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.     

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)     

Minimal kinetic mechanism for misincorporation by DNA polymerase I (Klenow fragment).

The minimal kinetic mechanism for misincorporation of a single nucleotide (dATP) into a short DNA primer/template (9/20-mer) by the Klenow fragment of DNA polymerase I [KF(exo+)] has been previously published [Kuchta, R. D., Benkovic, P., & Benkovic, S.J. (1988) Biochemistry 27, 6716-6725]. In this paper are presented refinements to this mechanism. Pre-steady-state measurements of correct nucleotide incorporation (dTTP) in the presence of a single incorrect nucleotide (dATP) with excess KF-(exo+) demonstrated that dATP binds to the KF(exo+)-9/20-mer complex in two steps preceding chemistry. Substitution of (alpha S)dATP for dATP yielded identical two-step binding kinetics, removing nucleotide binding as a cause of the elemental effect on the rate of misincorporation. Pyrophosphate release from the ternary species [KF'(exo+)-9A/20-mer-PPi] was found to occur following a rate-limiting conformational change, with this species partitioning equally to either nucleotide via internal pyrophosphorolysis or to misincorporated product. The rate of 9A/20-mer dissociation from the central ternary complex (KF'-9A/20-mer-PPi) was shown to be negligible relative to exonucleolytic editing. Pyrophosphorolysis of the misincorporated DNA product (9A/20-mer), in conjunction with measurement of the rate of dATP misincorporation, permitted determination of the overall equilibrium constant for dATP misincorporation and provided a value similar to that measured for correct incorporation. A step by step comparison of the polymerization catalyzed by the Klenow fragment for correct and incorrect nucleotide incorporation emphasizes that the major source of the enzyme's replicative fidelity arises from discrimination in the actual chemical step and from increased exonuclease activity on the ternary misincorporated product complex owing to its slower passage through the turnover sequence.     

Mechanism of DNA replication fidelity for three mutants of DNA polymerase I: Klenow fragment KF(exo+), KF(polA5), and KF(exo-)

Inhibition of the pre-steady-state burst of nucleotide incorporation by a single incorrect nucleotide (nucleotide discrimination) was measured with the Klenow fragment of DNA polymerase I [KF(exo+)]. For the eight mispairs studied on three DNA sequences, only low levels of discrimination ranging from none to 23-fold were found. The kinetics of dNTP incorporation into the 9/20-mer at low nucleotide concentrations was also determined. A limit of greater than or equal to 250 s-1 was placed on the nucleotide off-rate from the KF(exo+)-9/20-dTTP complex in accord with nucleotide binding being at equilibrium in the overall kinetic sequence. The influence of the relatively short length of the 9/20-mer on the mechanism of DNA replication fidelity was determined by remeasuring important kinetic parameters on a 30/M13-mer with high homology to the 9/20-mer. Pre-steady-state data on the nucleotide turnover rates, the dATP(alpha S) elemental effect, and the burst of dAMP misincorporation into the 30/M13-mer demonstrated that the kinetics were not affected by the length of the DNA primer/template. The effects on fidelity of two site-specific mutations, KF(polA5) and KF(exo-), were also examined. KF(polA5) showed an increased rate of DNA dissociation and a decreased rate of polymerization resulting in less processive DNA synthesis. Nevertheless, with at least one misincorporation event, that of dAMP into the 9/20-mer, KF(polA5) displays an increased replication fidelity.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Mechanism of primer-template-dependent conversion of dNTP leads to dNMP by T5 DNA polymerase

T5 DNA polymerase catalyzes both 5' leads to 3' polymerization and 3' leads to 5' hydrolysis in a processive fashion. This knowledge has been utilized to obtain evidence indicating that the enzyme has a single primer-template binding site which can function as either polymerase or exonuclease, perhaps with the cooperation of additional or different side groups. Template-dependent conversion of dNTP leads to dNMP was observed with an excess of either primer-template or enzyme. With primer-template excess, practically all the enzymes were functional as polymerase; with enzyme excess, all primer-templates were extended during the first cycle of catalysis. These observations suggest that turnover takes place at the points of chain growth. Evidence is also provided which demonstrates that the enzyme is capable of switching its direction of catalysis from 3' leads to 5' to 5' leads to 3' without leaving the primer-template. A clear correspondence between the relative amount of hydrolysis of a terminally labeled residue on the primer and the relative amount of turnover suggests that (a) the probability of hydrolysis of a given type of residue in contact with the "active site" is constant, and (b) during each turnover episode enzyme usually takes only one step in the 3' leads to 5' direction. A simple probabilistic model of turnover is discussed.     

