Photoactivated DNA analogs of substrates of the nucleotide excision repair system and their interaction with proteins of NER-competent HeLa cell extract.

Photoactivated DNA analogs of nucleotide excision repair (NER) substrates have been created that are 48-mer duplexes containing in internal positions pyrimidine nucleotides with bulky substituents imitating lesions. Fluorochloroazidopyridyl, anthracenyl, and pyrenyl groups introduced using spacer fragments at 4N and 5C positions of dCMP and dUMP were used as model damages. The gel retardation and photo-induced affinity modification techniques were used to study the interaction of modified DNA duplexes with proteins in HeLa cell extracts containing the main components of NER protein complexes. It is shown that the extract proteins selectively bind and form covalent adducts with the model DNA. The efficiency and selectivity of protein modification depend on the structure of used DNA duplex. Apparent molecular masses of extract proteins, undergoing modification, were estimated. Mutual influence of simultaneous presence of extract proteins and recombinant NER protein factors XPC-HR23B, XPA, and RPA on interaction with the model DNA was analyzed. The extract proteins and RPA competed for interaction with photoactive DNA, mutually decreasing the yield of modification products. In this case the presence of extract proteins at particular concentrations tripled the increase in yield of covalent adducts formed by XPC. It is supposed that the XPC subunit interaction with DNA is stimulated by endogenous HR23B present in the extract. Most likely, the mutual effect of XPA and extract proteins stimulating formation of covalent adducts with model DNA is due to the interaction of XPA with endogenous RPA of the extract. A technique based on the use of specific antibodies revealed that RPA present in the extract is a modification target for photoactive DNA imitating NER substrates.

3'-5' exonuclease activity of human apurinic/apyrimidinic endonuclease 1 towards DNAs containing dNMP and their modified analogs at the 3 end of single strand DNA break.

Human DNA apurinic/apyrimidinic (AP-) endonuclease 1 (APE1) is involved in the base excision repair (BER) pathway. The enzyme hydrolyzes DNA from the 5 side of the AP site. In addition to endonuclease activity, APE1 also possesses other slight activities including 3 -5 exonuclease activity. The latter is preferentially exhibited towards mispaired (non-canonical) nucleotides, this being the reason why APE1 is considered as a proofreading enzyme correcting the misincorporations introduced by DNA polymerase beta. We have studied 3 -5 exonuclease activity of APE1 towards dCMP and dTMP residues and modified dCMP analogs with photoreactive groups at the 3 end of the nicked DNA. Photoreactive dNMP residues were incorporated at the 3 end of the lesion using DNA polymerase beta and photoreactive dNTPs. The dependence of exonuclease activity on the "canonicity" of the base pair formed by dNMP flanking the nick at the 3 end, on the nature of the group flanking the nick at the 5 end, and on the reaction conditions has been determined. Optimal reaction conditions for the 3 -5 exonuclease hydrolysis reaction catalyzed by APE1 in vitro have been established, and conditions when photoreactive residues are not removed by APE1 have been chosen. These reaction conditions are suitable for using photoreactive nicked DNAs bearing 3 -photoreactive dNMP residues for photoaffinity labeling of proteins in cellular/nuclear extracts and model APE1-containing systems. We recommend using FAPdCTP for photoaffinity modification in APE1-containing systems because the FAPdCMP residue is less prone to exonuclease degradation, in contrast to FABOdCTP, which is not recommended.

Interaction of flap endonuclease-1 and replication protein A with photoreactive intermediates of DNA repair.

A new method for enzymatic synthesis of radioactive DNA flapped structures containing a photoreactive dCMP moiety at a branch point with 4-(4-azido-2,3,5,6-tetrafluorobenzylidene-hydrazinocarbonyl)butylcarbamoyl group attached at exo-N-position of cytosine was developed. The formation of complexes of flap endonuclease-1 (FEN-1) with flapped DNA was shown by photoaffinity modification and gel retardation assays. The substrate properties of the flapped structures with different flap lengths were studied in the reaction of endonuclease cleavage catalyzed by FEN-1. It was demonstrated that inhibition of FEN-1 activity by replication protein A (RPA) depends on the length of the single-stranded part of the flapped substrate. A significant inhibition of cleavage was observed when the flap length was sufficient for effective RPA binding, while for structures with short single-stranded part the efficiency of cleavage was independent of the presence of RPA. FEN-1 and RPA were modified by photoaffinity labeling using flap structures with single-stranded parts consisting of 8 and 21 nucleotides. Products of DNA photoattachment to FEN-1 were observed in both cases, while the covalent adducts with RPA were obtained only with the 21-nucleotide-long flap. Photoaffinity modification demonstrated that FEN-1 and RPA compete for the binding of the flapped substrates with long single-stranded parts.

