Mechanistic studies on deoxyribonucleic acid dependent ribonucleic acid polymerase from Escherichia coli using phosphorothioate analogues. 1. Initiation and pyrophosphate exchange reactions.

The diastereomers of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S) and adenosine 5'-O-(2-thiotriphosphate) (ATP beta S) can replace adenosine triphosphate (ATP) in the initiation reaction catalyzed by deoxyribonucleic acid (DNA) dependent ribonucleic acid (RNA) polymerase from Escherichia coli. In both cases, the Sp diastereomer is a better initiator than the Rp isomer. The diasteromers of 3'-uridyl 5'-adenosyl ,O-phosphorothioate [Up(S)A] can replace UpA in the primed initiation reaction catalyzed by RNA polymerase; however, the Rp diastereomer is a better initiator than the Sp isomer. By using ATP or CpA as initiator and UTP alpha S, isomer A, as substrate, we determined the stereochemical courses of both the initiation and primed initiation reactions, respectively, with T7 DNA template and found them to proceed with inversion of configuration. Determination of the stereochemical course of the pyrophosphate exchange reaction catalyzed by RNA polymerase provides evidence that this reaction is the reverse of the phosphodiester bond-forming reaction.     

Apparent stimulation of calf thymus DNA polymerase alpha by ATP.

The mechanism by which millimolar concentrations of ATP stimulate the activity and increase the processivity of calf thymus DNA polymerase alpha has been investigated with poly(dA)/oligo(dT) as template/primer to eliminate possible effects due to primer synthesis. The effect of ATP on the rate of DNA synthesis with this template/primer was found to be dependent upon whether or not the ATP was neutralized and the species of buffer used in the reaction. The present studies suggest that ATP stimulation of calf thymus DNA polymerase can be attributed to changes in the pH of the reaction mixture, a shift in the magnesium ion optimum, or both. Furthermore, effects of ATP on the processivity of DNA polymerase alpha could be mimicked by lowering the pH of the reaction mixture.     

A simple and sensitive ribonucleotide reductase assay

Ribonucleotide reductase (RR) is a key regulatory enzyme in the DNA synthesis pathway and is the target of the cancer chemotherapeutic agent hydroxyurea. The study of RR is significantly hindered by the tedious and labor-intensive nature of enzymatic assay. In this report, we present a novel RR assay in which detection of the deoxyribonucleotides produced by RR occurs via coupling to the DNA polymerase reaction, and is enhanced by using RNase to degrade endogenous RNA. Cell extracts from various cell lines were treated with RNase and then reacted with ATP and radioactive ribonucleotide diphosphate as the substrate. Incorporation of the radioactive substrate [¹⁴C]CDP into DNA was linear over 30 min and was linear with the amount of extract, which provided RR activity. The reaction was inhibited by hydroxyurea and required Mg²⁺ and ATP, suggesting that the assay is specific to RR activity. While RR activities determined by our method and by a conventional method were comparable, this novel method proved to be simpler, faster, more sensitive and less expensive. In addition, assay of the RR activity for multiple samples can easily be performed simultaneously. It is superior to other RR assays in all aspects.     

Characterization of 2'(3')-trinitrophenyl-ATP as an inhibitor of ATP-dependent initiation complex formation between the DNA polymerase III holoenzyme and primed DNA

We have identified 2'(3')-trinitrophenyl-ATP to be an inhibitor of the ATP-dependent initiation complex formation reaction between the Escherichia coli DNA polymerase III holoenzyme and primed DNA. The inhibitor is specific for the initiation stage; once initiation complexes are formed the subsequent elongation reaction is unaffected. Three ATP-dependent DNA polymerase III holoenzyme reactions can be independently assayed: the ATP-dependent formation of initiation complexes, ATP binding, and the primed DNA-dependent hydrolysis of ATP. Trinitrophenyl ATP inhibits all three reactions to a similar extent with an apparent Ki between 6 and 15 microM in the presence of 5 microM ATP. This suggests all of these reactions are related and that they proceed through a common ATP-binding site. We include an improved purification of the DNA polymerase III holoenzyme in this report.     

