A binary system of photoreagents for high-efficiency labeling of DNA polymerases

To increase the efficiency of photoaffinity labeling of DNA polymerases, a binary system of photoaffinity reagents was applied. Photoreactive radioactive primers were synthesized by DNA polymerases beta (pol beta) or DNA polymerase from Thermus thermophilus (pol Tte) using a template-primer duplex in the presence of a dTTP analogue containing 4-azidotetrafluorobenzoyl group linked via spacers of varying length to 5-position of uridine ring- 5-[N-(2,3,5,6-tetrafluoro-4-azidobenzoyl)-amino-trans-propenyl-1]-2'-deoxyuridine-5'-triphosphate (FAB-4-dUTP) or 5-[N-[[(2,3,5,6-tetrafluoro-4-azidobenzoyl)-butanoyl]-amino]-trans-3-aminopropenyl-1]-2'-deoxyuridine-5'-triphosphate (FAB-9-dUTP). The reaction mixtures were UV irradiated (lambda = 365-450 nm) in the absence or presence of a dTTP analog, containing a pyrene moiety-5-[N-(4-(1-pyrenyl)-butylcarbonyl)-amino-trans-propenyl-1]-2'-deoxyuridine-5'-triphosphate (Pyr- 8-dUTP) or 5-[N-(4-(1-pyrenyl)-ethylcarbonyl)-amino-trans-propenyl-1]-2'-deoxyuridine-5'-triphosphate (Pyr-6-dUTP). The most efficient crosslinking of both DNA polymerases was observed in the case of photoreactive DNA primer, carrying the FAB-4-dUMP moiety at the 3'-end, and Pyr-6-dUTP as a sensitizer. The binary system of photoaffinity reagents allows increasing photoaffinity labeling of the both DNA polymerases in comparison to the primer crosslinking without photosensitizer.     

Sensitized photomodification of mammalian DNA polymerase beta. A new approach for highly selective affinity labeling of polymerases

To enhance the specificity of polymerase photoaffinity labeling, a novel approach based on sensitized photomodification has been developed. A base-substituted analog of TTP containing a pyrene group (PyrdUTP) was synthesized and used as an active site-bound photosensitizer for photoaffinity modification of DNA polymerase beta (pol beta). 5'-[32P]-labeled primer was elongated in situ by pol beta with a photoreactive analog of TTP (FAB-4-dUTP). The pyrene sensitizer (PyrdUTP), excited by light (365-450 nm), can activate the photoreagent, cross-linking it to pol beta as a result of fluorescence resonance energy transfer. The initial rate of pol beta photomodification was shown to increase by a factor of ten. The selectivity of pol beta photosensitized modification was proved by adding human replication protein A.     

A Comparative Study of the Modification Efficiency of DNA Polymerases and DNA Template by the DNA Primers with Various Photoreactive Groups at Their 3"-Termini

The dependence of the modification efficiency of DNA polymerases and DNA template on the nature of photoreactive group and the length of the linker that joins the group with the heterocyclic base of the primer 3"-terminal nucleotide was studied. The primers that contained the photoreactive groups at their 3"-termini were obtained using the rat DNA polymerase or the DNA polymerase from Thermus thermophilus in the presence of one of the dTTP analogues carrying the photoreactive group in position 5 of thymidine residue. After irradiating the reaction mixture with UV light and separating the modification products, the level of covalent attachment of the [5"-³²P]primer to DNA polymerases and template was determined. The primers containing 4-azido-2,5-difluoro-3-chloropyridyl group were shown to be the most effective in the modification of DNA polymerases.     

A new binary system for photosensitized labeling of DNA polymerases in nuclear extract

A binary system of reagents was used for photosensitized labeling of proteins of bovine testis nuclear extract. A dUTP analog containing 4-azido-2,5-difluoro-3-chloropyridyl group (FAP-dUTP) was used for the first time as a component of the binary system, and a dUTP analog containing the pyrenyl group (Pyr-dUTP) was used as a photosensitizer. Photoaffinity labeling of proteins of nuclear extract was performed using the radioactively labeled DNA duplex with the photoreactive FAP group at the 3"-end of elongating DNA strand and analog of the deoxyribose phosphate residue (3-hydroxy-2-hydroxymethyltetrahydrofuran (F) 5"-phosphate) at the 5"-end of the nick. Such structure is formed by the action of nuclear extract enzymes from the initial DNA duplex containing a synthetic apurine/apyrimidine site and is a photoreactive analog of a long-patch base excision repair intermediate. UV-irradiation modified a limited number of proteins of the nuclear extract. As shown using specific antibodies, the new binary system of photoreagents increases the efficiency of DNA polymerase labeling.     