[Reagents for modification of protein-nucleic complexes. III. Site-specific photomodification of elongation complex of DNA polymerase beta with arylazide derivatives of primers sensitized with fluorescent ATP gamma-amide]. / Reagenty dlia modofikatsii belkovo-nukleinovykh kompleksov. III. Sait-spetsificheskaia fotomodifikatsiia élongiruiushchego kompleksa DNK-polimerazy beta arilazidnymi proizvodnymi praimerov, sensibilizirovannaia fluorestsentnymi gamma-amidami ATP.

ATP gamma-amides containing in gamma-N-position 1-methylpyrene, 9-methylanthracene, 10-chloro-9-methylanthracene, 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 beta. The photomodification was carried out with the use of photoaffine reagents, which were synthesized in situ by the 5'-(32)P-labeled primers extension with photoactive analogues of dCTP containing in the exo-N-position of cytosine various perfluoroarylazide groups. The effect of structures of the sensitizers and photoactive reagents on the efficiency and selectivity of photolinking of primers to the enzyme and template, 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 preparation of photolinks in such irradiation conditions when photomodification in their absence is not essentially observed.

[Reagents for modification of protein-nucleic acids complexes. II. Site-specific photomodification of DNA-polymerase beta complexes with primers elongated by the dCTP exo-N-substituted arylazido derivatives]. / Reagenty dlia modifikatsii belkovo-nukleinovykh kompleksov. II. Sait-spetsificheskaia fotomodofikatsiia kompleksov DNK-polimerazy beta praîmerami, élongirovannymi ékzo-N-zamashchennymi arilazidnymi proizvodnymi dCTP.

Substrate properties of the earlier synthesized and characterized dCTP derivatives bearing in the exo-N-position of cytosine 2-(4-azido-2,3,5,6-tetrafluorobenzoylamino)ethyl (I), 2-(2-nitro-5-azidobenzoylamino)ethyl (II), 2-(4-azido-2,3,5,6-tetrafluorobenzylideneaminooxymethylcarbonylamino)ethyl (III), 4-(4-azido-2,3,5,6-tetrafluorobenzylideneaminooxy)butyloxy (IV), or 4-(4-azido-2,3,5,6-tetrafluorobenzylidenehydrazinocarbonyl)butyl- carbonylamino (V) groups were studied in the primer extension reaction catalyzed by rat DNA polymerase beta. Unlike the earlier results obtained with HIV reverse transcriptase, dCTP derivatives (I)-(III) were not recognized by rat DNA polymerase beta as dTTP analogues, and all the five nucleotides were utilized as dCTP analogues. When compared with dCTP, Km values for the synthesized dCTP derivatives were higher by a factor of 4-20; Vmax were 1-2.3 times higher for (I)-(III) and (V) but 20-fold lower for derivative (IV). Site-specific photomodifications of the primer-template-DNA polymerase beta complexes were carried out using photoreactive reagents PRI-PRV, obtained in situ by extension of 5'-32P-labeled primers with dCTP analogues (I)-(V), respectively, when exposed to UV irradiation at 303-313 nm. Reagents PRI and PRIV provided the maximum photocrosslinking of the 5'-32P-labeled primer to the DNA template (56%) and to the enzyme (20%), respectively. The lowest efficiency of photocrosslinking was observed for PRII (about 1%).

Viral protein functions study by affinity modification.

The knowledge of virus reproduction is necessary to design new safe drugs for inhibition of infections. Ultra-violet irradiation of virus proteins with labeled virus genome fragments permits to identify specific nucleic acid binding proteins. Affinity modification of enzymes with nucleotide derivatives could help to determine NTP-binding proteins and those involved in viral genome replication. Photoreactive analogues of nucleic acids are among the tools used to detect elongation subunits of replicative complexes. Affinity modification approach has already resulted in successful treatment of virus diseases.

Photoaffinity labeling of DNA polymerase from Thermus thermophilus and DNA template by photoreactive analogs of dCTP.

Substrate properties of dCTP analogs N4-[2-(4-azidotetrafluorobenzoylamino)-ethyl]-2;-deoxycytidine-5; -triphosphate (FABdCTP), 5-[N-(4-azidotetrafluorobenzoyl)-3-amino-trans-propen-1-yl] -2;-deoxycytidine-5;-triphosphate (AlFABdCTP), and N4-[2-(2-nitro-5-azidobenzoylamino)-ethyl]-2;-deoxycytidine-5; -triphosphate (NABdCTP) were studied in the reaction of DNA synthesis catalyzed by DNA polymerase from the extremely thermophilic bacterium Thermus thermophilus B 35 (Tte DNA polymerase). The enzyme was photoaffinity labeled with the mentioned derivatives, NABdCTP being used for the first time. The photoreactive primers containing FABdCTP and AlFABdCTP were synthesized in situ by Tte DNA polymerase and used in the complementary addressed labeling of DNA template. The efficiency of DNA template labeling is shown to be a function of the structure of the photoactive group.

