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R D Kuchta

Publications and source records attributed to R D Kuchta.

33 records · Page 2Linked to original sources
PubMed1994

Misincorporation of nucleotides by calf thymus DNA primase and elongation of primers containing multiple noncognate nucleotides by DNA polymerase alpha.

Misincorporation of nucleotides by calf thymus DNA primase was examined using synthetic DNA templates of defined sequence. Primase seldom misincorporated NTPs during initiation of a new primer (i.e. polymerization of two NTPs to generate the dinucleotide). Following dinucleotide formation, however, primase readily misincorporated NTPs. Although the rate of misincorporation varied according to both the identity of the mismatch and the template sequence, primase is by far the least accurate nucleotide-polymerizing enzyme known. In some cases primase discriminated against incorrect NTPs by less than a factor of 100. After primase incorporated a noncognate nucleotide into the primer, the next correct NTP was readily added. Remarkably, primase could also polymerize consecutive noncognate nucleotides and generate primers containing multiple mismatches. Generation of a correctly base-paired primer-template negatively regulated further primer synthesis; however, generation of a primer-template containing multiple mismatches did not. After primase synthesized a primer containing multiple mismatches, the primer was transferred to the polymerase alpha active site via an intramolecular mechanism. Importantly, polymerase alpha readily elongated this primer if dNTPs were present. These data are discussed with respect to the question of why primase is required for DNA replication.

Animals↗
PubMed1994

3'-Azido-3'-deoxythymidine potently inhibits protein glycosylation. A novel mechanism for AZT cytotoxicity.

3'-Azido-3'-deoxythymidine (AZT) is one of the primary chemotherapeutic agents used in the treatment of human immunodeficiency virus (HIV) infection. Unfortunately, AZT therapy is accompanied by severe side effects. Using Golgi-enriched membrane fractions, we have determined that 3'-azido-3'-deoxythymidine monophosphate, the primary AZT metabolite in treated cells, potently inhibits protein glycosylation. This inhibition results from direct competition with several pyrimidine-sugars for transport into Golgi membranes. This potential mechanism of cytotoxicity does not involve 3'-azido-3'-deoxythymidine triphosphate, the AZT metabolite most likely responsible for its antiviral effects; thus, it may be possible to develop novel therapeutic strategies that prevent inhibition of glycosylation without affecting the anti-HIV properties of AZT.

Animals↗
PubMed1994

Calf thymus DNA polymerase alpha-primase: "communication" and primer-template movement between the two active sites.

The DNA polymerase alpha-primase complex replicates single-stranded DNA by first synthesizing a short RNA primer (primase) which is then further elongated by the incorporation of dNTPs (DNA polymerase alpha). While primase and pol alpha function independently prior to synthesis of an RNA primer, the two activities become coordinated after primer synthesis. After primase generates a primer-template, it moves from the primase active site to the pol alpha active site for further elongation without dissociating into solution. Intramolecular transfer occurs immediately after primer synthesis and is employed on both long templates such as poly(dT) and short synthetic templates (< or = 60 nucleotides). Primer-template transfer and elongation by pol alpha are rapid compared to primer synthesis. After pol alpha elongates the primer, primase reinitiates primer synthesis, and the cycle is repeated. However, if dNTPs are absent such that primer elongation cannot occur, further primase activity is inhibited after a single round of primer synthesis. This "negative regulation" of primase activity is mediated by the newly generated primer-template provided the following conditions are met: (1) Primase synthesizes the primer; (2) the primer is 7-10 nucleotides long and remains bound to the template; (3) the template is of sufficient length; (4) the primer-template dissociates slowly from the enzyme complex; and (5) the primer-template interacts with the pol alpha active site. Polymerization of multiple dNTPs by pol alpha rapidly reactivates primase; hence, negative regulation of primase activity likely ensures a new primer is not synthesized until the previous one has been elongated by pol alpha.

Animals↗
PubMed1993

DNA polymerase epsilon: aphidicolin inhibition and the relationship between polymerase and exonuclease activity.