A new highly efficient photoreactive analogue of dCTP. Synthesis, characterization, and application in photoaffinity modification of DNA binding proteins

A new base-substituted analogue of dCTP, exo-N-{2-[N-(4-azido-2,5-difluoro-3-chloropyridine-6-yl)-3-aminopropionyl]aminoethyl}-2'-deoxycytidine-5'-triphosphate (FAP-dCTP) has been synthesized and characterized. FAP-dCTP is an efficient substrate of mammalian DNA polymerase beta 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 beta. 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 beta, 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 primer-template 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 FAP-dCTP 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.     

Photoaffinity labeling of proteins in bovine testis nuclear extract

A binary system of photoaffinity reagents for selective affinity labeling of DNA polymerases has been developed. The photoreactive probe was formed in nuclear extract, using an end-labeled oligonucleotide containing a synthetic abasic site. This site was incised by apurinic/apyrimidinic endonuclease and then dNMPs carrying a photoreactive adduct were added to the 3(') hydroxyl using base-substituted arylazido derivatives of dUTP or dCTP. This results in the synthesis of photoreactive base excision repair (BER) intermediates. The photoreactive group was then activated, either directly (UV light exposure 320nm) or in the presence of the sensitizer of dTTP analog containing a pyrene group (Pyr-dUTP) under UV light 365nm. DNA polymerase beta was the main target crosslinked by photoreactive BER intermediates in this nuclear extract. In contrast, several proteins were labeled under the conditions of direct activation of arylazido group.     

Binary system for selective photoaffinity labeling of base excision repair DNA polymerases

A system of photoaffinity reagents for selective labeling of DNA polymerases in extracts has been examined. To create the photoreactive DNA probe in situ, DNA substrates containing a synthetic abasic site are incubated in mouse embryonic fibroblast (MEF) cellular extract in the presence of base-substituted arylazido derivatives of dNTPs. This results in synthesis of a photoreactive long patch base excision repair (BER) intermediate. The arylazido photoreactive group is then activated through energy transfer from the pyrene group of a dNTP analog (Pyr-dUTP), following 365 nm UV light exposure. Pyr-dUTP binds to the active site of DNA polymerases, and the pyrene group, when excited by 365 nm UV light, activates the nearby photoreactive group in the BER intermediate resulting in crosslinking of DNA-bound DNA polymerases. Under these conditions, various DNA binding proteins that are unable to bind Pyr-dUTP are not crosslinked to DNA. DNA polymerase β is the predominant crosslinked protein observed in the MEF extract. In contrast, several other DNA binding proteins are labeled under conditions of direct UV light activation of the photoreactive group at 312 nm. This study illustrates use of a new method of selective labeling of DNA polymerases in a crude cellular extract.     

The preparation of a photoreactive analogue of 2′,3′-dideoxyuridine 5′-triphosphate and its use for photoaffinity modification of human replication protein A

A new reagent for photoaffinity modification of biopolymers, 5-[E-N-(2-nitro-5-azidobenzoyl)-3-amino-1-propen-1-yl]-2′,3′-dideoxyuridine 5′-triphosphate (NAB-ddUTP), was synthesized. Like a similar derivative of 2′-deoxyuridine 5′-triphosphate (NAB-dUTP), it was shown to be able to effectively substitute for dTTP in the synthesis of DNA catalyzed by eukaryotic DNA polymerase β and to terminate DNA synthesis. A 5′-³²P-labeled primer with a photoreactive group at the 3′-terminus was derived from NAB-ddUTP and used for photoaffinity labeling of the human replication protein A (RPA). The covalent attachment of RPA p32 and p70 subunits to the labeled primers was demonstrated. NAB-ddUTP is a promising tool for studying the interaction of proteins of the replicative complex with NA in cellular extracts and living cells during the termination of DNA synthesis.     

