Rate-limiting steps in the DNA polymerase I reaction pathway

The initial rates of incorporation of dTTP and thymidine 5'-O-(3-thiotriphosphate) (dTTP alpha S) into poly(dA) X oligo(dT) during template-directed synthesis by the large fragment of DNA polymerase I have been measured by using a rapid-quench technique. The rates were initially equal, indicating a nonrate-limiting chemical step. However, the rate of thionucleotide incorporation steadily diminished to 10% of its initial value as the number of consecutive dTMP alpha S residues in the primer strand increased. This anomalous behavior can be attributed to the helix instability inherent in phosphorothioate-containing duplexes. Positional isotope exchange experiments employing the labeled substrate [alpha-18O2]dATP have revealed negligible alpha, beta-bridging----beta-nonbridging isotope exchange in template-directed reactions of Escherichia coli DNA polymerase I (Pol I) both in the presence and in the absence of added inorganic pyrophosphate (PPi), suggesting rapid PPi release following the chemical step. These observations are consistent with a rate-limiting step that is tentatively assigned to a conformational change of the E X DNA X dNTP complex immediately preceding the chemical step. In addition, the substrate analogue (Sp)-dATP alpha S has been employed to examine the mechanism of the PPi exchange reaction catalyzed by Pol I. The net retention of configuration at the alpha-P is interpreted in terms of two consecutive inversion reactions, namely, 3'-hydroxyl attack, followed by PPi attack on the newly formed primer terminus. Kinetic analysis has revealed that while alpha-phosphorothioate substitution has no effect upon the initial rate of polymerization, it does attenuate the PPi exchange reaction by a factor of 15-18 fold.(ABSTRACT TRUNCATED AT 250 WORDS)     

Non-canonical nucleotide exchange catalyzed by Escherichia coli DNA-polymerase I and the two-center model of the mechanism of action of this enzyme

It is shown, that DNA hydrolysis catalyzed by E. coli DNA polymerase I is inhibited, when a reaction mixture contains one type of deoxynucleoside 5'-triphosphate (dNTP). When the reaction mixture contains [32P]dNTP, then [32P] is incorporated into DNA and v. v. (32P) from DNA is transferred into dNTP. The nucleotide exchange between DNA and dNTP in the assay mixture is observed only in the case, when the chemical nature of nucleotide residue of dNTP and that of the 3'-terminus of DNA is the same. Analysis of products of DNA hydrolysis in the presence of one type of dNTP using electrophoresis in polyacrylamide gel shows that most of the DNA molecules are terminated at the 3'-termini by the dNMP residue of the same chemical nature as the dNTP in the assay mixture. However, in some cases DNA molecules contain one additional nucleotide residue. This phenomenon can be explained by incorporation of one additional dNMP residue originating from dNTP only in those cases, when a non-typical base pairing of this nucleotide residue with a template residue readily takes place. The above-mentioned facts can be interpreted within the model for DNA hydrolysis with involvement of two intermediate covalent forms of dNMP residues with DNA polymerase I; one dNMP-intermediate should be placed at the elongation center and the other--at the hydrolysis center. The DNA hydrolysis by 3'----5' exonuclease activity of DNA polymerase I proceeds through these two covalent forms. DNA polymerases alpha from calf thymus and T4 phage do not catalyze the nucleotide exchange between DNA and dNTP from the reaction media.     

Kinetic mechanism of DNA polymerase I (Klenow fragment): identification of a second conformational change and evaluation of the internal equilibrium constant.