Calf thymus DNA polymerase epsilon readily uses short, synthetic oligonucleotides as substrates for both polymerase and exonuclease activity. These substrates were used to examine the mechanism of inhibition by aphidicolin. Aphidicolin competes with each of the four dNTPs for binding to a pol epsilon.DNA complex. Importantly, aphidicolin binds equally well regardless of the identity of the next template base to be replicated (Ki approximately 0.6 microM). Hydrolysis of synthetic templates of defined sequence by the 3'-->5' exonuclease was examined. pol epsilon preferred to hydrolyze single-stranded DNA 3-fold better than double-stranded DNA (Vmax/KM), while under Vmax conditions single-stranded DNA was hydrolyzed 100-fold faster than double-stranded DNA. Aphidicolin did not inhibit exonuclease activity on single-stranded DNA; however, activity on double-stranded DNA was partially inhibited. Formation of an E.[template.primer].aphidicolin ternary complex inhibits exonuclease activity. However, even under conditions where the polymerase site is completely blocked by a template-primer, the exonuclease retains significant activity.

Animals↗
PubMed1993

Mechanism of calf thymus DNA primase: slow initiation, rapid polymerization, and intelligent termination.

The mechanism by which calf thymus DNA primase synthesizes RNA primers was examined. Primase first binds a single-stranded DNA template (KD << 100 nM) and can then slide along the DNA in order to find a start for initiating primer synthesis. NTP binding appears ordered, such that the NTP which eventually becomes the second nucleotide of the primer binds the E.DNA complex first. The NTP that becomes the second nucleotide of the primer thereby influences where primase initiates. Primer synthesis is remarkably slow (0.0027 s-1 at 20 microM NTP). The rate-limiting step is after formation of the E.DNA.NTP.NTP complex and before or during dinucleotide synthesis. After synthesis of the dinucleotide, additional NTPs are rapidly polymerized. Primase products are 2-10 nucleotides long. If the enzyme fails to synthesize a primer at least 7 nucleotides long, it reinitiates rather than dissociating from the template. Once a primer at least 7 nucleotides long has been generated, however, subsequent primase activity is inhibited. This inhibition is due to the generation of a stable primer-template complex, which likely remains associated with pol alpha.primase. The role of primase is to synthesize primers that pol alpha can elongate. The ability of primase to distinguish between primers at least 7 nucleotides long and shorter products therefore likely reflects the fact that pol alpha only utilizes primers at least 7 nucleotides long.

Adenosine Triphosphate↗
PubMed1992

Inhibition of DNA primase and polymerase alpha by arabinofuranosylnucleoside triphosphates and related compounds.

Inhibition of DNA primase and polymerase alpha from calf thymus was examined. DNA primase requires a 3'-hydroxyl on the incoming NTP in order to polymerize it, while the 2'-hydroxyl is advantageous, but not essential. Amazingly, primase prefers to polymerize araATP rather than ATP by 4-fold (kcat/KM). However, after incorporation of an araNMP into the growing primer, further synthesis is abolished. The 2'- and 3'-hydroxyls of the incoming nucleotide appear relatively unimportant for nucleotide binding to primase. Polymerization of nucleoside triphosphates by DNA polymerase alpha onto a DNA primer was similarly analyzed. Removing the 3'-hydroxyl of the incoming triphosphate decreases the polymerization rate greater than 1000-fold (kcat/KM), while a 2'-hydroxyl in the ribo configuration abolishes polymerization. If the 2'-hydroxyl is in the ara configuration, there is almost no effect on polymerization. An araCMP or ddCMP at the 3'-terminus of a DNA primer slightly decreased DNA binding as well as binding of the next correct 2'-dNTP. Changing the primer from DNA to RNA dramatically and unpredictably altered the interactions of pol alpha with araNTPs and ddNTPs. Compared to the identical DNA primer, pol alpha discriminated 4-fold better against araCTP polymerization when the primer was RNA, but 85-fold worse against ddCTP polymerization. Additionally, pol alpha elongated RNA primers containing 3'-terminal araNMPs more efficiently than the identical DNA substrate.

