Purification and characterization of DNA-dependent ATP phosphohydrolases from KB cells

DNA-dependent ATPases have been purified from logarithmically growing KB cells by chromatography on single-stranded DNA cellulose and phosphocellulose. Phosphocellulose resolved the DNA-dependent ATPases into three activities designated ATPase I, II and III, respectively. From gel filtration and sedimentation analysis ATPases II and III were found to be very similar, both with calculated molecular weights of 78,000. Due to the extreme lability these enzymes were not purified further. The molecular weight of ATPase I determined by gel filtration and sedimentation analysis was calculated to be 140,000. ATPase I was further purified by gradient elution on ATP-agarose, revealing two peaks of activity (IA and IB), and by sucrose gradient sedimentation. Analysis of the fractions from the sucrose gradient by sodium dodecylsulphate gel electrophoresis revealed only one broad polypeptide band co-sedimenting with both ATPase IA and ATPase IB. This band was composed of four closely spaced polypeptides with apparent molecular weights of 66,000, 68,000, 70,000 and 71,000. Comparison of the native molecule weight (140,000) with these results suggests that ATPase I is a dimer. ATPase IA and IB were indistinguishable in their structural and enzymatic properties and presumably represent the same enzyme. The purified enzyme has an apparent Km of 0.5 mM for ATP producing ADP + Pi. A maximum activity of 2,100 molecules of ATP hydrolyzed per enzyme molecular per minute was found. Hydrolysis of ATP requires the presence of divalent cations (Mg2+ greater than Ca2+ greater than Mn2+ greater than Co2+). A broad pH optimum (pH 6--8) was observed. The enzyme uses ATP or dATP preferentially as a substrate, while other deoxyribonucleoside or ribonucleoside triphosphates were inactive. ATPase I prefers denatured DNA as cofactor. The activity with native DNA is 40% of that with denatured DNA.     

A third DNA-dependent ATPase from Bacillus cereus free of ATP-dependent DNase activity

The purification of ATP-dependent DNase from Bacillus cereus led to the isolation and characterization of a third DNA-dependent ATPase. The enzyme called ATPase III has been purified free of nuclease activity. None of the expected ATPases proved to be identical with ATP-dependent DNase-DNA-dependent ATPase. Separation of ATPase I, II and III and a DNase specific for single-stranded DNA from the same source excludes the possibility of ATP-dependent DNase being the action of a single enzyme molecule.     

DNA-dependent adenosinetriphosphatase C1 from mouse FM3A cells has DNA helicase activity.

In our previous study, we identified four chromatographically distinct DNA-dependent ATPases, B, C1, C2, and C3, in mouse FM3A cells (Tawaragi, Y., Enomoto, T., Watanabe, Y., Hanaoka, F., and Yamada, M. (1984) Biochemistry 23, 529-533). The DNA-dependent ATPase C1 has been purified and characterized in detail. A divalent cation and a polynucleotide cofactor were required for the ATPase activity. Poly(dT), single-stranded circular DNA, and heat-denatured DNA were very effective. Almost no ATPase activity was observed with S1 nuclease-treated native DNA. ATPase C1 hydrolyzed ATP only among the ribo- and deoxyribonucleoside triphosphates tested, and this fact distinguished ATPase C1 from ATPases B, C2, and C3, because the latter enzymes are capable of hydrolyzing both ATP and dATP. The purified DNA-dependent ATPase C1 fraction was shown to have a DNA helicase activity that was dependent on hydrolysis of ATP. The helicase activity and DNA-dependent ATPase activity cosedimented at 5.2 S on glycerol gradient centrifugation. Both activities showed similar preferences for nucleoside 5'-triphosphates and similar requirements for divalent cations. The DNA helicase activity was inhibited by the addition of single-stranded DNAs that served as cofactor for the ATPase activity. The efficiency of a single-stranded DNA to inhibit DNA helicase activity correlated well with the capacity of the DNA to serve as cofactor for DNA-dependent ATPase activity. The helicase was shown to migrate along the DNA strand in the 5' to 3' direction, which is the same direction of migration of the mouse DNA helicase B (Seki, M., Enomoto, T., Yanagisawa, J., Hanaoka, F., and Ui, M. (1988) Biochemistry 27, 1766-1771).     