In a previously determined minimal kinetic scheme for DNA polymerization catalyzed by the Klenow fragment (KF) of Escherichia coli DNA polymerase I, a nonchemical step that interconverted the KF'.DNAn+1.PPi and KF.DNAn+1PPi complexes was not observed in correct incorporation [Kuchta, R. D., Mizrahi, V., Benkovic, P.A., Johnson, K.A., & Benkovic, S.J. (1987) Biochemistry 26, 8410-8417] but was detected in misincorporation [Kuchta, R. D., Benkovic, P.A., & Benkovic, S.J. (1988) Biochemistry 27, 6716-6725]. In a pulse-chase experiment in this study, a burst amplitude of 100% of the enzyme concentration is observed; under pulse-quench conditions, the burst amplitude is 80%, indicative of the accumulation of the KF'.DNA.dNTP species owing to a slow step subsequent to chemical bond formation. This latter step was unequivocally identified by single-turnover pyrophosphorolysis and pyrophosphate-exchange experiments as one interconverting KF'.DNAn+1.PPi and KF.DNAn+1.PPi. The rate constants for this step in both directions were established through the rate constants for processive synthesis and pyrophosphorolysis. Pyrophosphorolysis of a 3'-phosphorothioate DNA duplex confirmed that the large elemental effect observed previously [Mizrahi, V., Henrie, R. N., Marlier, J.F., Johnson, K.A., & Benkovic, S.J. (1985) Biochemistry 24, 4010-4018] in this direction but not in polymerization is due to a marked decrease in the affinity of KF for the phosphorothioate-substituted duplex and not to the chemical step. The combination of the experimentally measured equilibrium constant for the bound KF.DNA species with the collective kinetic measurements further extends previous insights into the dynamics of the polymerization process catalyzed by KF.     

Kinetic mechanism of DNA polymerase I (Klenow)

The minimal kinetic scheme for DNA polymerization catalyzed by the Klenow fragment of DNA polymerase I (KF) from Escherichia coli has been determined with short DNA oligomers of defined sequence. A key feature of this scheme is a minimal two-step sequence that interconverts the ternary KF.DNAn.dNTP and KF.DNAn+1.PPi complexes. The rate is not limited by the actual polymerization but by a separate step, possibly important in ensuring fidelity [Mizrahi, V., Henrie, R. N., Marlier, J. F., Johnson, K. A., & Benkovic, S. J. (1985) Biochemistry 24, 4010-4018]. Evidence for this sequence is supplied by the observation of biphasic kinetics in single-turnover pyrophosphorolysis experiments (the microscopic reverse of polymerization). Data analysis then provides an estimate of the internal equilibrium constant. The dissociations of DNA, dNTP, and PPi from the various binary and ternary complexes were measured by partitioning (isotope-trapping) experiments. The rate constant for DNA dissociation from KF is sequence dependent and is rate limiting during nonprocessive DNA synthesis. The combination of single-turnover (both directions) and isotope-trapping experiments provides sufficient information to permit a quantitative evaluation of the kinetic scheme for specific DNA sequences.     

DNA polymerase I, Klenow fragment of Escherichia coli: hydrolysis and pyrophosphorolysis of DNA containing phosphothioate groups

Reaction of DNA synthesis catalyzed by DNA polymerase I KF in the presence of 2'-deoxynucleoside 5'-alpha-thiotriphosphates (dNTP alpha S) was investigated. DNA with thiophosphate groups (DNA[P=S]) obtained by such a way was studied in reactions of hydrolysis and pyrophosphorolysis catalyzed by DNA polymerase I KF. It is shown that the rate of DNA elongation is decreased both on the step of incorporation of dNMP alpha S residues and on the step of incorporation of the next dNMP residue. The rate of pyrophosphorolysis of 3'-terminal dNMP alpha S was demonstrated to be one order of magnitude less in comparison with the corresponding reaction with the natural dNMP residue. Contrary, the rate of 3'----5'-exonuclease hydrolysis of both DNA[P=S] and DNA of the same structure revealed no distinguishable differences.     

