A New Highly Efficient Photoreactive Analogue of dCTP. Synthesis

Bioconjugate Chem. , 2005, 16 (1), pp 215–222 ... A. V. Ustinov , I. A. Stepanova , V. V. Dubnyakova , T. S. Zatsepin , E. V. Nozhevnikova , V. A. K...
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Bioconjugate Chem. 2005, 16, 215−222

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A New Highly Efficient Photoreactive Analogue of dCTP. Synthesis, Characterization, and Application in Photoaffinity Modification of DNA Binding Proteins Sergey V. Dezhurov,† Svetlana N. Khodyreva,† Ekaterina S. Plekhanova, and Olga I. Lavrik* Institute of Chemical Biology and Fundamental Medicine, Siberian Division, Russian Academy of Sciences, Prospect Lavrentieva 8, Novosibirsk, 630090 Russia. Received September 7, 2004; Revised Manuscript Received November 30, 2004

A new base-substituted analogue of dCTP, exo-N-{2-[N-(4-azido-2,5-difluoro-3-chloropyridine-6-yl)-3aminopropionyl]aminoethyl}-2′-deoxycytidine-5′-triphosphate (FAP-dCTP) has been synthesized and characterized. FAP-dCTP is an efficient substrate of mammalian DNA polymerase β in the reaction of primer elongation displaying substrate properties as an analogue of dCTP and dTTP. FAP-dCTP was used for the photoaffinity modification of mammalian DNA polymerase β. Two approaches to photoaffinity labeling were utilized. In one approach, photoreactive FAP-dCTP was first incorporated into radiolabeled primer-template, and photoreactive DNA was UV-irradiated in the presence of DNA polymerase β, which resulted in the polymerase labeling by photoreactive primer. In an alternate approach, FAP-dCTP was first UV-cross-linked to the enzyme; subsequently, radiolabeled primertemplate was added, and the enzyme-linked FAP-dCTP was incorporated into the 3′-end of radioactive primer. This “catalytic” modification pathway was shown to be less specific in recognition of FAPdCTP as an analogue of dCTP than dTTP. FAP-dCTP was used as substrate of endogenous DNA polymerases of HeLa cell extract to synthesize photoreactive DNAs for photoaffinity modification of cell proteins. UV irradiation results in modification of DNA binding proteins of cell extract. The level of photoaffinity labeling of protein targets in the cell extract was strongly dependent on the efficiency of synthesis of photoreactive DNA.

INTRODUCTION

The photoaffinity labeling technique in the study of DNA replication/repair machineries is based on UV-lightinduced cross-linking of photoreactive DNA to proteins. The most easy-to-use way to synthesize photoreactive probes is the in situ introduction of photoreactive dNMP moieties into the 3′ end of a DNA primer via the activity of DNA polymerases with photoreactive dNTP analogues used as substrates. A wide range of base-substituted dNTP analogues containing photoreactive groups of different photoreactivity and spacers of various lengths have been synthesized and characterized (1-3). Photoreactive dNTP analogues have been shown to be effective substrates of viral, bacterial, and eukaryotic DNA polymerases. These analogues were widely applied in study of proteins belonging to DNA replication/repair machineries in the systems reconstituted from purified proteins (4-6) and in cell extracts (7, 8). Photoreactive dNMP moieties varied substantially in their ability to form covalent cross-links with target protein depending on the type of photoreactive group used. The efficiency of covalent adduct formation may become a parameter restricting applicability of the method when applied in cell extracts for identification of unknown proteins because their concentrations are limited. Another factor that should be considered when photoreactive probes are synthesized in cell extracts via activity of the endogenous DNA polymerases is catalytic efficiency of dNTP deriva* To whom correspondence should be addressed. Tel: 7(3832) 344296. Fax: 7(3832) 333677. E-mail: [email protected]. † The authors wish it to be known that in their opinion, the first two authors should be regarded as joint first authors.