Purirication and characterization of two forms of DNA-dependent ATPase from yeast

Two forms of DNA-dependent ATPase activity have been purified from yeast extracts. The two ATPases differ from each other in chromatographic properties and heat stabilities but have similar molecular weight and reaction properties. DNA-dependent ATPase I has been purified to near homogeneity, while DNA-dependent ATPase II is only partially purified. The two ATPases from yeast are related structurally since antiserum raised against ATPase I cross-react against ATPase II. Yeast DNA-dependent ATPase I has a native molecular weight of about 68,000 and consists of a single polypeptide chain. ATPase II also sediments on sucrose gradient as a 68,000-dalton protein. Both yeast DNA-dependent ATPases hydrolyze dNTPs and rNTPs to their corresponding nucleoside diphosphates and orthophosphate, but dATP and ATP are preferred substrates. In addition to nucleoside triphosphates, both enzymes require a divalent cation and a polynucleotide for activity. Single-stranded DNAs and polydeoxynucleotides are the most effective co-substrates for yeast DNA-dependent ATPases. Addition of yeast DNA-dependent ATPases to DNA synthesis system containing yeast DNA polymerases does not significantly stimulate the rate of DNA synthesis.     

A cellular single-stranded DNA-dependent ATPase associated with simian virus 40 chromatin

A single-stranded DNA-dependent ATPase from monkey kidney tissue culture cells (CV-1) has been found associated with SV40 chromatin. This ATPase activity is distinguishable from the ATPase activity of T-antigen by the following properties: the Km for ATP, elution from phosphocellulose, and stimulation of the ATPase activity by single-stranded DNA but not by double-stranded DNA. The ATPase has been isolated and characterized from the nuclei of uninfected cells. ATP hydrolysis is dependent on single-stranded DNA and a divalent cation. The km values for ATP and single-stranded DNA are 0.024 mM and 0.09 microgram/ml, respectively. The affinity of the ATPase for single-stranded DNA is sufficiently high that the enzyme co-sediments with single-stranded DNA in glycerol gradients. The binding of single-stranded DNA is independent of ATP and MgCl2; however, ATP hydrolysis increases the exchange of enzyme between different DNA molecules. Form I (superhelical) SV40 DNA is also a substrate for ATPase binding, but relaxed Form I, Form II (nicked circular), and double-stranded linear SV40 DNAs are not substrates. Because the DNA helix within chromatin is not under the same kind of tortional strain as Form I DNA, we hypothesize that the ATPase is bound to the single-stranded regions of replication forks in the SV40 chromatin.     

DNA-dependent ATPases in Bacillus subtilis mutants and in competent cells

Three DNA-dependent ATPases (gamma phosphohydrolases) can be isolated from Bacillus subtilis cells. We studied these enzymes in a number of mutants deficient in recombination or repair functions (rec, uvr) and in competent cells. The recA mutant studied had lower ATPase II activity, while competent cells had higher ATPase I activity, in comparison with the parental strain not brought to competence.     

Chemiosmotic coupling in Methanobacterium thermoautotrophicum: hydrogen-dependent adenosine 5''-triphosphate synthesis by subcellular particles.

Hydrogenase and the adenosine 5'-triphosphate (ATP) synthetase complex, two enzymes essential in ATP generation in Methanobacterium thermoautotrophicum, were localized in internal membrane systems as shown by cytochemical techniques. Membrane vesicles from this organism possessed hydrogenase and adenosine triphosphatase (ATPase) activity and synthesized ATP driven by hydrogen oxidation or a potassium gradient. ATP synthesis depended on anaerobic conditions and could be inhibited in membrane vesicles by uncouplers, nigericin, or the ATPase inhibitor N,N'-dicyclohexylcarbodiimide. The presence of an adenosine 5'-diphosphate-ATP translocase was postulated. With fluorescent dyes, a membrane potential and pH gradient were demonstrated.     