Assay of RNA-linked nascent DNA pieces with polynucleotide kinase.

The 5′-OH end of DNA created upon alkaline hydrolysis of the RNA-linked nascent DNA pieces can be labeled with [γ-³²P]ATP using T4 polynucleotide kinase. However, it is difficult to use this method for the assay of these molecules in the presence of RNA-free DNA pieces because of the exchange reaction between the γ-phosphate of ATP and the 5′-phosphate of DNA catalyzed by the kinase. This difficulty can be circumvented by performing the polynucleotide kinase reaction at 0°C, where little exchange reaction occurs. Using these conditions, $\text{E. coli pol}$Aexl, a mutant defective in the 5′ → 3′ exonuclease activity of DNA polymerase I, is shown to contain several times as many RNA-linked DNA pieces as the wild type.     

Division of labor--sequential ATP hydrolysis drives assembly of a DNA polymerase sliding clamp around DNA.

The beta sliding clamp encircles DNA and enables processive replication of the Escherichia coli genome by DNA polymerase III holoenzyme. The clamp loader, gamma complex, assembles beta around DNA in an ATP-fueled reaction. Previous studies have shown that gamma complex opens the beta ring and also interacts with DNA on binding ATP. Here, a rapid kinetic analysis demonstrates that gamma complex hydrolyzes two ATP molecules sequentially when placing beta around DNA. The first ATP is hydrolyzed fast, at 25-30 s(-1), while the second ATP hydrolysis is limited to the steady-state rate of 2 s(-1). This step-wise reaction depends on both primed DNA and beta. DNA alone promotes rapid hydrolysis of two ATP molecules, while beta alone permits hydrolysis of only one ATP. These results suggest that beta inserts a slow step between the two ATP hydrolysis events in clamp assembly, during which the clamp loader may perform work on the clamp. Moreover, one ATP hydrolysis is sufficient for release of beta from the gamma complex. This implies that DNA-dependent hydrolysis of the other ATP is coupled to a separate function, perhaps involving work on DNA. A model is presented in which sequential ATP hydrolysis drives distinct events in the clamp-assembly pathway. We also discuss underlying principles of this step-wise mechanism that may apply to the workings of other ATP-fueled biological machines.     

ATP and the processivity of Xenopus laevis DNA polymerase α

We use specific restriction fragments as defined primers for DNA synthesis on single-stranded circular phage fd DNA. These structures are relatively poor templates for a highly purified DNA polymerase α from Xenopus laevis eggs. However, DNA synthesis is stimulated about 5-fold by addition of ATP to the reaction mixture. We show that the deoxynucleotide polymers, synthesized in the presence of ATP, are significantly longer than those produced in the absence of ATP. We also show that this effect is due to a more tenacious binding of DNA polymerase α to DNA and conclude that ATP increases the processivity of the enzyme.     

Determination of DNA polymerase and nuclease activities of DNA-dependent polymerases using fluorescence detection under real-time conditions

A new method is proposed for estimation of polymerase activities using fluorescence detection during isothermal reaction. The method allows simultaneous determination of DNA-dependent DNA polymerase and 5'-3'-exonuclease activities using amplifiers supplied with an optical module for fluorescence detection under real-time conditions. Different primer-template combinations used as polymerase substrates were compared. Primer elongation (polymerase reaction) is detected by changes in SYBR Green I fluorescence upon binding to dsDNA during reaction; nuclease activities are detected by changes in fluorescence due to cleavage of the probe, containing the reporter fluorophore and fluorescence quencher, and hybridized in advance to the template single-stranded region. It was also shown that the method can be used for determination of relative activities of DNA polymerase preparations, estimation of temperature-time dissociation parameters of polymerase complexes with specific antibodies to its active center, and analysis of effects of inhibitors and activators of different nature on reaction rates of dsDNA polymerization and 5'-3'-exonuclease cleavage by polymerase. The method can be also used for estimation of endonuclease activities of DNA polymerases.     

