Highly selective affinity labeling of DNA-polymerase from Thermus thermophilus B35 by a binary system of photoreactive agents

The thermostable DNA-polymerase from Thermus thermophilus B35 (Tte-polymerase) was affinity labeled by a binary system of photoreagents comprising base-substituted TTP analogs. The 5;-[32P]-labeled primer was elongated by Tte-polymerase in the presence of a TTP analog containing the photoreactive 2,3,5, 6-tetrafluoro-4-azidobenzoyl group (FAB-4-dUTP). Then the reaction mixture was UV-irradiated (365-450 nm) in the presence or the absence of a photosensitizer (TTP analog containing a pyrene moiety, Pyr-dUTP). The initial rate of the Pyr-dUTP-sensitized photomodification was almost 10-fold higher than the rate of direct photomodification (in the absence of Pyr-dUTP); in the case of the sensitized modification, the product of covalent cross-linking of the photoreactive primer with Tte-polymerase was apparently homogenous according to the data of electrophoresis. The enzyme was protected from the photosensitized modification by dNTP. To confirm the selectivity of the photosensitized modification of Tte-polymerase, another DNA-binding protein (human replication factor A, RPA) was added to the reaction mixture. In the presence of the photosensitizer (Pyr-dUTP), RPA was not labeled and only Tte-polymerase was modified, whereas in the case of direct modification, Tte-polymerase and the p32 and p70 subunits of RPA were labeled. The suggested method enables highly selective affinity modification of DNA-polymerases.     

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.     

Affinity capture of a mammalian DNA polymerase beta by inhibitors immobilized to resins used in solid-phase organic synthesis

The application of resins normally used in solid-phase organic synthesis to the affinity capture of a mammalian DNA polymerase beta (pol beta) is reported. Lithocholic acid (LCA), an inhibitor of pol beta, was immobilized on various solid supports, and the batch affinity purification of pol beta from a mixture of proteins using these LCA-immobilized resins was examined. Of the resins tested, TentaGel was the most effective at purifying pol beta and at resisting nonspecific absorption of proteins. The immobilized LCA recognized pol beta specifically, which resulted in pol beta binding to the resin. Using the LCA-immobilized resin, it was possible to purify pol beta from a mixture of proteins. Furthermore, it was possible to concentrate pol beta from a crude nuclear extract of human T lymphoma Molt4 cells. To facilitate the immobilization of compounds on TentaGel resins, we also designed and prepared photoaffinity beads containing a photoreactive group at the free termini of the TentaGel resin. The pol beta inhibitors LCA, C18-beta-SQDG, and epolactaene were immobilized on the photoaffinity beads by photoreaction. The batch affinity purification of pol beta from a protein mixture could be also achieved with these beads.     

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.     

Sensitized photomodification of DNA with binary systems of oligonucleotide conjugates. III. Double-quantum sensitization

Site-specific modification of single-stranded DNA by oligonucleotide derivatives of p-azido-O-(4-aminobutyl)tetrafluorobenzaldoxime sensitized by an oligonucleotide derivative of pyrenylethylamine was studied. Upon irradiation with the long-wave UV light (365-390 nm) of a DNA target-oligonucleotide reagent complementary complex, a considerable increase in the rate of sensitized photomodification at the G11 residue of the target relative to the direct photomodification was observed owing to the singlet-single energy transfer from the sensitizer onto the photoreagent. Upon simultaneous irradiation of the complex with UV and visible light in the region of the triplet-triplet absorption of pyrene (360-580 nm), an additional increase in the modification rate and a change in its site-direction (from the G11 to T13 residue) occurred through the two-photon triplet-triplet sensitization. The total extent of the structure photomodification amounted to 80%.     

