a heptamethylene usually leads to destabilization of the oligonucleotide complex. The complementary complex formed by the 3',5'-di-Phn derivative of the heptanucleotide was found to be the most stable among all duplexes investigated. Relative to the unmodified complex the increase in free energy was 4.96 +/- 0.04 kcal/mol. The association constant of this modified complex at 37 degrees C is 9.5 x 10(6) M-1, whereas the analogous value for the unmodified complex is only 3 x 10(3) M-1.

The dNTP binding pocket of human immunodeficiency virus type 1 reverse transcriptase (RT) and DNA polymerase ␤ (␤-pol) were labeled using a photoreactive analog of dCTP, exo-N-[␤-(p-azidotetrafluorobenzamido)-ethyl]-deoxycytidine-5-triphosphate (FABdCTP). Two approaches of photolabeling were utilized. In one approach, photoreactive FABdCTP and radiolabeled primer-template were UV-irradiated in the presence of each enzyme and resulted in polymerase radiolabeling. In an alternate approach, FABdCTP was first UV-cross-linked to enzyme; subsequently, radiolabeled primer-template was added, and the enzyme-linked dCTP analog was incorporated onto the 3-end of the radiolabeled primer. The results showed strong labeling of the p66 subunit of RT, with only minor labeling of p51. No difference in the intensity of cross-linking was observed with either approach. FABdCTP crosslinking was increased in the presence of a dideoxyterminated primer-template with RT, but not with ␤-pol, suggesting a significant influence of prior primer-template binding on dNTP binding for RT. Mutagenesis of ␤-pol residues observed to interact with the incoming dNTP in the crystal structure of the ternary complex resulted in labeling consistent with kinetic characterization of these mutants and indicated specific labeling of the dNTP binding pocket.DNA polymerases must recognize and incorporate the correct dNTP with high fidelity to maintain genetic stability. The interaction of the polymerase with the DNA primer-template is essentially nonspecific, since the enzyme must bind to an "infinite" number of sequences during replication, while dNTPtemplate base recognition must be highly specific for DNA replication of high fidelity. Since the role of the polymerase in dNTP recognition and selection is poorly understood, elucidation of polymerase-dNTP interactions under a variety of conditions will aid our understanding of this fundamental process.During the last several years our knowledge of the structure of DNA polymerases and their interactions with substrates has evolved significantly. The three-dimensional structures of several DNA polymerases have been solved by x-ray crystallography including the Klenow fragment of DNA polymerase I (1), HIV-1 1 reverse transcriptase (RT) (2-5), Taq polymerase (6), and rat/human DNA polymerase ␤ (␤-pol) (7-11). The overall architecture of these enzymes is similar, including a cleft that binds DNA. These polymerases have been described using analogy to the anatomical features of a hand as fingers, palm, and thumb subdomains (2). Catalytically essential carboxylates that bind to the incoming deoxynucleoside 5Ј-triphosphate (dNTP) via Mg 2ϩ are found in the palm subdomain. Whereas the palm subdomains of these polymerases are structurally similar, the finger and thumb subdomains are structurally distinct. The dNTP binding pocket for DNA polymerase ␤ has been defined by the x-ray structure of the ␤-pol⅐ddCTP⅐primer-template complex (9). The triphosphate moiety of the incoming ddCTP is observed to be interacting with re...

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