Characterization of a DNA primase from rat liver mitochondria

A DNA primase was partially purified from rat liver mitochondria and separated from the bulk of DNA polymerase gamma and mtRNA polymerase by heparin-agarose chromatography. The primase was distinguished from mtRNA polymerase by its response to pH, monoand divalent cations, and ATP concentrations. In the absence of an active DNA polymerase and using poly(dT) as template, primase synthesized mixed polynucleotide products consisting of units of oligo(A) 1-12 alternating with units of oligo(dA)25-40. Contributions to these products by contaminating DNA polymerase gamma were eliminated by the addition of dideoxy-ATP. Addition of 50 microM dATP to the primase reaction caused a 50% inhibition of AMP incorporation as compared to reactions containing low levels of dATP present only as a contaminant of the ATP added. The inhibition was due primarily to a reduction of new chain initiations. The dATP did not "lock" the primase reaction into the DNA mode of synthesis since the proportion of internal and 3'-terminal RNA segments was little affected. However, the addition of both 50 microM dATP and exogenous DNA polymerase to the primase reaction greatly reduced the amount of internal and 3'-terminal RNA segments, presumably due to the displacement of primase by DNA polymerase. Our data are consistent with the hypothesis (Hu, S.-Z., Wang, T.S.-F., and Korn, D. (1984) J. Biol. Chem. 259, 2602-2609) that the physiologically significant primer is a mixed 5'-oligoribonucleotide-3'-oligodeoxyribonucleotide and that the formation of the RNA to DNA junction is inherently a primase function.     

A novel MALDI-TOF based methodology for genotyping single nucleotide polymorphisms

A new MALDI-TOF based detection assay was developed for analysis of single nucleotide polymorphisms (SNPs). It is a significant modification on the classic three-step minisequencing method, which includes a polymerase chain reaction (PCR), removal of excess nucleotides and primers, followed by primer extension in the presence of dideoxynucleotides using modified thermostable DNA polymerase. The key feature of this novel assay is reliance upon deoxynucleotide mixes, lacking one of the nucleotides at the polymorphic position. During primer extension in the presence of depleted nucleotide mixes, standard thermostable DNA polymerases dissociate from the template at positions requiring a depleted nucleotide; this principal was harnessed to create a genotyping assay. The assay design requires a primer- extension primer having its 3'-end one nucleotide upstream from the interrogated site. The assay further utilizes the same DNA polymerase in both PCR and the primer extension step. This not only simplifies the assay but also greatly reduces the cost per genotype compared to minisequencing methodology. We demonstrate accurate genotyping using this methodology for two SNPs run in both singleplex and duplex reactions. We term this assay nucleotide depletion genotyping (NUDGE). Nucleotide depletion genotyping could be extended to other genotyping assays based on primer extension such as detection by gel or capillary electrophoresis.     

The ATP dependence of the incision and resynthesis steps of excision repair.

Escherichia coli made permeable by treatment with toluene can perform a mode of DNA synthesis that is stimulated by ultraviolet radiation and closely resembles the resynthesis step of excision repair. If ultraviolet-irradiated toulene-treated cells are incubated in an assay mixture with ATP but without the four deoxyribonucleoside triphosphates (dNTPs) or NAD, accumulations of single-strand breaks in the DNA are detected by alkaline sucrose gradient analysis. A second incubation with the dNTP'S and NAD but without ATP produces nonconservative DNA synthesis in strains with normal levels of DNA polymerase I. However, in PolA strains, ATP must be present during the second incubation in order to produce measurable amounts of ultraviolet-stimulated DNA synthesis. These results suggest that in strains deficient in DNA polymerase I there may be two ATP-dependent steps in this repair pathway, one required for incision and one associated with resynthesis.     