Inhibitors of DNA polymerase beta: activity and mechanism

Bioassay-guided fractionation of extracts prepared from Couepia polyandra and Edgeworthia gardneri resulted in the isolation of the DNA polymerase beta (pol beta) inhibitors oleanolic acid (1), edgeworin (2), betulinic acid (3), and stigmasterol (4). Study of these pol beta inhibitors revealed that three of them inhibited both the lyase and polymerase activities of DNA polymerase beta, while stigmasterol inhibited only the lyase activity. Further investigation indicated that the four inhibitors had substantially different effects on the DNA-pol beta binary complex that is believed to be an obligatory intermediate in the lyase reaction. It was found that the inhibitors potentiated the inhibitory action of the anticancer drug bleomycin in cultured A549 cells, without any influence on the expression of pol beta in the cells. The results of the unscheduled DNA synthesis assay support the thesis that the potentiation of bleomycin cytotoxicity by DNA pol beta inhibitors was a result of an inhibition of DNA repair synthesis.     

Sensitized photomodification of DNA with binary systems of oligonucleotide conjugates. V. Effect of the structure of the target DNA. Quantitative photomodification]

A sensitized photomodification of several single-stranded target DNAs by binary systems of oligonucleotide conjugates complementary to the adjacent regions of DNA was performed. One of the conjugates contained a sensitizer (pyrene, anthracene, or 1,2-benzanthracene), and another conjugate contained a photoreagent 4-azidotetrafluorobenzalhydrazone. The sensitized photomodification is initiated by irradiation at 365-580 nm due to effective energy transfer from the excited sensitizer to the photoreagent in a complementary complex of the binary system with the target DNA where the sensitizer and photoreagent are brought sterically together. Conditions for the quantitative photomodification of a single-stranded DNA by the binary system of oligonucleotide conjugates were found. The maximum degree of photomodification depends on the number of guanosine residues in the (pG)n sequence of the target DNA at the modification site: at n = 1 the yield of covalent adducts was 62-68%, at n = 2, 75-82%, and at n = 4, 98-99%.     

A photoreactive analogue of 2'3'-dideoxyuridine 5'-triphosphate: preparation and 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 beta 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.     

Misincorporation of dAMP opposite 2-hydroxyadenine, an oxidative form of adenine.

Nucleotide incorporation opposite an oxidative form of adenine, 2-hydroxyadenine (2-OH-Ade) was investigated. When a primed template with 2-OH-Ade was treated with an exonuclease-deficient Klenow fragment of Escherichia coli DNA polymerase I (KFexo-), recombinant rat DNA polymerase beta (pol beta) or calf thymus DNA polymerase alpha (pol alpha), incorporation of dTMP and dAMP was observed. In addition, KFexo- inserted dGMP as well. A steady-state kinetic study indicated that the insertion of dAMP and dTMP opposite the DNA lesion occurred with similar frequency with KFexo- and pol beta. Insertion of dTMP opposite 2-OH-Ade was favored to that of dAMP by pol alpha. Chain extension from the A.2-OH-Ade pair is less favored than that from the T.2-OH-Ade pair by all three DNA polymerase. Analysis of full-length products of in vitro DNA synthesis showed that dTMP and dAMP were incorporated by DNA polymerases and that exonuclease-proficient and -deficient Klenow fragments also inserted dGMP opposite 2-OH-Ade. These results suggest that formation of 2-OH-Ade from A in DNA will induce A-->T and A-->C transversions in cells.     

Reagents for Modification of Protein–Nucleic Acid Complexes: III.1Site-Specific Photomodification of Elongation Complex of DNA Polymerase β with Arylazide Derivatives of Primers Sensitized with Fluorescent ATP γ-Amides

ATP -amides containing in -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 . The photomodification was carried out with the use of photoaffinity reagents, which were synthesized in situby the 5"-³²P-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 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 one to obtain photocrosslinks under such irradiation conditions when photomodification in their absence is not essentially observed.     