Animals↗
PubMed1991

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)

Base Sequence↗
PubMed1991

Inhibition of DNA primase by 9-beta-D-arabinofuranosyladenosine triphosphate.

9-beta-D-Arabinofuranosyladenosine triphosphate (araATP) is a potent inhibitor of DNA primase. Primase readily incorporates araATP into primers, and primers containing araAMP are then elongated by DNA polymerase alpha (pol alpha) upon addition of dNTPs. AraATP did not inhibit utilization of primers under conditions where the ability of pol alpha to elongate primers was independent of the dATP concentration. The fraction of primers elongated by pol alpha was reduced by araATP only when elongation was dependent upon the dATP concentration. When the Ki for primase was measured in terms of the inhibition of the synthesis of primers that can be utilized by pol alpha, we obtained Ki = 2.7 microM (37 degrees C) and 2.0 microM (25 degrees C). Inhibition was competitive with ATP. Inhibition of pol alpha activity by araATP was measured under conditions where primase-catalyzed primer synthesis was required for the pol alpha activity. The decreased pol alpha activity was due to primase inhibition, and at constant dATP, araATP inhibition was competitive with ATP and gave Ki = 1.2 microM, similar to the Ki for primase alone. Increasing the dATP concentration had no effect on inhibition. In combination with previously reported in vivo data, we conclude that DNA primase is the primary in vivo target of the arabinofuranosyl nucleotides, not pol alpha.

Animals↗
PubMed1990

DNA primase. Processivity and the primase to polymerase alpha activity switch.

Calf thymus DNA primase was examined to determine the kinetic parameters that define its unusual processivity. At 37 degrees C, the major products were 8-9 and 2-3 nucleotides long. The 2-mer was the predominant product when considered on a molar basis. At each polymerization cycle en route to synthesis of a unit length primer (7-10 nucleotides), processivity was defined by competition of enzyme dissociation with ATP binding as well as an ATP independent step(s). Reducing the temperature to 25 degrees C had relatively little effect on the production of primers less than or equal to 6 nucleotides long, but greatly enhanced production of primers twice (16-18 nucleotides) the normal unit length. Kinetic analysis revealed that synthesis of these longer primers largely involves dissociation of the primase after completion of the unit length primer. After synthesis of a primer, the primase-polymerase complex normally switches to polymerase activity. Only primers greater than or equal to 7 nucleotides long were utilized by the polymerase regardless of the dNTP concentration, indicating that the signal for the primase to polymerase activity switch is primer completion. During the switch, either the primer-template does not dissociate from the complex or the complex has extraordinarily high affinity for the primers. At 25 degrees C and physiological dNTP concentrations the activity switch is very efficient, greater than 90% of the primers are elongated. However, at 37 degrees C the switch is much less efficient, likely due to primer-template denaturation.

Adenosine Triphosphate↗
PubMed1988

Kinetic mechanism whereby DNA polymerase I (Klenow) replicates DNA with high fidelity.

A complete kinetic scheme describing the polymerization of correct and incorrect dNTPs by the Klenow fragment (KF) of DNA polymerase I has been developed by using short DNA oligomers of defined sequence. The high fidelity arises from a three-stage mechanism. The first stage of discrimination [(1.1 X 10(4-) greater than 1.2 X 10(6]-fold] comes primarily from a dramatically reduced rate of phosphodiester bond formation for incorrect nucleotides, but it also gains a smaller contribution from selective dNTP binding. After phosphodiester bond formation, a conformational change slows dissociation of the incorrect DNA products from KF and, in conjunction with editing by the 3'----5'-exonuclease, increases fidelity 4- greater than 61-fold. Finally, KF polymerizes the next correct dNTP onto a mismatch very slowly, providing a further 6- greater than 340-fold increase in fidelity. Surprisingly, the 3'----5'-exonuclease did not in its hydrolysis reaction differentiate between correctly and incorrectly base-paired nucleotides; rather, an increased lifetime of the enzyme-DNA complex containing the misincorporated base is responsible for discrimination.