Oligonucleotide synthesis by Escherichia coli dnaG primase in conjunction with phage P22 gene 12 protein

The phage P22 gene 12 protein was found to be like the Escherichia coli dnaB protein in that it stimulated phiX174 DNA synthesis in heat-inactivated extracts of dnaB temperature-sensitive cells (see preceding paper, Wickner, S. (1984) J. Biol. Chem. 259, 14038-14043). phiX174 replication catalyzed by the purified P22 12 protein also by-passed the normal requirement for dnaC protein. However, synthesis still required dnaG primase and the DNA polymerase III holoenzyme components. This DNA synthesis reaction has been reconstituted with purified proteins and found to require P22 12 protein, dnaG protein, DNA polymerase III holoenzyme components, 4 dNTPs, Mg2+, any one of ATP, GTP, UTP, or CTP and single-stranded DNA. The reaction has been dissected into partial reactions: (a) in a prepriming reaction, P22 12 protein binds to single-stranded DNA in an ATP-dependent reaction (Wickner, S. (1984) J. Biol. Chem. 259, 14038-14043); (b) in a priming reaction requiring at least one rNTP and the other dNTPs or rNTPs, dnaG primase catalyzes oligonucleotide synthesis dependent on the P22 12 protein-DNA complex; (c) finally, DNA polymerase III holoenzyme components catalyze DNA elongation of the primer.     

Proofreading activity of DNA polymerase III responds like elongation activity to auxiliary subunits

A comparison of the 3'----5' proofreading properties between Escherichia coli DNA polymerase III holoenzyme and DNA polymerase III' was conducted. This study indicated that the influence of the holoenzyme auxiliary subunits on the proofreading exonuclease parallels their effect on the elongation reaction. At physiological ionic strengths the auxiliary subunits markedly stimulated the exonuclease rate in an ATP-dependent reaction, while the exonuclease rate of DNA polymerase III' was not affected by ATP. E. coli single-stranded DNA binding protein stimulated the 3'----5' exonuclease activity of holoenzyme and inhibited DNA polymerase III'. Similarly, the auxiliary subunits and ATP converted the proofreading activity to a highly processive exonuclease. Our observation, that the exonuclease activity of the DNA polymerase III holoenzyme responded to ATP, salt, and E. coli single-stranded DNA-binding protein like the elongation activity, is consistent with the polymerase and exonuclease subunits acting within the same complex in a coordinated reaction.     

Exploring whole genome amplification as a DNA recovery tool for molecular genetic studies.

The whole genome amplification (WGA) protocol evaluated during this study, GenomiPhi DNA amplification kit, is a novel method that is not based on polymerase chain reaction but rather relies on the highly processive and high fidelity Phi29 DNA polymerase to replicate linear genomic DNA by multiple strand displacement amplification. As little as 1 ng of genomic DNA template is sufficient to produce microgram quantities of high molecular weight DNA. The question explored during this study is whether such a WGA method is appropriate to reliably replenish and even recover depleted DNA samples that can be used for downstream genetic analysis. A series of human DNA samples was tested in our laboratory and validated using such analytical methods as gene-specific polymerase chain reaction, direct sequencing, microsatellite marker analysis, and single nucleotide polymorphism allelic discrimination using TaqMan and Pyrosequencing chemistries. Although degraded genomic DNA is not a good template for Phi29 WGA, this method is a powerful tool to replenish depleted DNA stocks and to increase the amount of sample for which biological tissue availability is scarce. The testing performed during the validation phase of the study indicates no discernable difference between WGA samples and the original DNA templates. Thus, GenomiPhi WGA can be used to increase precious or depleted DNA stocks, thereby extending the life of a family-based linkage analysis project and increasing statistical power.     

Analysis of the ATPase subassembly which initiates processive DNA synthesis by DNA polymerase III holoenzyme.