Dideoxynucleoside triphosphate-sensitive DNA polymerase from rice is involved in base excision repair and immunologically similar to mammalian DNA pol beta

A single polypeptide with ddNTP-sensitive DNA polymerase activity was purified to near homogeneity from the shoot tips of rice seedlings and analysis of the preparations by SDS-PAGE followed by silver staining showed a polypeptide of 67 kDa size. The DNA polymerase activity was found to be inhibitory by ddNTP in both in vitro DNA polymerase activity assay and activity gel analysis. Aphidicolin, an inhibitor of other types of DNA polymerases, had no effect on plant enzyme. The 67 kDa rice DNA polymerase was found to be recognized by the polyclonal antibody (purified IgG) made against rat DNA polymerase beta (pol beta) both in solution and also on Western blot. The recognition was found to be very specific as the activity of Klenow enzyme was unaffected by the antibody. The ability of rice nuclear extract to correct G:U mismatch of oligo-duplex was observed when oligo-duplex with 32P-labeled lower strand containing U (at 22nd position) was used as substrate. Differential appearance of bands at 21-mer, 22-mer, and 51-mer position in presence of dCTP was visible only with G:U mismatch oligo-duplex, but not with G:C oligo-duplex. While ddCTP or polyclonal antibody against rat-DNA pol beta inhibits base excision repair (BER), aphidicolin had no effect. These results for the first time clearly demonstrate the ability of rice nuclear extract to run BER and the involvement of ddNTP-sensitive pol beta type DNA polymerase. Immunological similarity of the ddNTP-sensitive DNA polymerase beta of rice and rat and its involvement in BER revealed the conservation of structure and function of ddNTP-sensitive DNA pol beta in plant and animal.     

Specific amino acid residues in the beta sliding clamp establish a DNA polymerase usage hierarchy in Escherichia coli

Escherichia coli dnaN159 strains encode a mutant form of the beta sliding clamp (beta159), causing them to display altered DNA polymerase (pol) usage. In order to better understand mechanisms of pol selection/switching in E. coli, we have further characterized pol usage in the dnaN159 strain. The dnaN159 allele contains two amino acid substitutions: G66E (glycine-66 to glutamic acid) and G174A (glycine-174 to alanine). Our results indicated that the G174A substitution impaired interaction of the beta clamp with the alpha catalytic subunit of pol III. In light of this finding, we designed two additional dnaN alleles. One of these dnaN alleles contained a G174A substitution (beta-G174A), while the other contained D173A, G174A and H175A substitutions (beta-173-175). Examination of strains bearing these different dnaN alleles indicated that each conferred a distinct UV sensitive phenotype that was dependent upon a unique combination of Delta polB (pol II), Delta dinB (pol IV) and/or Delta umuDC (pol V) alleles. Taken together, these findings indicate that mutations in the beta clamp differentially affect the functions of these three pols, and suggest that pol II, pol IV and pol V are capable of influencing each others' abilities to gain access to the replication fork. These findings are discussed in terms of a model whereby amino acid residues in the vicinity of those mutated in beta159 (G66 and G174) help to define a DNA polymerase usage hierarchy in E. coli following UV irradiation.     

Butylphenyl dGTP: a selective and potent inhibitor of mammalian DNA polymerase alpha.

BuPdGTP , the 2'-deoxyribonucleoside 5'-triphosphate of the DNA polymerase alpha (pol alpha)-specific inhibitor, N2-(p-n- butylphenyl )guanine, was examined with respect to its mechanism and its capacity to inhibit the mammalian DNA polymerases, pol alpha, pol beta, and pol gamma. BuP dGTP was specifically inhibitory for pol alpha, with no discernible activity on pol beta and pol gamma. The potency of BuP dGTP is unprecedented, with an apparent Ki less than 10 nanomolar. The unusual potency of the BuP dGTP is derived primarily from the 5' alpha and beta phosphoryl moieties, whose binding to enzyme complements that of the base-linked butylphenyl substituent. BuP dGTP is competitive with dGTP and apparently not subject to polymerization. Experiments employing BuP dGTP in the presence of a non-complementary template suggest that the core polymerase or an associated coprotein contains dNTP binding sites which recognize specific nucleic acid bases. The partial sensitivity of selected, non-mammalian DNA polymerases suggests that modification of the N2 substituent of dGTP will be a useful route to the design of novel, polymerase-specific affinity-probes.     