Comparison of DIG-11-dUTP utilization by Geobacillus caldoxylosilyticus TK4, Mycobacterium tuberculosis and Escherichia coli DNA polymerases

DNA polymerases are used for many applications and we comparatively investigated DNA synthesis activity of DNA polymerase I enzymes of Geobacillus caldoxylosilyticus TK4, Escherichia coli and Mycobacterium tuberculosis with DIG-11-dUTP using synthetic DNA substrates. We showed that Gca polymerase I and Klenow Fragment (KF) used DIG-11-dUTP instead of dTTP almost at the same ratio, but more efficiently than Mtb polymerase I. We considered that Gca polymerase I could be efficiently used to label a DNA oligonucleotide either internally or at the 3′-terminus by DIG-11-dUTP for the generation of non-radioactive labeled DNA substrates at higher temperature than KF. All three polymerases could not elongate the primer terminus after adding ddNTPs into DNA that is characteristic for all known DNA polymerase I enzymes.     

Affinity labeling of flap-endonuclease FEN-1 by photoreactive DNAs

Eukaryotic flap-endonuclease (FEN-1) is 42-kD single-subunit structure-specific nuclease that cleaves 5"-flap strands of the branched DNA structure and possesses 5"-exonuclease activity. FEN-1 participates in DNA replication, repair, and recombination. The interaction of FEN-1 with DNA structures generated during replication and repair was studied using two types of photoreactive oligonucleotides. Oligonucleotides bearing a photoreactive arylazido group at the 3"-end of the primer were synthesized in situ by the action of DNA polymerase using base-substituted photoreactive dUTP analogs as the substrates. The photoreactive group was also bound to the 5"-end phosphate group of the oligonucleotide by chemical synthesis. Interaction of FEN-1 with both 5"- and 3"-ends of the nick or with primer–template systems containing 5"- or 3"-protruding DNA strands was shown. Formation of a structure with the 5"-flap containing the photoreactive group results in decrease of the level of protein labeling caused by cleavage of the photoreactive group due to FEN-1 endonuclease activity. Photoaffinity labeling of proteins of mouse fibroblast cell extract was performed using the radioactively labeled DNA duplex with the photoreactive group at the 3"-end and the apurine/apyrimidine site at the 5"-end of the nick. This structure is a photoreactive analog of an intermediate formed during DNA repair and was generated by the action of cell enzymes from the initial DNA duplex containing the 3-hydroxy-2-hydroxymethyltetrahydrofurane residue. FEN-1 is shown to be one of the photolabeled proteins; this indicates possible participation of this enzyme in base excision repair.     

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 derivatives, (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]-trans-3-aminopropenyl-1}-2"-deoxyuridine 5"-triphosphates, were used as substrates in the DNA polymerase -catalyzed reaction. The resulting nick, containing 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 generated photoreactive DNA duplexes, the photoaffinity modifications of DNA polymerase and human replication protein A (hRPA) were carried out. It was shown that DNA polymerase 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 preferentially labeled, implying a crucial role of this subunit in the protein–DNA interaction.     

Ferrocene conjugates of dUTP for enzymatic redox labelling of DNA

Two ferrocene-labelled analogues of dTTP, 5-(3-ferrocenecarboxamidopropenyl-1) 2′-deoxyuridine 5′-triphosphate (Fc1-dUTP) and 5-(3-ferroceneacet-amidopropenyl-1) 2′-deoxyuridine 5′-triphosphate (Fc2-dUTP) have been produced to demonstrate the incorporation of redox labels into DNA by polymerases. Cyclic voltammetry indicates that the ferrocenyl moieties display reversible redox behaviour in aqueous buffer with E_1/2 values of 398 (Fc1-dUTP) and 260 mV (Fc2-dUTP) versus Ag/AgCl. Primer extension by the proofreading enzymes Klenow fragment and T4 DNA polymerase shows that Fc1-dUTP is efficiently incorporated into DNA during synthesis, including incorporation of two successive modified nucleotides. Production of a 998 bp amplicon by Tth DNA polymerase demonstrates that Fc1-dUTP is also a satisfactory substrate for PCR. Despite its structural similarity, Fc2-dUTP acts predominantly as a terminator with the polymerases employed here. UV melting analysis of a 37mer duplex containing five Fc1-dU residues reveals that the labelled nucleotide introduces only a modest helix destabilisation, with T_m = 71 versus 75°C for the corresponding natural construct. Modified DNA is detected at femtomole levels using a HPLC system with a coulometric detector. The availability of simple and effective enzymatic labelling strategies should promote the further development of electrochemical detection in nucleic acid analysis.     