tives since the concentrations of DNA polymerases are limited, which may restrict the yield of photoreactive DNA. It should be noticed that photoreactive dNTP analogues displayed different kinetic parameters in the primer elongation catalyzed by Pol β1 (9). In our previous studies, we clearly recognized the factors that influence the efficiency of protein cross-linking. The most significant factors are the nature of photoreactive group and its position at the heterocyclic base. For example, among dUTP derivatives bearing substituents at the 5th position of the uridine ring, the most efficient in protein crosslinking was shown to be DNA containing FAP-dUMP moieties (10). As far as dCTP derivatives, the most efficient in protein cross-linking were FABO- and FABCcontaining photoreactive DNA when these analogues were introduced opposite to an adenine of the template (11). As has been previously shown, dCTP derivatives 1 Abbreviations: FAP-dCTP, exo-N-{2-[N-(4-azido-2,5-difluoro3-chloropyridine-6-yl)-3-aminopropionyl]aminoethyl}-2′-deoxycitidine-5′-triphosphate (lithium salt); FABO-dCTP, exo-N-[(4azido-2,3,5,6-tetrafluorobenzylideneaminooxy)-butyloxy]-2′deoxycytidine-5′-triphosphate (lithium salt); FABC-dCTP, exoN-[2-(4-azido-2,3,5,6-tetrafluorobenzylideneaminooxymethylcarbonyl)-aminoethyl]-2′- deoxycytidine-5′-triphosphate (lithium salt); FAB-dCTP, exo-N-[2-(4-azidotetrafluorobenzoyl)-aminoethyl]-2′-deoxycytidine-5′-triphosphate (lithium salt); FAP-dUTP, 5-{N-[N-(4-azido-2,5-difluoro-3-chloropyridine-6-yl)-3-aminopropionyl]-trans-3-aminopropenyl-1}-2′-deoxyuridine-5′-triphosphate (lithium salt); FABC-dUTP, 5-[N-(4-azido-2,3,5,6-tetrafluorobenzylideneaminooxymethylcarbonyl)-methylcarbamoyl]-trans3-aminopropenyl-1]-2′-deoxyuridine-5′-triphosphate (lithium salt); Pol β, DNA polymerase β; N3dATP, 2-azidoadenosine triphosphate.

10.1021/bc0497867 CCC: $30.25 © 2005 American Chemical Society Published on Web 12/23/2004

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Figure 1. Structures of photoreactive analogues of dNTP.

were recognized by DNA polymerases as dTTP due to the shift of tautomeric equilibrium from the amino to the imino form cased by introduction of the photoreactive group (9). A characteristic feature of FABO- and FABCdCTP derivatives is an electron-deficient atom of nitrogen, which is involved in π-system of the arylazido group (Figure 1). Comparison of the efficiencies of photoreagent crosslinking to Pol β when DNA probes contain the same FABC group attached to either the 4th or 5th positions of the pyrimidine ring (moieties of FABC-dCMP and FABC-dUMP, respectively) clearly demonstrated that the former was more effective (11). FAB-dCTP used to study the interactions of base excision repair proteins in mouse embryonic fibroblast extract with DNA intermediates synthesized in situ by endogenous DNA polymerases was efficiently incorporated in the primer (12). This analogue displayed rather good parameters in primer elongation catalyzed by Pol β but provided a low level of enzyme modification being incorporated in the primer chain. The main target for cross-linking of the photoreactive dCMP-containing primer was shown to be the complementary template chain (9). The corresponding efficiencies of the photoreactive primer cross-linking to Pol β and the template were 2% and 84%, respectively. Thus, taking together all these data prompted us to synthesize a dCTP analogue substituted at the 4th position with a FAP group, which provided rather effective modification of Pol β as a part of a FAP-dUMP containing primer. We expected the new analogue to combine good substrate properties in DNA synthesis with effective cross-linking to proteins when introduced into DNA that would make it suitable for photoaffinity modification both in reconstituted systems and in cellular extracts. We tested FAP-dCTP as a substrate in the primer elongation catalyzed by Pol β and endogenous DNA polymerases of HeLa cell extract. Photoreactive DNA with FAP-dCMP moieties synthesized in situ was used for photoaffinity modification of Pol β and DNA binding proteins in the cell extract.