Study of interaction of XRCC1 with DNA and proteins of base excision repair by photoaffinity labeling technique

The X-ray repair cross-complementing group 1 (XRCC1) protein plays a central role in base excision repair (BER) interacting with and modulating activity of key BER proteins. To estimate the influence of XRCC1 on interactions of BER proteins poly(ADP-ribose) polymerase 1 (PARP1), apurinic/apyrimidinic endonuclease 1 (APE1), flap endonuclease 1 (FEN1), and DNA polymerase beta (Pol beta) with DNA intermediates, photoaffinity labeling using different photoreactive DNA was carried out in the presence or absence of XRCC1. XRCC1 competes with APE1, FEN1, and PARP1 for DNA binding, while Pol beta increases the efficiency of XRCC1 modification. To study the interactions of XRCC1 with DNA and proteins at the initial stages of BER, DNA duplexes containing a photoreactive group in the template strand opposite the damage were designed. DNA duplexes with 8-oxoguanine or dihydrothymine opposite the photoreactive group were recognized and cleaved by specific DNA glycosylases (OGG1 or NTH1, correspondingly), although the rate of oxidized base excision in the photoreactive structures was lower than in normal substrates. XRCC1 does not display any specificity in recognition of DNA duplexes with damaged bases compared to regular DNA. A photoreactive group opposite a synthetic apurinic/apyrimidinic (AP) site (3-hydroxy-2-hydroxymethyltetrahydrofuran) weakly influences the incision efficiency of AP site analog by APE1. In the absence of magnesium ions, i.e. when incision of AP sites cannot occur, APE1 and XRCC1 compete for DNA binding when present together. However, in the presence of magnesium ions the level of XRCC1 modification increased upon APE1 addition, since APE1 creates nicked DNA duplex, which interacts with XRCC1 more efficiently.     

Three-dimensional structural model analysis of the binding site of lithocholic acid, an inhibitor of DNA polymerase beta and DNA topoisomerase II.

The molecular action of lithocholic acid (LCA), a selective inhibitor of mammalian DNA polymerase beta (pol beta), was investigated. We found that LCA could also strongly inhibit the activity of human DNA topoisomerase II (topo II). No other DNA metabolic enzymes tested were affected by LCA. Therefore, LCA should be classified as an inhibitor of both pol beta and topo II. Here, we report the molecular interaction of LCA with pol beta and topo II. By three-dimensional structural model analysis and by comparison with the spatial positioning of specific amino acids binding to LCA on pol beta (Lys60, Leu77, and Thr79), we obtained supplementary information that allowed us to build a structural model of topo II. Modeling analysis revealed that the LCA-interaction interface in both enzymes has a pocket comprised of three amino acids in common, which binds to the LCA molecule. In topo II, the three amino acid residues were Lys720, Leu760, and Thr791. These results suggested that the LCA binding domains of pol beta and topo II are three-dimensionally very similar.     

Mismatched base-pair simulations for ASFV Pol X/DNA complexes help interpret frequent G*G misincorporation