A nuclear mutant of Chlamydomonas reinhardtii defective in photosynthetic photophosphorylation. Characterization of the algal coupling factor ATPase

Use of the Flagyl selection procedure [Schmidt et al. (1977) Proc. Natl Acad. Sci. USA, 74, 610-614] led to the isolation of a nuclear mutant of Chlamydomonas reinhardtii designated thm-24. This mutant displays normal electron transport rates in vitro, possesses high latent ATPase activity bound to the thylakoid membrane, but is incapable of photophosphorylation. Decay of the transmembrane potential, as indicated by the kinetics of the 520-nm absorption change after illumination, is unusually slow and markedly biphasic. Sodium dodecylsulfate/polyacrylamide gel electrophoresis of purified thylakoid membranes shows mutant thm-24 to be lacking a number of polypeptides including those previously designated 4.1, 4.2 and 8.1. Treatment of purified thylakoid membranes of wild-type and mutant algae, using the chloroform-release procedure of Beechey et al. [(1975) Biochem. J. 148, 533-537] resulted in the removal of ATPase activity from each strain. In wild-type cells, the ATPase activity was of heterogeneous enzymatic origin; fractionation of the chloroform-release extracts by non-denaturing polyacrylamide gel electrophoresis yielded three distinct bands displaying ATPase activity, designated ATPases I, II and III. In contrast, extracts from membranes of mutant thm-24 yielded only one ATPase-containing fraction, co-migrating with ATPase I from wild-type. Use of electrophoretic, immunological and enzymatic methods established a correspondence of the polypeptide subunits of ATPases II and III and those of spinach coupling factor, CF1. ATPase I from either algal strain was shown to be structurally distinct from high plant CF1 and to C. reinhardtii ATPases II and III.     

The p-type ATPase superfamily

P-type ATPases function to provide homeostasis in higher eukaryotes, but they are essentially ubiquitous, being found in all domains of life. Thever and Saier [J Memb Biol 2009;229:115-130] recently reported analyses of eukaryotic P-type ATPases, dividing them into nine functionally characterized and 13 functionally uncharacterized (FUPA) families. In this report, we analyze P-type ATPases in all major prokaryotic phyla for which complete genome sequence data are available, and we compare the results with those for eukaryotic P-type ATPases. Topological type I (heavy metal) P-type ATPases predominate in prokaryotes (approx. tenfold) while type II ATPases (specific for Na(+),K(+), H(+) Ca(2+), Mg(2+) and phospholipids) predominate in eukaryotes (approx. twofold). Many P-type ATPase families are found exclusively in prokaryotes (e.g. Kdp-type K(+) uptake ATPases (type III) and all ten prokaryotic FUPA familes), while others are restricted to eukaryotes (e.g. phospholipid flippases and all 13 eukaryotic FUPA families). Horizontal gene transfer has occurred frequently among bacteria and archaea, which have similar distributions of these enzymes, but rarely between most eukaryotic kingdoms, and even more rarely between eukaryotes and prokaryotes. In some bacterial phyla (e.g. Bacteroidetes, Flavobacteria and Fusobacteria), ATPase gene gain and loss as well as horizontal transfer occurred seldom in contrast to most other bacterial phyla. Some families (i.e. Kdp-type ATPases) underwent far less horizontal gene transfer than other prokaryotic families, possibly due to their multisubunit characteristics. Functional motifs are better conserved across family lines than across organismal lines, and these motifs can be family specific, facilitating functional predictions. In some cases, gene fusion events created P-type ATPases covalently linked to regulatory catalytic enzymes. In one family (FUPA Family 24), a type I ATPase gene (N-terminal) is fused to a type II ATPase gene (C-terminal) with retention of function only for the latter. Several pseudogene-encoded nonfunctional ATPases were identified. Genome minimalization led to preferential loss of P-type ATPase genes. We suggest that in prokaryotes and some unicellular eukaryotes, the primary function of P-type ATPases is protection from extreme environmental stress conditions. The classification of P-type ATPases of unknown function into phylogenetic families provides guides for future molecular biological studies.     