Influence of template primary and secondary structure on the rate and fidelity of DNA synthesis

High resolution gel electrophoresis was used to monitor the successive addition of dNMP residues onto the 3'-OH ends of discrete 5'-32P-primers, during DNA synthesis on natural templates. Resulting autoradiographic banding patterns revealed considerable variation in the relative rates of incorporation at different positions along the template. The pattern of "pause sites" along the template was unique for each of three different DNA polymerases (polymerase I (the "large fragment" form of Escherichia coli), T4 polymerase (encoded by bacteriophage T4), and AMV polymerase (DNA polymerase of avian myeloblastosis virus]. Most pause sites were not caused by attenuation of polymerization at regions of local secondary structure in the template. Assays of the accuracy of incorporation at different positions along the template (in which elongation was monitored in the presence of only 3 of the 4 2'-deoxynucleoside 5'-triphosphates) strongly suggested that the relative fidelity of DNA synthesis catalyzed by different polymerases depends on the position on the template at which the comparison is made. Primer-templates were constructed that permitted comparison of elongation during synthesis on a single-stranded template with that during polymerization through a double-stranded region (wherein elongation required concomitant displacement of a strand annealed adjacent to the 5'-32P-primer). Although strand displacement DNA synthesis catalyzed by polymerase I occurred approximately ten times more slowly than synthesis in the same region of a single-stranded viral template, most of the pause sites were the same in the presence or absence of "tandem" primer. Electrophoretic assays of the fidelity of DNA synthesis suggested that an increased tendency toward misincorporational "hotspots" occurred when elongation required concomitant strand displacement.     

Dynamics of termination during in vitro replication of ultraviolet-irradiated DNA with DNA polymerase III holoenzyme of Escherichia coli

During in vitro replication of UV-irradiated single-stranded DNA with Escherichia coli DNA polymerase III holoenzyme termination frequently occurs at pyrimidine photodimers. The termination stage is dynamic and characterized by at least three different events: repeated dissociation-reinitiation cycles of the polymerase at the blocked termini; extensive hydrolysis of ATP to ADP and inorganic phosphate; turnover of dNTPs into dNMP. The reinitiation events are nonproductive and are not followed by further elongation. The turnover of dNTPs into dNMPs is likely to result from repeated cycles of insertion of dNMP residues opposite the blocking lesions followed by their excision by the 3'----5' exonucleolytic activity of the polymerase. Although all dNTPs are turned over, there is a preference for dATP, indicating that DNA polymerase III holoenzyme has a preference for inserting a dAMP residue opposite blocking pyrimidine photodimers. We suggest that the inability of the polymerase to bypass photodimers during termination is due to the formation of defective initiation-like complexes with reduced stability at the blocked termini.     

Evidence for an intermediate in DNA synthesis involving pyrophosphate exchange. A possible role in fidelity

The incorporation of exogenous deoxyribonucleotide monophates (dNMP) was measured under conditions of ongoing DNA synthesis, providing arguments for the existence of a [DNAn X dNMP X PPi] intermediate in the nucleotide incorporation step of DNA synthesis: (formula; see text). The existence of such an intermediate is suggested by an apparent exchange of both dNMP and pyrophosphate (PPi) moieties of the deoxyribonucleotide triphosphate (dNTP) substrate with exogenous molecules. Such exchange and the incorporation of exogenous dNMP into DNA, strictly require ongoing DNA synthesis, suggesting that the energy for exchange reactions is provided by the cleavage of dNTP substrate. We propose that nucleotide selection during ongoing DNA synthesis results largely from the different relative rates of forward (beta) and backward (-alpha) reactions involving the [DNAn X dNMP X PPi] intermediate: the forward (incorporation) reaction is expected to predominate for the correct nucleotide, whereas the backward (abortive) reaction is expected to predominate for incorrect nucleotides.     

Action of 5-trifluoromethyl-2'-deoxyuridine on DNA synthesis.

The action of 5-trifluoromethyl-2'-deoxyuridine (CF3dUrd) on DNA synthesis was investigated in vitro assay systems with purified DNA polymerases. CF3dUrd was incorporated into the DNA of mammalian cells in culture. We studied the incorporation of CF3dUrd 5'-triphosphate (CF3dUTP) into DNA and effect of CF3dUrd residue on DNA synthesis. Therefore, we synthesized oligonucleotides that allow site specific introduction of a CF3dUrd residue into a synthetic DNA oligonucleotide. After CF3dUTP incorporation, the primer was extended for human DNA polymerase alpha (pol. alpha). When CF3dUrd residue was located at an internucleotide site in the template, however, pol. alpha was exhibited a strong arrest band one nucleotide after the CF3dUrd residue site, and Escherichia coli polymerase I (Klenow fragment) also exhibited a weaker arrest band one nucleotide before the CF3dUrd residue. These results suggested that a mechanism of antitumor activity of CF3dUrd is inhibition of DNA replication.     