DNA polymerase X (pol X) from the African swine fever virus is a 174-amino-acid repair polymerase that likely participates in a viral base excision repair mechanism, characterized by low fidelity. Surprisingly, pol X's insertion rate of the G*G mispair is comparable to that of the four Watson-Crick base pairs. This behavior is in contrast with another X-family polymerase, DNA polymerase beta (pol beta), which inserts G*G mismatches poorly, and has higher DNA repair fidelity. Using molecular dynamics simulations, we previously provided support for an induced-fit mechanism for pol X in the presence of the correct incoming nucleotide. Here, we perform molecular dynamics simulations of pol X/DNA complexes with different incoming incorrect nucleotides in various orientations [C*C, A*G, and G*G (anti) and A*G and G*G (syn)] and compare the results to available kinetic data and prior modeling. Intriguingly, the simulations reveal that the G*G mispair with the incoming nucleotide in the syn configuration undergoes large-scale conformational changes similar to that observed in the presence of correct base pair (G*C). The base pairing in the G*G mispair is achieved via Hoogsteen hydrogen bonding with an overall geometry that is well poised for catalysis. Simulations for other mismatched base pairs show that an intermediate closed state is achieved for the A*G and G*G mispair with the incoming dGTP in anti conformation, while the protein remains near the open conformation for the C*C and the A*G syn mismatches. In addition, catalytic site geometry and base pairing at the nascent template-incoming nucleotide interaction reveal distortions and misalignments that range from moderate for A*G anti to worst for the C*C complex. These results agree well with kinetic data for pol X and provide a structural/dynamic basis to explain, at atomic level, the fidelity of this polymerase compared with other members of the X family. In particular, the more open and pliant active site of pol X, compared to pol beta, allows pol X to accommodate bulkier mismatches such as guanine opposite guanine, while the more structured and organized pol beta active site imposes higher discrimination, which results in higher fidelity. The possibility of syn conformers resonates with other low-fidelity enzymes such as Dpo4 (from the Y family), which readily accommodate oxidative lesions.     

Translesion synthesis past 2'-deoxyxanthosine, a nitric oxide-derived DNA adduct, by mammalian DNA polymerases

Cellular DNA is damaged by nitric oxide (NO), a multifunctional bioregulator and an environmental pollutant that has been implicated in diseases associated with cancer and chronic inflammation. 2'-Deoxyxanthosine (dX) is a major NO-derived DNA lesion. To explore the mutagenic potential of dX, a 38-mer oligodeoxynucleotide ((5')CATGCTGATGAATTCCTTCXCTTCTTTCCTCTCCCTTT) modified site-specifically with dX at the X position was prepared post-synthetically and used as a DNA template in primer extension reactions catalyzed by calf thymus DNA polymerase (pol) alpha and human DNA pol beta, eta, and kappa. Primer extension reactions catalyzed by pol alpha or beta in the presence of four dNTPs were retarded at the dX lesion while pol eta and kappa readily bypassed the lesion. The fully extended products were analyzed to quantify the miscoding specificity and frequency of dX using two-phase polyacrylamide gel electrophoresis (PAGE). With pol alpha, eta and kappa, incorrect dTMP was preferentially incorporated opposite the lesion, along with lesser amounts of dCMP, the correct base. When pol beta was used, direct incorporation of correct dCMP was primarily observed, accompanied by small amounts of misincorporation of dTMP, dAMP and dGMP. Steady-state kinetic analyses supported the results obtained from the two-phase PAGE assay. dX is a miscoding lesion capable of preferentially generating G-->A mutations. The miscoding frequency varied depending on DNA polymerase used.     

Taxol derivatives are selective inhibitors of DNA polymerase alpha

During screening for mammalian DNA polymerase inhibitors, we found and succeeded in isolating a potent inhibitor from a higher plant, Taxus cuspidate. The compound was unexpectedly determined to be taxinine, an intermediate of paclitaxel (taxol) metabolism. Taxinine was found to selectively inhibit DNA polymerase alpha (pol.alpha) and beta (pol.beta). We therefore, tested taxol and other derivatives and found that taxol itself had no such inhibitory effect, and only taxinine could inhibit both pol.alpha and beta. The other compounds used, one derivative, cephalomannine, and five intermediates synthesized chemically inhibited only the pol.alpha activity in vitro. None of the compounds, including taxinine, influenced the activities of the other DNA polymerases, which are reportedly targeted by many pol.beta inhibitors. With both pol.alpha and beta, all of the compounds tested noncompetitively inhibited with respect to both the DNA template-primer and the dNTP substrate.