EXPERIMENTAL PROCEDURES

Materials. Ethylenediaminetetraacetic acid (EDTA), dCTP, glycine, Tris, N,N,N′,N′-tetramethylethylenediamine (TEMED), and Coomassie G-250 were from Sigma (St. Louis, MO); MgCl2 and formamide were from Fluka (Buchs, Switzerland); β-mercaptoethanol was from Serva (Heidelberg, Germany); acrylamide, N,N′-methylenebisacrylamide, and urea were from ICN (Aurora, OH); γ-[32P]ATP (>110 TBq/mmol) and T4 polynucleotide kinase were from Biosan (Novosibirsk, Russia). The 1H, 19F, and 31P NMR spectra were recorded with a Bruker AM-400 spectrometer. UV spectra were recorded with UV 2100 Shimadzu (Kyoto, Japan). Oligonucleotides 5′-GGAAGACCCTGACGTTGCCCAACTTAATCGCC-3′ (36-mer template I), 5′-GGAAGACCCTGACGTTACCCAACTTAATCGCC-3′ (36-mer template II), and 5′-GGCGATTAAGTTGGG-3′ (15-mer primer) Sibenzyme (Novosibirsk, Russia) were used for synthesis of photoreactive DNA. The primers were the 5′-endlabeled with [32P] using T4 polynucleotide kinase and γ-[32P]ATP (13) and then purified by denaturing PAGE in 7 M urea (14). The radioactive primer was mixed with the template at 1:1.5 molar ratio, and the DNA duplexes were formed by heating to 90 °C and slowly cooling to room temperature. Recombinant rat DNA polymerase β (EC 2.7.7.7) was purified as described (15). Plasmid pRSET containing the cDNA of rat DNA polymerase β was the kind gift of Dr. S. H. Wilson (National Institute of Environmental Health Sciences, USA). HeLa cells were kindly provided by Dr. M. A. Zenkova (Institute of Chemical Biology and Fundamental Medicine, Novosibirsk). The whole cell extract was prepared as described (16). FABO-dCTP and FABC-dCTP synthesized as described (1) were a generous gift of Dr. I. V. Safronov (Institute of Chemical Biology and Fundamental Medicine, Novosibirsk). Synthesis and Characterization of FAP-dCTP. Synthesis of exo-N-{2-[N-(4-azido-2,5-difluoro-3-chloropyridine-6-yl)-3-aminopropionyl]aminoethyl}-2′-deoxyciti-

Highly Efficient Photoreactive Analogue of dCTP

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Scheme 1. Synthesis of FAP-dCTP

dine-5′-triphosphate (lithium salt) was carried out according to the standard method of the introduction of photoreactive groups (1). N-(4-Azido-2,5-difluoro-3-chloropyridine-6-yl)-3-aminopropionic acid N-hydroxysuccinimidyl ester (0.5 mmol) dissolved in DMFA (1 M, 500 µL) was mixed with 114 µmol (16 µL) of triethylamine and 0.4 mmol of triphosphate 1 (Scheme 1) dissolved in 2 mL of DMFA. When the reaction was completed (2 h, 37 °C), 50 mL of a 2% solution of LiClO4 in acetone was added to the reaction mixture. The resulting precipitate was separated by centrifugation and washed with acetone and Et2O. FAP-dCTP was purified by large scale HPLC using chromatographic complex Waters and column Lichroprep RP-18 (5-20 µm). Solvent was removed under reduced pressure; the residue was precipitated with a 2% solution of LiClO4 in acetone and washed with Et2O (61 mg, 20%). The purity of the analogue was 95% as estimated by analytical HPLC. The product was homogeneous according to TLC with Rf ) 0.21 (dioxane-ammonia-water, 6:4:0.3); UV (H2O) λmax ) 237 ( ) 26 000), λmax ) 322 ( ) 4000), λmin ) 280 ( ) 1450); NMR spectra (D2O), 31P (δ, ppm) -5.95 (d, J ) 20 Hz, Pγ, 1P,), -10.21 (d, J ) 20 Hz, PR, 1P), -20.31 (t, J ) 20 Hz, Pβ, 1P), 1H (δ, ppm) 2.2-2.4 (d, bs, H2′, 2H), 2.4-2.6 (t, bs, H9, 2H), 3.3-3.7 (m, H7, H8, H10, 6H), 4.1-4.3 (m, bs, H4′, H5′, 3H), 4.5 (s, H3′, 1H), 6.1 (d, H5, 1H), 6.4 (d, H1′, 1H), 7.9 (d, H6, 1H), 19F (δ, ppm) 7.9 (d, J ) 23 Hz, F5, 1F), 86.7 (d, J ) 23 Hz, F2, 1F). Mass Spectrometry. The mass spectra were recorded by MALDI-TOF mass spectrometer REFLEX III (Bruker Daltonics, Bremen, Germany), equipped with a single stage reflector giving an effective 3.0 m flight path and using the instrument’s standard 337 nm nitrogen laser and microchannel plate detector. Range of operational parameters was 20 kV acceleration voltage, 0 ns extraction delay, and 90% grid voltage. Generally the extraction delay and grid voltage were set to optimize resolving power. Spectra were obtained as averages of 60 laser shots, and the laser energy was generally set 25% above the ionization threshold. The matrix was 3-hydroxypicolinic acid. Saturated solution of matrix (50 mg/mL) was prepared in 1:1 acetonitrile/water containing 1% diammonium hydrogen citrate. Substrate Properties of FAP-dCTP. DNA synthesis catalyzed by Pol β was carried out in buffer containing 50 mM Tris-HCl (pH 8.8), 50 mM KCl, and 10 mM MgCl2. In addition, the reaction mixture contained 1.5 µM template, 1 µM 5′-[32P]-primer, and FAP-dCTP in variable concentrations (10-300 µM). The reaction was started by the enzyme addition to the final concentration of 20 or 2000 nM depending on the primer-template system and performed at 37 °C. Aliquots of 10 µL were taken out of the 200 µL reaction mixture at different time intervals (0.167-15 min) and quenched by the addition of loading buffer containing 80% formamide and 50 mM EDTA. After being heated (5 min, 90 °C), the samples were analyzed by electrophoresis in 20% PAG under denaturing conditions (14). Distribution of radioactivity