DNA polymerase III from Saccharomyces cerevisiae. II. Inhibitor studies and comparison with DNA polymerases I and II

The newly identified yeast DNA polymerase III was compared to DNA polymerases I and II and the mitochondrial DNA polymerase. Inhibition by aphidicolin (I50) of DNA polymerases I, II, and III was 4, 6, and 0.6 micrograms/ml, respectively. The mitochondrial enzyme was insensitive to the drug. N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate strongly inhibited DNA polymerase I (I50 = 0.3 microM), whereas DNA polymerase III was less sensitive (I50 = 80 microM). Conditions that allowed proteolysis to proceed during the preparation of extracts converted DNA polymerase II from a sensitive form (I50 = 2.4 microM) to a resistant form (I50 = 2 mM). The mitochondrial DNA polymerase is insensitive (I50 greater than 5 mM). With most other inhibitors tested (N-ethylmaleimide, heparin, salt) only small differences were observed between the three nuclear DNA polymerases. Polyclonal antibodies to DNA polymerase III did not inhibit DNA polymerases I and II, nor were those polymerases recognized by Western blotting. Monoclonal antibodies to DNA polymerase I did not crossreact with DNA polymerases II and III. The results show that DNA polymerase III is distinct from DNA polymerase I and II.     

Structural and functional studies of the dnaB protein using limited proteolysis. Characterization of domains for DNA-dependent ATP hydrolysis and for protein association in the primosome

Two separable structural domains were identified in the Escherichia coli dnaB protein (Mr = 52,000) by partial proteolytic cleavage under nondenaturing conditions. The hydrolysis of dnaB protein by trypsin proceeded in two distinct stages in the presence of ATP or ADP. In the first stage, 14 amino acid residues at the NH2-terminal end were removed and dnaB protein was converted into a fragment with a molecular weight of 50,000 (Fragment I). Fragment I retained about 60% of the original activity in priming DNA replication and was fully active in DNA-dependent ATPase activity. In the second stage, Fragment I was further cleaved into two separable polypeptides with molecular weights of 33,000 (Fragment II) and 12,000 (Fragment III), respectively. Fragment II, as a hexamer, retained DNA-dependent ATPase activity comparable to the intact protein but was totally inactive in priming DNA replication. No known activity of dnaB protein was detected in Fragment III alone. NH2 termini of Fragments I and III and COOH termini of dnaB protein and Fragment II were identical indicating that Fragments III and II were located at the NH2 and COOH termini of Fragment I, respectively. These results indicate that dnaB protein is composed of at least two distinct domains. 1) Fragment III, the rigid domain, is essential for protein interaction, i.e. association with dnaC protein and primase in priming DNA replication in the primosome. 2) A 14-amino acid residue fragment, at the NH2-terminal end adjacent to Fragment III, probably required to stabilize the protein interaction involved in priming DNA replication. 3) Fragment II, the flexible COOH-terminal domain, contains the active sites for DNA binding, ATP binding, and protein oligomerization. Fragment II is cleaved by trypsin at many sites in the absence of ATP or ADP ligands. The rate of conversion of Fragment I into the yield of Fragments II and III was decreased approximately by 2 orders of magnitude by changing the ligand from ADP to the nonhydrolyzed ATP analog, adenosine 5'-O-(3-thiotriphosphate). These results indicate that the conformation of the COOH-terminal domain in the dnaB protein is stabilized by ATP or ADP. Such a nucleotide-induced conformational change was also demonstrated by circular dichroism spectroscopy. Moreover, the data suggest that the conformation of the dnaB protein complexed with adenosine 5'-O-(3-thiotriphosphate) is different from that complexed with ADP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Coupling DNA-binding and ATP hydrolysis in Escherichia coli RecQ: role of a highly conserved aromatic-rich sequence

RecQ enzymes are broadly conserved Superfamily-2 (SF-2) DNA helicases that play critical roles in DNA metabolism. RecQ proteins use the energy of ATP hydrolysis to drive DNA unwinding; however, the mechanisms by which RecQ links ATPase activity to DNA-binding/unwinding are unknown. In many Superfamily-1 (SF-1) DNA helicases, helicase sequence motif III links these activities by binding both single-stranded (ss) DNA and ATP. However, the ssDNA-binding aromatic-rich element in motif III present in these enzymes is missing from SF-2 helicases, raising the question of how these enzymes link ATP hydrolysis to DNA-binding/unwinding. We show that Escherichia coli RecQ contains a conserved aromatic-rich loop in its helicase domain between motifs II and III. Although placement of the RecQ aromatic-rich loop is topologically distinct relative to the SF-1 enzymes, both loops map to similar tertiary structural positions. We examined the functions of the E.coli RecQ aromatic-rich loop using RecQ variants with single amino acid substitutions within the segment. Our results indicate that the aromatic-rich loop in RecQ is critical for coupling ATPase and DNA-binding/unwinding activities. Our studies also suggest that RecQ's aromatic-rich loop might couple ATP hydrolysis to DNA-binding in a mechanistically distinct manner from SF-1 helicases.     