Effectiveness of complex-formation of nucleotides with human DNA polymerase alpha from data of enzyme modification by reactive nucleotide analogs

The modification of the human placenta DNA polymerase alpha by the imidazolides of dNMP was investigated. The modification was shown to occur only in the simultaneous presence of the template and the primer. This process, however, doesn't depend on the complementary interaction of the nucleotide base with the template. The Kd values of the complexes between the different nucleotides and DNA polymerase alpha were estimated. The affinity of Im-dTMP was determined from the dependence of the Kapp of the enzyme inactivation rate on the reagent concentration. The Kd values for dNMP, dNDP, dNTP were estimated using the protective effect of these nucleotides under the enzyme modification by Im-dTMP. The comparison of the interaction efficiency between the polymerase and dNMP, dNDP, dNTP (complementary or non-complementary to the template) allow to conclude that the nucleotide discrimination occurs on the dNTP level, i. e. dNMP and dNDP upon forming the complex with the enzyme, don't interact complementarily with the template. The additional contacts between the enzyme and the nucleotide terminal phosphate were supposed to form only for the complementary dNTP. The studies allowed to put forward a hypothetical model of the template complementary dNTP binding to the polymerases. The role of the hydrophobic interaction of the nucleotides with the enzyme as well as the possible influence of the nucleotide gamma-phosphate group on the template--dNTP complement formation. The Watson-Crick bound formation of the nucleotide with the template was supposed to be followed by the additional conformational rearrangement of the nucleotide triphosphate chain. The latter process leads to the formation of additional contacts between the enzyme and the nucleotide gamma-phosphate.     

The activation of ribonucleotide reductase in animal organs as the cellular response against the treatment with DNA-damaging factors and the influence of radioprotectors on this effect

Cellular requirements for deoxyribonucleotide (dNTP) pools during DNA synthesis are related to ensuring of the accuracy of DNA copying during replication and repair. This paper covers some problems on the reactions of dNTP synthesis system in organs of animals against the treatment with DNA-damaging agents. Ribonucleoside diphosphate reductase (NDPR) is the key enzyme for the synthesis of dNTP, since it catalyses the reductive conversion of ribonucleotides to deoxyribonucleotides. The results obtained show that the rapid and transient increase in NDPR activity in animal organs occurs as cellular response against the treatment with DNA-damaging agents (SOS-type activation). We have also found the intensive radioprotector-stimulated activation of deoxyribonucleotide synthesis as well as DNA and protein synthesis in mice organs within 3 days after the administration of two radioprotectors, indralin and indometaphen, that provide the high animal survival. Our studies suggest that these effects are the most important steps in the protective mechanism of the radioprotectors and are responsible for the high animal survival.     

Pathways of pyrimidine deoxyribonucleotide biosynthesis in Mycoplasma mycoides subsp. mycoides.

By measuring the specific activity of deoxyribonucleotides isolated from DNA after the incorporation of 14C-labeled precursors with and without competition from other nucleotide precursors, we defined the major pathways of pyrimidine deoxyribonucleotide synthesis in Mycoplasma mycoides subsp. mycoides. Uracil, guanine, and thymine are required for the synthesis of nucleotides. Cytidine competed effectively with uracil to provide all of the deoxycytidine nucleotide, as well as most of the deoxyribose-1-phosphate, for the synthesis of thymidylate from thymine via thymidine phosphorylase. Each of dUMP, dCMP, and dTMP competed with cytidine for incorporation into DNA thymidylate. Appreciable incorporation of exogenous deoxyribonucleoside 5'-monophosphates into DNA without prior dephosphorylation was observed. Dephosphorylation also occurred since the added deoxyribonucleotide provided phosphate for the synthesis of the other nucleotides in DNA in competition with the 32Pi in the growth medium. Hydroxyurea inhibited cell growth and decreased the intracellular level of dATP, consistent with the action of a ribonucleoside diphosphate reductase with regulatory properties similar to those of the Escherichia coli enzyme.     

In vitro DNA replication implicates O2-ethyldeoxythymidine in transversion mutagenesis by ethylating agents.