in the gel was quantified using Molecular Imager (BioRad, USA) and software “Quantity One”. For each of the reaction mixtures, the efficiency of the primer elongation was determined. The data obtained were used for calculation of the Km and kcat values using the software Origin 6.0 (Microcal Software Inc., USA). Determination of the Modification Rate Constant (k) and the Time of Half-Modification (τ1/2) of Pol β by Photoreactive Primers. The reaction mixtures for DNA synthesis (200 µL) contained standard components described above, but the concentration of Pol β and FAPdCTP were 3 and 10 µM, respectively. The reaction mixtures were incubated for 30 min at 37 °C to introduce a photoreactive FAP-dCMP moiety into the 3′-end of the primer by the activity of Pol β. After primer elongation, the reaction mixtures were placed in water bath (25 °C). Photolysis was induced by UV light using a DRK-120 VIO-1 UV lamp (‘LOMO’, Saint Petersburg, Russia) supplemented with glass filter UFS-6 (λ ) 313-365 nm) at a distance of 110 mm. During the irradiation, 15 µL aliquots were taken at definite time intervals (1-30 min). The reactions were stopped by adding the Laemmli loading buffer and heating at 90 °C for 5 min. The covalent adducts of Pol β with the [5′-32P] DNA primer were separated from the free DNA primer and the products of template modification by 12% PAGE as described (17). Distribution of radioactivity in the gel was quantified as described above. The rate constants of modification (k) were calculated from the time dependence of the amount of modification products, assuming that the modification reaction is of the first order. The half-time of modification was calculated by the formula τ1/2 ) (ln 2)/k, which corresponds to the formation of the half of the maximum amount of adducts under these conditions. Determination of the Efficiency of Modification of Pol β (WR). Pol β was cross-linked to photoreactive DNA as described in the previous sections by irradiation for the time t . τ1/2 (15 min). The covalent adducts of the [5′-32P] DNA primer with DNA polymerase were separated from the other components of the reaction mixture by 12% PAGE (17). Distribution of radioactivity in the gel was quantified as describe above. The efficiency of modification was calculated from the equations WR ) Wr/x, Wr ) 100%[A1/(A1 + A2)] where A1 is the amount of the primer covalently linked to Pol β, A2 is the amount of the unbound radioactive DNA primer, and x is the part of extended primer. The A1 and A2 values were determined by counting the corresponding areas of the gel. Determination of the Efficiency of DNA Template Modification. The modification of DNA template, which occurred along with the modification of Pol β, was determined quantitatively after the separation of the products by 20% PAGE under denaturing conditions (14). Distribution of radioactivity in the gel was quantified as describe above. The modification efficiency was calculated as the ratio of the template modification products to the total amount of the reactive [5′-32P] DNA primer.

