1068-1620/01/2705- $25.00 © 2001 MAIK “Nauka /Interperiodica” 0330 Russian Journal of Bioorganic Chemistry, Vol. 27, No. 5, 2001, pp. 330–339. Translated from Bioorganicheskaya Khimiya, Vol. 27, No. 5, 2001, pp. 372–382. Original Russian Text Copyright © 2001 by Safronov, Drachkova, Petruseva, Khodyreva, Dobrikov, Ivanova, Shishkin, Lavrik. 1 2 INTRODUCTION Recently, a method for sensitized affinity photo- modification of DNA polymerases with binary systems photoaffinity reagent–sensitizer was developed [2] 3 . As photoaffinity reagents F , primers extended by DNA polymerases in situ with the base-substituted dUTP 4- azidotetrafluorobenzoylaminoderivative and as sensi- tizer a base-substituted dUTP pyrene derivative were used. In such enzyme complexes F is located in DNA- binding channel whereas the sensitizer is in the dNTP- binding pocket of the enzyme. Upon UV-irradiation at 365–390 nm, the energy initially absorbed by the pyrene group of the sensitizer can be transferred onto the arylazide group of F and lead to photocrosslinking of the primer to the enzyme. It was shown that under these conditions of irradiation the rate of formation of photocrosslinks between 5'- 32 P-labeled primers and DNA polymerase β in the presence of the dUTP deriv- ative was 10 times higher than upon the photomodifica- tion without sensitizer [2]. Similar results were 1 For Part II, see [1]. 2 To whom correspondence should be addressed; phone: +7 (3832) 333-762; e-mail: safron@niboch.nsc.ru. 3 Abbreviations: NA, nucleic acids; p/t, primer–template system; F , photoaffinity reagent; MCC, microcolumn chromatography. achieved with DNA polymerases of Thermus thermo- philus [3]. RESULTS AND DISCUSSION The main goal of this study was to enhance the ratio of rates of photomodification in the protein–nucleic acid primer–template complexes in the presence and in the absence of sensitizers, dNTP derivatives. To this end, we employed the approach earlier realized in the complementary addressed sensitized photomodifica- tion of single-stranded DNA templates with binary sys- tems of oligonucleotide conjugates [4–7]. Replacement of the pyrene fluorescent groups by the anthracene and perylene groups in oligonucleotide sensitizers and of 4-azidotetrafluorobenzoyl groups by 4-azidotetrafluo- robenzylidene groups in photoreactive derivatives of oligonucleotides allowed irradiation with visible light to be used and resulted in an enhancement of the ratio of rates of the sensitized and direct photomodifications from ~100 to ~300000 and in an increase in efficiency of photomodification of single-stranded DNA template from 58% [7] to 90–100% [4–6]. In addition, we intended to study the effect of struc- tures of sensitizers and F on the direction of photomod- Reagents for Modification of Protein–Nucleic Acid Complexes: III. 1 Site-Specific Photomodification of Elongation Complex of DNA Polymerase b with Arylazide Derivatives of Primers Sensitized with Fluorescent ATP g-Amides I. V. Safronov*, 2 I. A. Drachkova**, I. O. Petruseva*, S. N. Khodyreva* , **, M. I. Dobrikov*, T. M. Ivanova*, G. V. Shishkin*, and O. I. Lavrik* , ** *Novosibirsk Institute of Bioorganic Chemistry, Siberian Division, Russian Academy of Sciences, pr. Akad. Lavrentieva 8, Novosibirsk, 630090 Russia **Novosibirsk State University, ul. Pirogova 2, Novosibirsk, 630090 Russia Received September 8, 2000; in final form, March 7, 2001 Abstract—ATP γ-amides containing in γ-N-position 1-methylpyrene, 9-methylanthracene, 10-chloro-9-methyl- anthracene, and 3-methylperylene residues were synthesized and characterized. These compounds were used as sensitizers of site-specific photomodification of the reconstituted elongating complex of the mammalian DNA polymerase β. The photomodification was carried out with the use of photoaffinity reagents, which were synthesized in situ by the 5'- 32 P-labeled primers extension with photoreactive analogues of dCTP containing in the exo-N-position of cytosine various perfluoroarylazide groups. The effect of structures of the sensitizers and photoreactive reagents on the efficiency and selectivity of photocrosslinking of primers to the enzyme and tem- plate, as well as formation of a number of other photomodification products was studied. It was shown that the sensitizers containing 10-chloro-9-methylanthracene and 3-methylperylene residues allow one to obtain pho- tocrosslinks under such irradiation conditions when photomodification in their absence is not essentially observed. Key words: anthracene, pyrene, perylene, perfluoroazide, DNA polymerase β, affinity modification