A 36-nucleotide oligomer containing a single O2-ethyldeoxythymidine (O2-Et-dT) adduct at a specific site was synthesized. The oligomer, which corresponds to a specific DNA sequence in gene G of bacteriophage phi X174, was used as a template by T7 DNA polymerase to investigate the in vitro mutagenic specificity of O2-Et-dT. At 10 microM dNTP and 5 mM Mg++, the progress of T7 DNA polymerase was interrupted by O2-Et-dT: 80% 3' to O2-Et-dT and 14% after incorporating a nucleotide opposite O2-Et-dT (incorporation-dependent blocked product). DNA synthesis past the lesion was low (6%). Incorporation of a nucleotide opposite O2-Et-dT and subsequent postlesion synthesis were enhanced by increasing the dNTP concentration, with postlesion synthesis reaching 30% at 200 microM. Postlesion synthesis was further increased to 45% by addition of 10 mM dAMP to the polymerization reactions. DNA sequencing revealed that both dA and dT were incorporated opposite O2-Et-dT with dA incorporation impeding the progress of DNA synthesis. dT incorporation was efficiently extended implicating O2-Et-dT in transversion mutagenesis in vivo. These studies provide a basis for understanding the molecular mechanisms by which ethylating agents contribute to cytotoxicity, A.T transversion mutagenesis and activation of the oncogene neu by an A.T----T.A transversion event in rat neuroblastomas.     

Altered order of substrate binding by DNA polymerase X from African Swine Fever virus

A sequential ordered substrate binding established previously for several DNA polymerases is generally extended to all DNA polymerases, and the characterization of novel polymerases is often based on the assumption that the enzymes should productively bind DNA substrate first, followed by template-directed dNTP binding. The comprehensive kinetic study of DNA polymerase X (Pol X) from African swine fever virus reported here is the first analysis of the substrate binding order performed for a low-fidelity DNA polymerase. A classical steady-state kinetic approach using substrate analogue inhibition assays demonstrates that Pol X does not follow the bi-bi ordered mechanism established for other DNA polymerases. Further, using isotope-trapping experiments and stopped-flow fluorescence assays, we show that Pol X can bind Mg (2+).dNTPs in a productive manner in the absence of DNA substrate. We also show that DNA binding to Pol X, although rapid, may not always be productive. Furthermore, we show that binding of Mg (2+).dNTP to Pol X facilitates subsequent formation of the catalytically competent Pol X.DNA.dNTP ternary complex, whereas DNA binding prior to dNTP binding brings the enzyme into a nonproductive conformation where subsequent nucleotide substrate binding is hindered. Together, our results suggest that Pol X prefers an ordered sequential mechanism with Mg (2+).dNTP as the first substrate.     

Comparative kinetics of nucleotide analog incorporation by vent DNA polymerase

Comparative kinetic and structural analyses of a variety of polymerases have revealed both common and divergent elements of nucleotide discrimination. Although the parameters for dNTP incorporation by the hyperthermophilic archaeal Family B Vent DNA polymerase are similar to those previously derived for Family A and B DNA polymerases, parameters for analog incorporation reveal alternative strategies for discrimination by this enzyme. Discrimination against ribonucleotides was characterized by a decrease in the affinity of NTP binding and a lower rate of phosphoryl transfer, whereas discrimination against ddNTPs was almost exclusively due to a slower rate of phosphodiester bond formation. Unlike Family A DNA polymerases, incorporation of 9-[(2-hydroxyethoxy)methyl]X triphosphates (where X is adenine, cytosine, guanine, or thymine; acyNTPs) by Vent DNA polymerase was enhanced over ddNTPs via a 50-fold increase in phosphoryl transfer rate. Furthermore, a mutant with increased propensity for nucleotide analog incorporation (Vent(A488L) DNA polymerase) had unaltered dNTP incorporation while displaying enhanced nucleotide analog binding affinity and rates of phosphoryl transfer. Based on kinetic data and available structural information from other DNA polymerases, we propose active site models for dNTP, ddNTP, and acyNTP selection by hyperthermophilic archaeal DNA polymerases to rationalize structural and functional differences between polymerases.     