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Catalytic Modification of Pol β. Modification of Pol β by the catalytic pathway was realized via initial UV irradiation of the enzyme in the presence of FAP-dCTP for the time t . τ1/2 followed by addition of primertemplate and further incubation for 30 min at 37 °C. Concentrations of components were the same as for direct modification described in previous section. Photoaffinity Modification of DNA Binding Proteins in Cellular Extract. Photoreactive dCMP moieties were incorporated in the primer by endogenous DNA polymerases of cell extract. The reaction mixtures (15 µL) contained standard components described above, but DNA duplex was 0.1 µM. One of the photoreactive dCTP analogues was added to the final concentration of 30 µM. The reaction mixtures contained 33 µg of total HeLa cell proteins. The reaction of incorporation of photoreactive dCMP residue was started by addition of cellular extract and allowed to proceed for 15 min at 37 °C. Two microliter aliquots were taken after completing DNA synthesis to analyze the efficiency of primer elongation. The rest of the reaction mixtures were placed on ice and irradiated for 15 min as described above. The efficiencies of primer elongation and cross-linking to proteins were quantified as described above. RESULTS AND DISCUSSION

Characterization of FAP-dCTP. FAP-dCTP (Figure 1) was synthesized according to the standard method of the introduction of photoreactive groups (1) as shown in Scheme 1. FAP-dCTP was purified by large scale HPLC. The purity of the analogue was 95% as estimated by analytical HPLC. Data of NMR and UV spectroscopy confirmed the structure of the obtained compound. Characterization of photoreactive derivatives of nucleoside triphosphates by MALDI-MS is poorly represented in the literature. We have found a single example, where the authors have determined MALDI-TOF spectra to characterize N3dATP and a few nonnucleoside aryl azide derivatives (18). In the negative ion MALDI spectra of these ATP analogues, “fingerprint” peaks corresponding to [M - 10 - 1], [M - 12 - 1], [M - 16 - 1], [M -26 - 1], [M -28 - 1], [M - 41 - 1], and [M - 42 - 1] were observed. The model of a fragmentation of cited photoreactive compounds induced by ultraviolet laser has been suggested. It seems clear that [M - 28 - 1] corresponds to the nitrene and its isomers and [M - 42 - 1] indicates that it is possible for the azide to lose N2 and N to form a free radical, for example. The MALDI-MS analysis for FAP-dCTP revealed analogous products [M - 1], [M 41 - 1], and [M - 42 - 1], but the main peaks [M - 1], [M - 26 - 1], and [M - 28 - 1] were shifted (+7) in m/z values; these products represent the adducts containing Li+ ions (Figure 2). Substrate Properties. Substrate properties of FAPdCTP in reaction of DNA primer elongation catalyzed by Pol β was characterized for two DNA duplexes, in which FAP-dCTP is incorporated either as a dCTP (PT1) or a dTTP analogue (PT2). Under conditions used, a single FAP-dCMP moiety was incorporated on both PT1 and PT2. Data on kinetic parameters of primer elongation are listed in Table 1. It should be emphasized that on PT2 when FAP-dCTP was used as a dTTP analogue the efficiency of incorporation was substantially lower in comparison with PT1. In the former case, the concentration of Pol β in the reaction mixture was enhanced about 1000 times to register incorporation of the analogue. Since the concentrations of Pol β and template-primer

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Figure 2. MALDI-TOF analysis of FAP-dCTP: peaks [M - 1], [M - 1 - 41], and [M - 1 - 42] (corresponding m/z values are 767.8, 727.7, and 726.7) and peaks of Li+-containing adducts [M - 1 + Li], [M - 1 - 26 + Li], and [M - 1 - 28 + Li] (corresponding m/z values are 774.7, 748.5, and 746.5). Table 1. Kinetic Parameters of Primer Elongation Catalyzed by DNA Polymerase β case

kcat, s-1

Km × 105, M

as analogue of dCTP (PT1) as analogue of dTTP (PT2)