Affinity labelling of the tick-borne encephalitis virus RNA replicase proteins by 4-N-exo-base-substituted photoreactive CTP analogs.

4-N-exo-base-substituted photoreactive analogs of CTP were designed and synthesized. Two flavivirus proteins NS5 and NS3 are shown to be labelled after RNA synthesis in the presence of the analogs, irradiation by UV-light (313 nm) and subsequent [alpha-32P]NTP incorporation.

[New photoreactive N(4)-substituted dCTP analogues:synthesis, photochemical characteristics, and substrate properties in HIV-1 reverse transcriptase catalyzed DNA synthesis]. / Novye fotoaktivnye N(4)-zameshchennye analogi dCTP: poluchenie, fiziko-khimicheskie i substratnye svoistva pri sinteze DNK, kataliziruemoi obratnoi transkriptazoi VICh-1.

Photochemical characteristics and substrate properties of four newly synthesized dCTP analogues: N4-[2-(2-nitro-5-azidobenzoylamino)ethyl]-, N4-[2-(4-azidotetrafluorobenzylideneaminooxymethylcarbamoyl)ethyl] -, N4-[4-(4-azidotetrafluorobenzylideneaminooxy)butyloxy]-, and N4-[4-(4-azidotetrafluorobenzylidene hydrazinocarbonyl)butylcarbamoyl]-, and N4-[4-(4-azidotetrafluorobenzylideneaminooxy)butyloxy]-2'-de oxycytidine 5'-triphosphates as well as those of the earlier described N4-[2-(4-azidotetrafluorobenzoylamino)ethyl]- and 5-[E-3-(4-azidotetrafluorobenzoylamino)-1-propenyl)]-2'-deoxycytid ine 5'-triphosphates were compared. When being irradiated with UV light at a wavelength of 303-313 nm, the new analogues demonstrated greater than 10-fold higher photoactivity as compared with the old compounds. The first three new compounds were utilized by HIV-1 reverse transcriptase as dCTP and dTTP, while the last derivative was recognized only as dTTP. Once incorporated into the primer 3'-terminus, none of the analogues synthesized terminated further primer elongation with natural triphosphates.

dNTP binding to HIV-1 reverse transcriptase and mammalian DNA polymerase beta as revealed by affinity labeling with a photoreactive dNTP analog.

The dNTP binding pocket of human immunodeficiency virus type 1 reverse transcriptase (RT) and DNA polymerase beta (beta-pol) were labeled using a photoreactive analog of dCTP, exo-N-[beta-(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 cross-linking was increased in the presence of a dideoxyterminated primer-template with RT, but not with beta-pol, suggesting a significant influence of prior primer-template binding on dNTP binding for RT. Mutagenesis of beta-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.

Synthesis and characterization of (d)NTP derivatives substituted with residues of different photoreagents.

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.

[Use of non-radioactive biotin-labelled probes for detecting hepatitis A virus]. / Ispol'zovanie neradioaktivnykh biotin-mechennykh zondov dlia detektsii virusa gepatita A.

The biotin-labeled DNA probes were constructed on the basis of the hybrid bacteriophage M13nip 9 single-stranded DNA containing the fragments of the hepatitis A viral cDNA. The probes were biotin treated by chemical modification of the DNA by the peraminating reagent or photochemically. The labeled DNA probes were used in molecular hybridization experiments with the nuclear acids fixed on the nitrocellulose filters. The biotin treated DNA was determined by the avidin-gold colloid conjugate with the subsequent physical silver amplification or by the streptavidin-alkaline phosphatase conjugate. The sensitivity of both probes was identical and permitted the determination of 5 x 10(-11)-5 x 10(-12) g of the control DNA and 10(-9) g of the hepatitis A virus. The developed test systems were used for detection of the viral RNA in blood from patients.

[Localization of the antigenic segment of the tick-borne encephalitis virus envelope protein using monoclonal antibodies]. / Lokalizatsiia antigennogo uchstka belka obolochki virusa kleshchevogo éntsefalita s ispol'zovaniem monoklonal'nykh antitel.

The largest cyanogen bromide fragment (GP-14,5; coordinates 78-176) of E protein belonging to the envelope of the tick-borne encephalitis (TBE) virus (Far Eastern subtype, strain Sofjin) interacted with five out of twelve E-specific monoclonal antibodies (MAbs). Having compared; efficiencies of some MAbs binding to the antigens of TBE viruses of Far Eastern and West European subtypes and primary structures of analogous peptides of these viruses, we suggested the epitopes of these MAbs to be located in the vicinity of 89 and/or 116-th amino acid residues of E protein. Effect of denaturing agents and reduction followed by carboxymethylation on the protein E antigenic properties was studied.