Is ATP a substrate for 15-lipoxygenase?

Lipoxygenases catalyze peroxidation of polyunsaturated fatty acids containing the 1-cis, 4-cis pentadiene structure. Linoleic (18:2), linolenic (18:3), and arachidonic (20:4) acids are the predominant substrates for this class of enzymes. Effects of 15-lipoxygenase on the hydrolysis of adenosine 5'-triphosphate were investigated in vitro using soybean lipoxygenase and adenosine 5'-[gamma-32P]triphosphate. The amount of inorganic phosphate released from adenosine 5'-triphosphate was dependent upon enzyme as well as substrate concentrations, pH, and the duration of incubation. The ATPase activity with a Vmax value of 3.3 mumol.mg protein-1.h-1 and a Km value of 5.9 mM was noted in the presence of different concentrations of ATP at pH = 7.4. Phenidone, a lipoxygenase inhibitor, had no effect on this reaction. These findings suggest that soybean lipoxygenase catalyzes the release of inorganic phosphate from ATP primarily via hydrolysis.     

The hyperthermophilic archaeon Pyrodictium occultum has two alpha-like DNA polymerases.

We cloned two genes encoding DNA polymerases from the hyperthermophilic archaeon Pyrodictium occultum. The deduced primary structures of the two gene products have several amino acid sequences which are conserved in the alpha-like (family B) DNA polymerases. Both genes were expressed in Escherichia coli, and highly purified gene products, DNA polymerases I and II (pol I and pol II), were biochemically characterized. Both DNA polymerase activities were heat stable, but only pol II was sensitive to aphidicolin. Both pol I and pol II have associated 5'-->3' and 3'-->5' exonuclease activities. In addition, these DNA polymerases have higher affinity to single-primed single-stranded DNA than to activated DNA; even their primer extension abilities by themselves were very weak. A comparison of the complete amino acid sequences of pol I and pol II with two alpha-like DNA polymerases from yeast cells showed that both pol I and pol II were more similar to yeast DNA polymerase III (ypol III) than to yeast DNA polymerase II (ypol II), in particular in the regions from exo II to exo III and from motif A to motif C. However, comparisons region by region of each polymerase showed that pol I was similar to ypol II and pol II was similar to ypol III from motif C to the C terminus. In contrast, pol I and pol II were similar to ypol III and ypol II, respectively, in the region from exo III to motif A. These findings suggest that both enzymes from P. occultum play a role in the replication of the genomic DNA of this organism and, furthermore, that the study of DNA replication in this thermophilic archaeon may lead to an understanding of the prototypical mechanism of eukaryotic DNA replication.     

Single-stranded DNA binding and methylation by EcoP1I DNA methyltransferase

EcoP1I methyltransferase (M.EcoP1I) belongs to the type III restriction-modification system encoded by prophage P1 that infects Escherichia coli. Binding of M.EcoP1I to double-stranded DNA and single-stranded DNA has been characterized. Binding to both single- and double-stranded DNA could be competed out by unlabeled single-stranded DNA. Metal ions did not influence DNA binding. Interestingly, M.EcoP1I was able to methylate single-stranded DNA. Kinetic parameters were determined for single- and double-stranded DNA methylation. This feature of the enzyme probably functions in protecting the phage genome from restriction by type III restriction enzymes and thus could be considered as an anti-restriction system. This study describing in vitro methylation of single-stranded DNA by the type III methyltransferase EcoP1I allows understanding of the mechanism of action of these enzymes and also their role in the biology of single-stranded phages.     