0.33 ( 0.04 0.005 ( 0.0005

1 ( 0.5 3 ( 1.5

in this case were comparable, that is, the MichaelisMenten approximation does not hold, the kinetic parameters of FAP-dCTP incorporation as an analogue of dTTP should be regarded only as a crude estimate. Km values for FAP-dCTP are rather close on both template-primer duplexes and differ insignificantly from Km values for dCTP and dTTP, which were determined in refs 9 and 11, respectively. Km values for FABO-dCTP and FABC-dCTP were 10-fold higher than that of dCTP (9). When these analogues were used as dTTP, a 10-fold increase in Km values in comparison with that for dTTP was also detected (11). Unlike dCTP derivatives bearing FABO or FABC groups, FAP-dCTP appears not to face steric hindrance in the interactions with the enzyme binding site. kcat values for FAP-dCTP differ 65-fold depending on the template-primer duplex. When FAPdCTP replaced dCTP, the kcat value was in close agreement with that of dCTP (9). Thus, FAP-dCTP displays the best substrate properties among all photoreactive dCTP derivatives synthesized by us until now. When FAP-dCTP was used as dTTP the kcat value was substantially lowered. DNA polymerase β appears to recognize FAP-dCTP in this case as an incorrect dNTP. Such a behavior conforms to the mechanism that is responsible for Pol β fidelity. DNA polymerase β provides dNTP selection mainly at the step of phosphodiester bond formation, but this enzyme can also discriminate between the correct and incorrect dNTP during stage of nucleotide binding (19). Photoaffinity Modification of Pol β. We characterized all previously synthesized photoreactive dNTP analogues in their ability to cross-link Pol β when they were incorporated at the 3′ end of primers (9, 10, 11). To study the efficiency of FAP-dCMP-containing DNA as a photoreagent for protein modification, we used the same approach. We studied kinetics of the covalent attachment of photoreactive primer to Pol β. FAP-dCMP moieties were introduced in situ by Pol β using both template-

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Highly Efficient Photoreactive Analogue of dCTP Scheme 2. Catalytic and Direct Pathways of Photoaffinity Modification of Pol β

Figure 3. SDS-PAGE analysis of products of Pol β photoaffinity labeling: lane 1, no Pol β; lanes 2 and 3, direct and catalytic adduct formation, respectively; lane 4, FAP-dCTP irradiated (15 min) prior to addition to the reaction mixture; lane 5, no FAP-dCTP. Table 2. Parameters of FAP-dCTP as Photoreagent case

primer duplexes PT1 and PT2. Molar ratio of templateprimer duplex to Pol β and their concentrations provided practically complete binding of DNA duplex by Pol β. Under these conditions, the unproductive photolysis of photoreactive DNA in solution, which does not lead to formation of covalent adducts with Pol β, is minimized. When FAP-dCTP was used as dCTP analogue, that is, on PT1, the efficiency of primer elongation prior to UVirradiation was more than 90%. The kinetics of formation of covalent adducts of photoreactive primer with Pol β and the template strand fitted the exponential equation of the first order as was the case with other photoreactive dNTP derivatives (10, 11). We have estimated the rate constant of formation of covalent adducts and half-time of Pol β modification. The obtained values were shown to be similar to those for photoreactive primers with FAPdUMP moiety bearing the same photoreactive group (10). When FAP-dCTP was used as dTTP, that is, on PT2, the yield of cross-linking of photoreactive primer to Pol β was abnormal. The level of cross-linking (Wr) was 43% and exceeded the extent of conversion of the initial primer to the photoreactive form, which was less than 25%. This fact prompted us to propose that the additional amount of the covalent adducts of Pol β with the photoreactive primer was formed by an unusual pathway. The conventional pathway proceeds via prior incorporation of photoreactive dNMP moiety into the 3′ end of the primer followed by UV-light-induced cross-linking of photoreactive DNA to the protein. But, in the last unusual case, the process appears to proceed via the following steps (Scheme 2): first, cross-linking of photoreactive dCTP analogue to dNTP binding site of Pol β and, second, rather fast transfer of dNMP moiety onto the 3′ end of the primer from the dCTP analogue, which is covalently attached to the enzyme surface. Such a way of Pol β labeling by using a similar photoreactive analogue of dCTP was suggested by us earlier (20). This pathway appears to provide the surplus in the level of covalent adduct formation. To prove this proposal, we compared the rates of the primer elongation by Pol β and the cross-linking of the photoreactive primer to the enzyme. When FAP-dCTP was used as dTTP analogue, the rate of the primer elongation was approximately 10 times lower than the