Hydrolytic approach for production of deoxyribonucleoside-and ribonucleoside-5′-monophosphates and enzymatic synthesis of their polyphosphates

A new, simple, and ingenious method for enzymatic synthesis of deoxy- and ribonucleoside-5 -triphosphates (dNTP and NTP, respectively) has been developed. The method includes the following stages: hydrolysis of DNA with DNase and immobilized S1-nuclease, phosphorylation of the resulting deoxy- and ribonucleoside-5 -monophosphates (dNMP and NMP, respectively) with nucleotidyl kinase from Escherichia coli, and purification by chromatography of the synthesized dNTP and NTP. dNMP was phosphorylated using an ATP-regenerating system based on acetokinase from E. coli and lithium acetylphosphate.     

Quantification of pyrophosphate as a universal approach to determine polymerase activity and assay polymerase inhibitors

The importance of DNA polymerases in biology and biotechnology, and their recognition as potential therapeutic targets, drives development of methods for deriving kinetic characteristics of polymerases and their propensity to perform polynucleotide synthesis over modified DNA templates. Among various polymerases, translesion synthesis (TLS) polymerases enable cells to avoid the cytotoxic stalling of replicative DNA polymerases at chemotherapy-induced DNA lesions, thereby leading to drug resistance. Identification of TLS inhibitors to overcome drug-resistance necessitates the development of appropriate high-throughput assays. Since polymerase-mediated DNA synthesis involves the release of inorganic pyrophosphate (PPi), we established a universal and fast method for monitoring the progress of DNA polymerases based on the quantification of PPi with a fluorescence-based assay that we coupled to in vitro primer extension reactions. The established assay has a nanomolar detection limit in PPi and enables the evaluation of single nucleotide incorporation and DNA synthesis progression kinetics. The results demonstrated that the developed assay is a reliable method for monitoring TLS and identifying nucleoside and nucleotide-based TLS inhibitors.     

The catalytic cycle for ribonucleotide incorporation by human DNA Pol λ

Although most DNA polymerases discriminate against ribonucleotide triphosphaets (rNTPs) during DNA synthesis, recent studies have shown that large numbers of ribonucleotides are incorporated into the eukaryotic nuclear genome. Here, we investigate how a DNA polymerase can stably incorporate an rNTP. The X-ray crystal structure of a variant of human DNA polymerase λ reveals that the rNTP occupies the nucleotide binding pocket without distortion of the active site, despite an unfavorable interaction between the 2'-O and Tyr505 backbone carbonyl. This indicates an energetically unstable binding state for the rNTP, stabilized by additional protein-nucleotide interactions. Supporting this idea is the 200-fold lower catalytic efficiency for rNTP relative to deoxyribonucleotide triphosphate (dNTP) incorporation, reflecting a higher apparent Km value for the rNTP. Furthermore, distortion observed in the structure of the post-catalytic product complex suggests that once the bond between the α- and β-phosphates of the rNTP is broken, the unfavorable binding state of the ribonucleotide cannot be maintained. Finally, structural and biochemical evaluation of dNTP insertion onto an ribonucleotide monophosphate (rNMP)-terminated primer indicates that a primer-terminal rNMP does not impede extension. The results are relevant to how ribonucleotides are incorporated into DNA in vivo, during replication and during repair, perhaps especially in non-proliferating cells when rNTP:dNTP ratios are high.     

In vitro mispairing specificity of O2-ethylthymidine.