Photoaffinity labeling of functional states of the nicotinic acetylcholine receptor

The nicotinic acetycholine receptor was subjected to photoaffinity labeling in different conformational and functional states. The photolabel used was the ion-channel blocker [³H]-TPMP⁺. A procedure is described for isolating labeled -polypeptide chains from the receptor complex by preparative SDS-polyacrylamide gel electrophoresis. The photolabel was localized in the primary structure of the -chain. The site of labeling was found to be identical when photoaffinity labeling was performed in the resting, desensitized, or antagonist state, respectively.     

Study of interaction of human replication factor A with DNA using new photoreactive analogs of dTTP

Replication factor A (RPA) is a protein that binds single-stranded DNA in eukaryotic cells; it participates in replication, repair, and recombination of DNA. RPA is composed of three subunits with molecular masses 70 (p70), 32 (p32), and 14 kD (p14). The photoaffinity labeling method was used to study the interaction of RPA with the 3;-end of duplex DNA containing extended 5;-end of a single strand. We have synthesized dTTP analogs containing photoreactive 2,3,5,6-tetrafluoro-4-azidobenzoyl group attached to the 5th position of the uracil residue with linkers of variable length (9, 11, and 13 atom chains). Using these analogs and dTTP analog containing the same photoreactive residue attached to the 5th position of the uracil residue with a 4-atom linker, a number of oligonucleotide primers carrying a single photoreactive group on the 3;-end were enzymatically synthesized. Using the complex of the photoreactive primers with DNA template containing extended 19-base 5;-end, human RPA was photoaffinity modified. The primers were covalently bound to the p70 and p32 subunits of RPA and the p14 subunit was not labeled by the primers. The data are discussed considering the previously suggested model of interaction of RPA with DNA during replication.     

Interaction of APE1 and other repair proteins with DNA duplexes imitating intermediates of DNA repair and replication

Interactions of APE1 (human apurinic/apyrimidinic endonuclease 1) and DNA polymerase beta with various DNA structures imitating intermediates of DNA repair and replication were investigated by gel retardation and photoaffinity labeling. Photoaffinity labeling of APE1 and DNA polymerase beta was accomplished by DNA containing photoreactive group at the 3 -end in mouse embryonic fibroblast (MEF) cell extract or for purified proteins. On the whole, modification efficiency was the same for MEF-extract proteins and for purified APE1 and DNA polymerase beta depending on the nature of the 5 -group of a nick/gap in the DNA substrate. Some of DNA duplexes used in this work can be considered as short-patch (DNA with the 5 -phosphate group in the nick/gap) or long-patch (DNA containing 5 -sugar phosphate or 5 -flap) base excision repair (BER) intermediates. Other DNA duplexes (3 -recessed DNA and DNA with the 5 -hydroxyl group in the nick/gap) have no relation to intermediates forming in the course of BER. As shown by both methods, APE1 binds with the highest efficiency to DNA substrate containing 5 -sugar phosphate group in the nick/gap, whereas DNA polymerase beta binds to DNA duplex with a mononucleotide gap flanked by the 5 -p group. When APE1 and DNA polymerase beta are both present, a ternary complex APE1-DNA polymerase beta-DNA is formed with the highest efficiency with DNA product of APE1 endonuclease activity and with DNA containing 5 -flap or mononucleotide-gapped DNA with 5 -p group. It was found that APE1 stimulates DNA synthesis catalyzed by DNA polymerase beta, and a human X-ray repair cross-complementing group 1 protein (XRCC1) stimulates APE1 3 -5 exonuclease activity on 3 -recessed DNA duplex.     