k, min-1

as analogue of 0.23 ( 0.05 dCTP (PT1) as analogue of 0.55 ( 0.2 dTTP (PT2) a

x, %

WR, %

W r, %

70 ( 2

57 ( 5

40 ( 5

20 ( 2

b

43 ( 2

WDNA,% a 10 ( 2

Not determined. b Not applicable.

rate of cross-linking (data not shown). The rate of the primer elongation when FAP-dCTP was used as dCTP analogue was too high at the used concentration of Pol β to register the contribution of the second pathway. Thus, the more efficient covalent adducts Pol β-DNA primer formation appears to occur via saturation of dNTP binding site with the covalently attached dCTP analogue, from which a dCMP residue is transferred to the 3′ end of the primer. The additional argument for the proposed mechanism comes from the experiments where Pol β was UVirradiated in the presence of FAP-dCTP with the reaction mixtures being devoid of DNA duplex during UV-irradiation. After completion of irradiation, the reaction mixtures were supplemented with DNA duplex and further incubated. The following analysis revealed the efficient formation of the covalent adducts. The data are presented in Figure 3. The amounts of DNA-protein adducts formed in each of the pathways are comparable, which speaks in favor of the essential contribution of catalytic labeling of Pol β in the total level of its modification. Covalent fixation of a dNTP analogue in the active site of Pol β probably allows the enzyme to skip the stage of selection of the complementary dNTP. The transfer of the NAB-dUMP moiety from the dUTP analogue covalently fixed in the active site of Pol β onto the 3′ end of the oligonucleotide in a DNA duplex with a blunt end, that is, without a templating base, was shown by us earlier (21). The data on the efficiency of Pol β modification are listed in Table 2. The relative efficiency (WR) of crosslinking, that is, the part of photoreactive DNA primer cross-linked to Pol β, was the highest achieved so far with any photoreactive analogue used by us, although when FAP-dCTP was used as dTTP, the value of the relative efficiency is ill-defined due to the considerable contribution of catalytic modification of Pol β in the total level of the modification. Therefore, the WR value (the amount of photoreactive primer cross-linked to Pol β normalized

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Figure 4. Efficiencies of incorporation of different dCTP analogues by endogenous DNA polymerases of HeLa cell extract. Letters in parentheses designate as what deoxynucleoside triphosphate dCTP or dTTP the analogue was used.

Figure 5. SDS-PAGE analysis (A) of products of photo-cross-linking of DNA binding proteins in HeLa cell extract by photoreactive DNAs synthesized in situ by endogenous DNA polymerases with different dCTP analogues as substrates and efficiencies of protein labeling (B) in HeLa cell extract by photoreactive DNAs (a.u. ) arbitrary units). Letters in parentheses designate as what deoxynucleoside triphosphate dCTP or dTTP the analogue was used.

on the amount of the photoreactive primer synthesized prior to irradiation) is not shown in the Table 2. However, the Wr value directly determined from the experiment is rather high in comparison with the primers bearing other photoreactive dCMP moieties. When FAP-dCMP was placed opposite an adenosine of the template chain, the modification of the template did not practically occurred. Location of FAP-dCMP moiety opposite the template guanosine residue resulted in less than 10% cross-linking of the photoreactive primer to the complementary template chain. The levels of the template modification containing A or G opposite FAPdCMP correlates with the nucleophilicity of these bases. Rather low levels of the template modification were also observed in the case of FABC-dCMP and FABO-dCMP primers (11). Thus, these analogues can be considered as protein-directed reagents. Thus, newly synthesized FAP-dCTP displays the best substrate characteristics among dCTP analogues in primer elongation catalyzed by Pol β and provides the