Synthesis of dihydrothymidine and thymidine glycol 5'-triphosphates and their ability to serve as substrates for Escherichia coli DNA polymerase I

5,6-Dihydrothymidine 5'-triphosphate (DHdTTP) was synthesized by catalytic hydrogenation of thymidine 5'-triphosphate (dTTP). Thymidine glycol 5'-triphosphate (dTTP-GLY) was prepared by bromination of dTTP followed by treatment with Ag2O. The modified nucleotides were extensively purified by anion-exchange high-performance liquid chromatography (HPLC). Alkaline phosphatase digestion of DHdTTP and dTTP-GLY gave the expected products (5,6-dihydrothymidine and cis-thymidine glycol), the identities of which were confirmed by reverse-phase HPLC using authentic markers. HPLC analysis of the alkaline phosphatase digested DHdTTP revealed that DHdTTP was a mixture of C5 diastereoisomers [(5S)- and (5R)-DHdTTP]. Despite the significant distortion of the pyrimidine ring in DHdTTP, it was incorporated in place of dTTP during primer elongation catalyzed by Escherichia coli DNA polymerase I Klenow fragment. The rate of incorporation of DHdTTP was about 10-25-fold lower than that of dTTP. On the other hand, dTTP-GLY, which also has a distorted pyrimidine ring, did not replace dTTP, and no elongation of the primer was observed. In order to study the preference of incorporation of the diastereoisomers of DHdTTP into DNA, salmon testes DNA, activated by exonuclease III, was used as a template for DNA polymerase I Klenow fragment in the presence of [3H]DHdTTP (S and R mixture) and normal nucleotides. After enzymatic digestion of the DNA to nucleosides, the products were analyzed by HPLC. The ratio of the isomers incorporated into DNA (S:R = 73.27) was virtually the same as that of the [3H]DHdTTP substrates (S:R = 79.21).(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential inhibition of DNA polymerases of calf thymus by 9-beta-D-arabinofuranosyladenine-5'-triphosphate.

The effect of 9-beta-D-arabinofuranosyladenine-5'-triphosphate (araATP) on the reactions of DNA polymerases alpha and beta [E.C. 2.7.7.7] purified from calf thymus was examined. The reaction of DNA polymerase alpha was shown to be more sensitive to the inhibition than that of DNA polymerase beta. The K1 value of DNA polymerase beta for araATP was 45 micrometer; 15 times higher than that of DNA polymerase alpha (3 micrometer). The mode of inhibition by araATP was essentially competitive to deoxyadenosine triphosphate (dATP) in the reactions catalyzed by both DNA polymerase alpha and beta using activated DNA as a template-primer. However, in the reactions of the alpha-enzyme, araATP also inhibited the incorporation of deoxyribonucleotides othan than dATP non-competitively.     

Characterization of 3''----5'' exonuclease associated with DNA polymerase of silkworm nuclear polyhedrosis virus.

3'----5' Exonuclease specific for single-stranded DNA copurified with DNA polymerase of nuclear polyhedrosis virus of silkworm Bombyx mori (BmNPV Pol). BmNPV Pol has no detectable 5'----3' exonuclease activity on single-stranded or duplex DNA. Analysis of the products of 3'----5' exonucleolytic reaction showed that deoxynucleoside monophosphates were released during the hydrolysis of single-stranded DNA. The exonuclease activity cosedimented with the polymerase activity during ultracentrifugation of BmNPV Pol in glycerol gradient. The polymerase and the exonuclease activities of BmNPV Pol were inactivated by heat with nearly identical kinetics. The mode of the hydrolysis of single-stranded DNA by BmNPV Pol-associated exonuclease was strictly distributive. The enzyme dissociated from single-stranded DNA after the release of a single dNMP and then reassociated with a next polynucleotide being degradated.     

Inhibitory effects of various 3'-deoxyribonucleotides on DNA polymerase alpha 2-primase from developing cherry salmon (Oncorhynchus masou) testes

DNA polymerase alpha 2-primase has been purified 2750 fold from developing cherry salmon (Oncorhynchus masou) testes by the following purification steps: fractional extraction, phosphocellulose (1st), ammonium sulfate fractionation, DEAE-cellulose, phosphocellulose (2nd), hydroxylapatite and single-stranded DNA-cellulose column chromatographies. Final preparation of this enzyme has a specific activity of 107,000 units/mg protein (activated salmon sperm DNA as template-primer). DNA primase activity (rGTP dependent incorporation of labelled dGMP into poly (dC) or rNTP dependent incorporation of dNMP into M13 single-stranded DNA) was tightly associated with DNA polymerase alpha activity during all stage of this purification process. Inhibition of DNA primase activity by six kinds of 3'-deoxyribonucleotides was studied by using rNTP dependent DNA synthesis on M13 DNA as template. The inhibition constants (Ki) were larger than those of DNA-dependent RNA polymerases I and II. However, Ki/Km values were very close.