The Eukaryotic Replication Complex and Its Affinity Modification Analysis

Replication of eukaryotic DNA is performed by a protein complex in which the central part is played by DNA polymerases. Synthesis with eukaryotic DNA polymerases , , and involves various replication factors, including the replication protein A, replication factor C, proliferating cell nuclear antigen, etc. Replication enzymes and factors also participate in DNA repair, which is interrelated with DNA replication. The function of the entire multicomponent system is regulated by protein–nucleic acid and protein–protein interactions. The eukaryotic replication complex was not isolated as a stable supramolecular structure, suggesting its dynamic organization. Hence X-ray analysis and other instrumental techniques are hardly suitable for studying this system. An alternative approach is affinity modification. Its most promising version involves in situ generation of photoreactive DNA replication intermediates. The review considers the recent progress in photoaffinity modification studies of DNA polymerases, eukaryotic replication factors, and their interactions with DNA replication intermediates.     

New reagents for affinity modification of biopolymers. Photoaffinity modification of Tte-DNA polymerase]

Arylazides N-(4-azido-2,5-difluoro-3-chloropyridinyl-6)-beta-alanine (Ia) and N-(4-azido-2,5-difluoro-3-chloropyridinyl-6)-glycine (Ib) were synthesized and covalently attached to 5-(3-aminopropenyl-1)-dUTP through the amino group to give 5'-triphosphate (IIa) and 5'-triphosphate (IIb). The resulting azides were subjected to photolysis in aqueous solution. The spectral and photochemical characteristics of azides (I) and (II) imply that their use for the modification of biopolymers holds promise. Compounds (IIa, b) effectively substituted dTTP in DNA polymerization catalyzed by thermostable DNA polymerase from Thermus thermophilus B-35 (Tte DNA polymerase). Photoaffinity modification of Tte DNA polymerase was carried out by dTTP analogues (IIa, b) and by earlier obtained 5-[N-(5-azido-2-nitrobenzoyl)-trans-3-aminopropenyl-1]deoxyuridine 5'-triphosphate (III) and 5-[N-(4-azido-2,3,5,6-tetrafluorobenzyol)-trans-3- aminopropenyl-1]deoxyuridine 5'-triphosphate (IV) using two variants of labeling. All four dTTP analogues were shown to modify Tte DNA polymerase.     

Photoreactive dTTP Analogues as Substrates for Thermostable DNA Polymerase from Thermus thermophilus B35

Substrate properties of several dTTP analogues bearing a photoreactive 2-nitro-5-azidobenzoyl (NAB) group attached at position 5 of uracil through linkers of various lengths, dTTP–NAB-x-dUTP (where x = 2, 4, 7–13 is the number of atoms in the linker), were studied. All the analogues are substrates for thermostable Thermus thermophilus B35 DNA polymerase in the elongation reaction of the 5-³²P-labeled primer–template complex. The kinetic parameters of some of the analogues were determined and compared with those of natural dTTP. It was shown that an increase in the linker length results in a higher efficiency of the analogue. The incorporation of NAB-x-dUP residues into the 3 primer end did not impede further elongation of the chain in the presence of natural dNTP.     

Interaction of Replication Protein A and Flap Endonuclease 1 with DNA Duplexes Containing a Nick or a Flap

Nicks and flaps are intermediates in various processes of DNA metabolism, including replication and repair. Photoaffinity modification was employed in studying the interaction of the replication protein A (RPA) and flap endonuclease 1 (FEN-1) with DNA duplexes similar to structures arising during long-patch base excision repair. The proteins were also tested for effect on DNA polymerase (Pol) interaction with DNA. Using Pol, a photoreactive dTTP analog was added to the 3" end of an oligonucleotide flanking a nick or a flap in DNA intermediates. The character and intensity of protein labeling depended on the type of intermediates and on the presence of the phosphate or tetrahydrofuran at the 5" end of a nick or a flap. Photoaffinity labeling of Pol substantially (up to three times) increased in the presence of RPA or FEN-1. Various DNA substrates were used to study the effects of RPA and FEN-1 on Pol-mediated DNA synthesis with displacement of a downstream primer. In contrast to FEN-1, RPA had no effect on DNA repair synthesis by Pol during long-patch base excision repair.