Base Sequence↗
PubMed1987

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↗
PubMed1987

Implications for enzymic catalysis from free-energy reaction coordinate profiles.

The constraints on the internal ground and transition states of enzyme-bound intermediates are mandated by the overall free-energy change in the direction of flux (Chin 1983; Stackhouse et al. 1985; Raines 1986) and the barrier for combination with reagent. Within these confines there are many solutions to transit the reaction coordinate. The path taken may reflect the function of the enzyme in question; particularly, whether in addition to catalysis there is a need to optimize reaction accuracy (Cramer and Freist 1987), to perform mechanochemical coupling (Jencks 1980), or to create metabolic control (Newsholme and Crabtree 1981; Koshland 1984).

Catalysis↗
PubMed1986

Fe-S centers in lactyl-CoA dehydratase.

Lactyl-CoA dehydratase consists of two enzymes, E1 and E2, and requires catalytic quantities of ATP for activity [Kuchta, R. D., & Abeles, R. H. (1985) J. Biol. Chem. 260, 13181-13189]. In contrast to E1, which contains no Fe, E2 contains 8.20 +/- 0.04 mol of Fe/mol of E2, one of which can be removed by 1,10-phenanthroline. E2 also contains 7.33 +/- 0.68 mol of inorganic sulfur/mol of E2, indicating that at least seven of the Fe atoms are present as Fe-S clusters. E1 and E2 contain less than 0.14 mol of Cu, Co, Zn, Mn, and Ni/mol of E1 or E2. Both reduced and oxidized E1 are EPR silent over a 10,000-G scan range at 4 K, while two signals in E2 are observable at 4 K. Identical spectra were obtained with E2 containing either seven or eight Fe atoms, and both signals were only observable at T less than 30 K. Signal 1 has axial symmetry with g = 2.0232 and g = 2.0006. Signal 2 is orthorhombic with g1 = 1.982, g2 = 1.995, and g3 = 2.019. Computer simulation of these spectra with a S = 1/2 spin Hamiltonian was used to extract the g matrices. The intensity of both signals decreases when E2 is reduced with Na2S2O4. We propose that signal 1 is due to an unusual [4Fe-4S] cluster and signal 2 to a [3Fe-3/4S] cluster. Addition of either acrylyl-CoA or lactyl-CoA dramatically alters signal 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Electron Spin Resonance Spectroscopy↗
PubMed1985

Lactate reduction in Clostridium propionicum. Purification and properties of lactyl-CoA dehydratase.

Clostridium propionicum converts lactate to propionate (Cardon, B.P., and Barker, H.A. (1947) Arch. Biochem. Biophys. 12, 165-171). We have obtained a soluble system that carries out this conversion as well as the hydration of acrylate to lactate and the reduction of acrylate to propionate. 3-Pentynyl-CoA inhibits reduction of acrylate and lactate to propionate, but not hydration of acrylate to lactate by cell extracts. The conversion probably involves CoA esters. When [beta-2H3] lactate is used as a substrate, the rate of propionate formation is reduced 1.8-fold, and the methyl group of the resulting propionate has lost 1.4 deuterium atoms. These results are consistent with the intermediate formation of acrylate (acrylyl-CoA) in the conversion of D-lactate to propionate. Two proteins, which we designate E I and E II, were purified to greater than 90% homogeneity. Together, they catalyze the hydration of acrylyl-CoA to lactyl-CoA. E I has an apparent molecular mass of 27,000 daltons and is rapidly and irreversibly inactivated by O2. E II consists of two subunits of molecular mass 41,000 and 48,000 daltons and contains equal amounts of riboflavin and flavin mononucleotide. Hydration of acrylyl-CoA to lactyl-CoA requires Mg2+ and catalytic quantities of ATP. GTP can replace ATP, but ADP and adenylyl imidodiphosphate cannot. We were unable to detect any stable intermediate during acrylyl-CoA hydration. Finally, we proposed a mechanism for this reaction.

Acrylates↗