highest level of Pol β modification achieved for any photoreactive dNTP derivatives synthesized and used by us until now. Application of FAP-dCTP in Cell Extract. The most easy-to-use and adequate way to synthesize photoreactive probes is the in situ introduction of photoreactive dNMP moieties into DNA via activity of endogenous DNA polymerases using photoreactive dNTP analogues as substrates. In cell extracts, where the concentrations of DNA polymerases are rather low, the poor substrate properties of dNTP analogues may become the limiting factor in DNA synthesis, which, in turn, restricts the applicability of the analogue in these systems. We demonstrated good substrate properties of FAP-dCTP in the primer elongation catalyzed by Pol β, but in cell extracts the efficiency of incorporation of photoreactive moieties into DNA can be modulated by the presence of other DNA polymerases and proteins, which can influence the activity of Pol β. Therefore, the most adequate way to estimate the potential of the analogue as a reagent

Highly Efficient Photoreactive Analogue of dCTP

for the in situ synthesis of photoreactive DNA probes in cell extracts is the direct measurement of the efficiency of its incorporation. We compared the efficiencies of incorporation of photoreactive dCMP moieties into DNA in HeLa cell extract using FAP-dCTP, FABO-dCTP, and FABC-dCTP as substrates. DNA containing FABO-dCMP and FABC-dCMP residues provide efficient cross-linking to Pol β, but corresponding dCTP analogues vary substantially in their substrate properties as dCTP or dTTP analogues in DNA synthesis catalyzed by Pol β (9, 11). Both FABC-dCTP and FABO-dCTP were used as dTTP to synthesize DNA probes for photoaffinity modification of proteins in HeLa cell extract (11). FABC-dCTP was shown to be less efficient in protein labeling than FABOdCTP, but the data were not quantified. Here we quantitatively compare the levels of incorporation of these three analogues and efficiencies of photoaffinity modification of DNA binding proteins in HeLa cell extract by DNA synthesized in situ. The data on the efficiencies of analogue incorporation both as dCTP and dTTP by endogenous DNA polymerases in HeLa cell extract are presented in Figure 4. FAP-dCTP and FABC-dCTP were rather efficiently incorporated as dCTP analogues, but considerably worse when used as dTTP. FABO-dCTP was rather inefficient in the extract both as dCTP and dTTP. Photoreactive DNAs synthesized in situ were used for photoaffinity modification of DNA binding proteins in HeLa extract. A pattern of protein modification in HeLa cell extract is shown in Figure 5A. Quantitative estimation of the efficiencies of protein cross-linking to photoreactive DNA synthesized on the basis of different photoreactive dCTP analogues clearly demonstrates that the efficiencies are determined by the levels of analogue incorporation (Figure 5B). The yield of cross-linking products also depends on the type of photoreactive group. For example, FAP-dCTP and FABC-dCTP were incorporated as dCTP with comparable efficiencies, but DNA primer containing FAP-dCMP provided at least 2.5-fold higher level of protein labeling. Analogous dependencies of the efficiencies of protein labeling in HeLa cell extract on substrate properties of the analogues and the types of photoreactive groups were demonstrated for the set of photoreactive dUTP analogues (11). The patterns of protein labeling were rather similar for all three analogues except the efficiency of crosslinking. Thus, the performance requirements for photoreactive dNTP analogues depend on the system, in which the analogues are planned to be used; and in the systems with limited DNA polymerase activities, the substrate characteristics of the analogues in DNA synthesis become very important. Taking together, the data clearly shows that newly synthesized FAP-dCTP possessing the best substrate and photochemical characteristics among the photoreactive dNTP analogues synthesized by us until now meets the requirements to be applied to the systems reconstituted from purified proteins and crude cellular extracts. ACKNOWLEDGMENT

The work was supported by grants from the Russian Foundation for Basic Research, Project Nos. 04-04-48525, 03-04-48562, and 04-03-32490, and by a grant from HFSP, No. RG0007/2004-C104. We thank Dr. Vladimir Koval for recording the MALDI-MS. LITERATURE CITED (1) Safronov, I. V., Shcherbik, N. V., Khodyreva, S. N., Vlasov, V. A., Dobrikov, M. I, Shishkin, G. V., and Lavrik, O. I. (1997)New photoreactive N4-substituted dCTP analogues: Prepara-

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