Restriction enzyme digestion of hemimethylated DNA.

Hemimethylated duplex DNA of the bacteriophage phi X 174 was synthesized using primed repair synthesis is in vitro with E. coli DNA polymerase I followed by ligation to produce the covalently closed circular duplex (RFI). Single-stranded phi X DNA was used as a template, a synthetic oligonucleotide as primer and 5-methyldeoxycytidine-5'-triphosphate (5mdCTP) was used in place of dCTP. The hemimethylated product was used as substrate for cleavage by various restriction enzymes. Out of the 17 enzymes tested, only 5 (BstN I, Taq I, Hinc II, Hinf I and Hpa I) cleaved the hemimethylated DNA. Two enzymes (Msp I and Hae III) were able to produce nicks on the unmethylated strand of the cleavage site. Msp I, which is known to cleave at CCGG when the internal cytosine residue is methylated, does not cleave when both cytosines are methylated. Another enzyme, Apy I, cleaves at the sequence CCTAGG when the internal cytosine is methylated, but is inactive on hemimethylated DNA in which both cytosines are methylated. Hemimethylated molecules should be useful for studying DNA methylation both in vivo and in vitro.     

Motif III in Superfamily 2 “Helicases” Helps Convert the Binding Energy of ATP into a High-Affinity RNA Binding Site in the Yeast DEAD-Box Protein Ded1

Motif III in the putative helicases of superfamily 2 is highly conserved in both its sequence and its structural context. It typically consists of the sequence alcohol-alanine-alcohol (S/T-A-S/T). Historically, it was thought to link ATPase activity with a “helicase” strand displacement activity that disrupts RNA or DNA duplexes. DEAD-box proteins constitute the largest family of superfamily 2; they are RNA-dependent ATPases and ATP-dependent RNA binding proteins that, in some cases, are able to disrupt short RNA duplexes. We made mutations of motif III (S-A-T) in the yeast DEAD-box protein Ded1 and analyzed in vivo phenotypes and in vitro properties. Moreover, we made a tertiary model of Ded1 based on the solved structure of Vasa. We used Ded1 because it has relatively high ATPase and RNA binding activities; it is able to displace moderately stable duplexes at a large excess of substrate. We find that the alanine and the threonine in the second and third positions of motif III are more important than the serine, but that mutations of all three residues have strong phenotypes. We purified the wild-type and various mutants expressed in Escherichia coli. We found that motif III mutations affect the RNA-dependent hydrolysis of ATP (k_cat), but not the affinity for ATP (K_m). Moreover, mutations alter and reduce the affinity for single-stranded RNA and subsequently reduce the ability to disrupt duplexes. We obtained intragenic suppressors of the S-A-C mutant that compensate for the mutation by enhancing the affinity for ATP and RNA. We conclude that motif III and the binding energy of γ-PO₄ of ATP are used to coordinate motifs I, II, and VI and the two RecA-like domains to create a high-affinity single-stranded RNA binding site. It also may help activate the β,γ-phosphoanhydride bond of ATP.     

DNA-dependent adenosinetriphosphatase A is the eukaryotic analogue of the bacteriophage T4 gene 44 protein: immunological identity of DNA replication-associated ATPases.

We report the construction of three stable murine hybridomas that secrete monoclonal antibodies which recognize calf thymus DNA-dependent adenosinetriphosphatase A. All three of the antibodies react specifically with calf thymus ATPase A and the gene 44 protein from the bacteriophage T4 DNA-dependent ATPase. Each of the three anti-ATPase A antibodies appears to recognize a different epitope and none of the antibodies inhibit DNA-dependent ATP hydrolysis by ATPase A. Furthermore, one of the antibodies has been shown to react with two different preparations of HeLa cell DNA-dependent ATPases and a yeast DNA-dependent ATPase, all of which have been implicated in the enzymology of DNA replication. These findings provide strong evidence for the role of ATPase A in DNA replication. These observations lead us to conclude that, apart from the nucleotide binding sites, there are at least three epitopes common to both the bacteriophage and eukaryotic DNA-dependent ATPases that we have examined and that the different preparations of the eukaryotic ATPases contain the same DNA-dependent ATPase.     

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.