The O2-position of thymine is a major site of base alkylation by N-nitroso-alkylating agents, and its biological relevance remains obscure. The potential significance of this DNA damage was ascertained by studying in vitro DNA replication properties of O2-ethylthymidine (O2-Et-dT) site-specifically incorporated into a 36-nucleotide template. DNA replication was initiated eight nucleotides away from the O2-Et-dT lesion by Escherichia coli polymerase I (Klenow fragment) using a 17-nucleotide primer. In the presence of 10 microM dNTP and Mg2+, O2-Et-dT blocked DNA replication predominantly (94%) 3' to O2-Et-dT, with the remainder (5%) blocked after incorporation of a nucleotide opposite O2-Et-dT (incorporation-dependent blocked product). Postlesion synthesis was negligible (less than 1%). Nucleotide incorporation opposite O2-Et-dT increased to 23% at 200 microM dNTP. Postlesion synthesis remained negligible (less than 2%). DNA sequencing revealed dA present opposite O2-Et-dT in the incorporation-dependent blocked product. Negligible postlesion synthesis suggests that incorporation of dA opposite O2-Et-dT inhibits in vitro DNA synthesis. The O2-Et-dT.dA base pair may also impede DNA synthesis in vivo, contributing to the cytotoxicity of the ethylating agents. Substitution of Mn2+ for Mg2+ enhanced nucleotide incorporation opposite O2-Et-dT and produced postlesion synthesis (16%) at 10 microM dNTP, which increased to 39% at 200 microM dNTP. DNA sequence analysis showed that while dA was present opposite O2-Et-dT in the incorporation-dependent blocked product, both dA and dT were present opposite this lesion in the postlesion synthesis product.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reactions of deoxyribonucleotide synthesis system to irradiation and their modification by radioprotectors

The paper covers the problem on reactions of deoxyribonucleotide (dNTP) synthesis system in blood-forming organs of animals induced by irradiation. The synthesis of dNTP is a rate-limiting stage for DNA synthesis. Cellular requirements for dNTP pools during DNA synthesis are related with ensuring of the accuracy of DNA copying during replication and repair. It has been shown that organism defence mechanisms against irradiation include the following stages: 1. The prompt SOS-activation of dNTP synthesis 30 min later after irradiation, playing the important role in protecting of cell's genetic apparatus from damage. 2. The inhibition of dNTP synthesis within 3-24 h after irradiation resulting to the imbalance of four dNTP and the decrease of their pools. As result of that, the abnormal repair is observed due to depurinations, errors of base incorporations and "misrepair". 3. The restore of dNTP synthesis occurred 2 days later after irradiation. The increase of dNTP pools promotes the increase of DNA synthesis rate as well as proliferative activity of cells. Confirming the fact that the alterations in dNTP pools play essential role in the production of DNA lesions became an important step in understanding of the multistage process leading to radioprotection. To get high and balanced pools of dNTP needed for the increase in the volume of repair of DNA lesions the radioprotectors with high efficiency relative to the survival test were used in experiments. They induced the elevated dNTP synthesis in bone marrow and spleen during the time when the irradiation alone caused the essential prolonged suppression of dNTP synthesis as well as DNA and protein synthesis in organs of nonprotected animals. It has been shown that substances with antioxidant and antiradical activity induced the dNTP synthesis, too. In vivo regulatory factors of dNTP synthesis have been studied to elucidate the mechanisms of getting of high and balanced dNTP pools by using of different substances.     

Protein-nucleic acid interaction in reactions catalyzed with DNA polymerases

The affinities of oligothymidylates and of some analogs for the template site, of a set of oligodeoxyribo- and oligoribonucleotides for the primer site, and of dNTPs and some analogs for the substrate sites of DNA polymerase I Klenow fragment and of human placenta DNA polymerase alpha were measured using them either as competitors of affinity modification or as substrates. The data obtained enable us to hypothesize that the Me2+-dependent electrostatic contact and hydrogen bond of a single internucleotide phosphate and the hydrophobic interactions of the other nucleotide units determine the formation of oligonucleotide-template site complexes. Interaction of the primer's 3'-terminal hydroxy group and of the negatively charged adjacent phosphate with the enzyme, and Watson-Crick base pairing with the template are of crucial importance for the formation of the ternary enzyme-template-primer complex. dNTP and dNMP imidazolides inactivate enzymes via an affinity modification mechanism only in the presence of the template-primer complex. dNTP affinities exceed those of dNDPs and dNMPs, the enhancement being most significant for the substrate that is complementary to the template, thus suggesting the participation of the gamma-phosphate of dNTP in the substrate selection step.