The gamma complex (gamma delta delta' chi psi) subassembly of DNA polymerase III holoenzyme transfers the beta subunit onto primed DNA in a reaction which requires ATP hydrolysis. Once on DNA, beta is a "sliding clamp" which tethers the polymerase to DNA for highly processive synthesis. We have examined beta and the gamma complex to identify which subunit(s) hydrolyzes ATP. We find the gamma complex is a DNA dependent ATPase. The beta subunit, which lacks ATPase activity, enhances the gamma complex ATPase when primed DNA is used as an effector. Hence, the gamma complex recognizes DNA and couples ATP hydrolysis to clamp beta onto primed DNA. Study of gamma complex subunits showed no single subunit contained significant ATPase activity. However, the heterodimers, gamma delta and gamma delta', were both DNA-dependent ATPases. Only the gamma delta ATPase was stimulated by beta and was functional in transferring the beta from solution to primed DNA. Similarity in ATPase activity of DNA polymerase III holoenzyme accessory proteins to accessory proteins of phage T4 DNA polymerase and mammalian DNA polymerase delta suggests the basic strategy of chromosome duplication has been conserved throughout evolution.     

The adenovirus DNA binding protein and adenovirus DNA polymerase interact to catalyze elongation of primed DNA templates

The adenovirus-encoded 140-kDa DNA polymerase (Ad Pol) and the 59-kDa DNA binding protein (Ad DBP) are both required for the replication of viral DNA in vivo and in vitro. Previous studies demonstrated that, when poly(dT).oligo(dA) was used as a template-primer, both proteins were required for poly(dA) synthesis. In this report, the interaction between the Ad Pol and Ad DBP was further investigated using poly(dT).oligo(dA) as well as a linear duplex molecule containing 3' poly(dT) tails. DNA synthesis with the tailed template required Ad Pol, Ad DBP, and an oligo(dA) primer hydrogen bonded to the poly(dT) tails. Incorporation was stimulated 8-10-fold by ATP; however, no evidence of ATP hydrolysis to ADP was observed. Synthesis was initiated at either end of the tailed molecule and proceeded through the duplex region to the end of the molecule. This ability to translocate through duplex DNA and to synthesize long poly(dA) chains suggests that the Ad Pol.Ad DBP complex can act efficiently in the elongation reactions involved in the replication of Ad DNA (both type I and type II). During the replication reaction, substantial hydrolysis of deoxynucleoside triphosphates to the corresponding deoxynucleoside monophosphates occurred. This reaction required DNA synthesis and most likely reflects an idling reaction similar to that observed with other DNA polymerases containing 3'----5' exonuclease activity in which the polymerase first incorporates and then hydrolyzes a dNMP.     

Mammalian DNA helicase.

A forked DNA was constructed to serve as a substrate for DNA helicases. It contains features closely resembling a natural replication fork. The DNA was prepared in large amounts and was used to assay displacement activity during isolation from calf thymus DNA polymerases alpha holoenzyme. One form of DNA polymerase alpha holoenzyme is possibly involved leading strand replication at the replication fork and possesses DNA dependent ATPase activity (Ottiger, H.-P. and Hübscher, U. (1984) Proc. Natl. Acad. Sci. USA 81, 3993-3997). The enzyme can be separated from DNA polymerase alpha by velocity sedimentation in conditions of very low ionic strength and then be purified by chromatography on Sephacryl S-200 and ATP-agarose. At all stages of purification, DNA dependent ATPase and displacement activity profiles were virtually superimposable. The DNA dependent ATPase can displace a hybridized DNA fragment with a short single-stranded tail at its 3'hydroxyl end only in the presence of ATP, and this displacement relies on ATP hydrolysis. Furthermore, homogeneous single-stranded binding proteins from calf thymus as well as from other tissues cannot perform this displacement reaction. By all this token the DNA dependent ATPase appears to be a DNA helicase. It is suggested that this DNA helicase might act in concert with DNA polymerase alpha at the leading strand, possibly pushing the replication fork ahead of the polymerase.     

Enhanced processivity of nuclear matrix bound DNA polymerase alpha from regenerating rat liver

Translocation of DNA during in vitro DNA synthesis on nuclear matrix bound replicational assemblies from regenerating rat liver was determined by measuring the processivity (average number of nucleotides added following one productive binding event of the polymerase to the DNA template) of nuclear matrix bound DNA polymerase alpha with poly(dT).oligo(A)10 as template primer. The matrix-bound polymerase had an average processivity (28.4 nucleotides) that was severalfold higher than the bulk nuclear DNA polymerase alpha activity extracted during nuclear matrix preparation (8.9 nucleotides). ATP at 1 mM markedly enhanced the activity and processivity of the matrix-bound polymerase but not the corresponding salt-soluble enzyme. The majority of the ATP-dependent activity and processivity enhancement was completed by 100 microM ATP and included products ranging up to full template length (1000-1200 nucleotides). Average processivity of the net ATP-stimulated polymerase activity exceeded 80 nucleotides with virtually all the DNA products greater than 50 nucleotides. Release of nuclear matrix bound DNA polymerase alpha by sonication resulted in a loss of ATP stimulation of activity and a corresponding decrease in processivity to a level similar to that of the salt-soluble polymerase (6.8 nucleotides). All nucleoside di- and triphosphates were as effective as ATP. Stimulation of both activity and processivity by the nonhydrolyzable ATP analogues adenosine 5'-O-(3-thiotriphosphate), 5'-adenylyl imidodiphosphate, and adenosine 5'-O-(1-thiotriphosphate) further suggested that the hydrolysis of ATP is not required for enhancement to occur.(ABSTRACT TRUNCATED AT 250 WORDS)     

Apparent suicidal inactivation of DNA polymerase by adenosine 2',3'-riboepoxide 5'-triphosphate.

Adenosine 2',3'-riboepoxide 5'-triphosphate (epoxyATP) has been found to be a suicidal inactivator of DNA polymerase I from Escherichia coli by the following criteria. Inactivation is complete, is first order in enzyme activity, and shows saturation kinetics with an apparent KD of 30 +/- 10 micron for epoxy ATP. This KD is comparable to the KM of the substrate dATP. The t1/2 for inactivation is 1.3 min. Inactivation requires Mg2+ and the complementary template. The enzyme is protected by dATP but not by an excess of template. Gel filtration of the reaction mixture after inactivation with [3H]epoxy ATP results in the comigration of E. coli DNA polymerase I, the tritium-labeled inactivator, and the DNA template. The stoichiometry of binding approaches 1 mol of [3H]epoxy nucleotide per mol of inactivated enzyme. These results are consistent with the hypothesis that epoxy ATP initially serves as a substrate for the polymerase reaction, elongating the DNA chain by a nucleotidyl unit, and subsequently alkylates an essential base at the primer terminus binding site of the enzyme. Epoxy ATP also inactivates human and viral DNA polymerases but not E. coli RNA polymerase or rabbit muscle pyruvate kinase. Hence epoxy ATP may be a specific suicide reagent for DNA polymerases.     

An ATP stimulation of T4 DNA polymerase mediated via T4 gene 44/62 and 45 proteins. The requirement for ATP hydrolysis.

An in vitro replication system reconstituted from six purified T4 bacteriophage proteins, each of which is essential for T4 DNA replication in vivo, requires ATP. Because of the complexity of the complete system, we examine in this report the involvement of ATP in two subsystems of the overall DNA synthesis reaction. One subsystem consists of the T4 DNA polymerase (gene 43 protein) and its "accessory proteins," the gene 44/62 and 45 products. An even simpler subsystem consists of the gene 44/62 and 45 proteins alone, which together have a DNA-dependent ATPase activity. The combination of the 44/62 and 45 proteins hydrolyze ATP to ADP and inorganic phosphate in the presence of DNA. These essential accessory proteins have been previously shown to increase T4 DNA polymerase activity on primed, single-stranded DNA templates. In this report we use nucleotide analogues to demonstrate that this polymerase stimulation requires hydrolysis of the beta,gamma-phosphate bond of ATP. However, our data suggest that the mechanism of accessory protein stimulation is such that less than 1 ATP molecule need be hydrolyzed per 10 deoxyribonucleotides incorporated by the DNA polymerase into DNA.