Biomedical subjects

M A Griep

Publications and source records attributed to M A Griep.

16 recordsLinked to original sources

PubMed1997

The tyrosine photophysics of a primase-derived peptide are sensitive to the peptide's zinc-bound state: proof that the bacterial primase hypothetical zinc finger sequence binds zinc.

A 35-amino acid peptide corresponding to the putative "zinc finger" sequence of primase was prepared to study its zinc binding properties. When zinc was added to the peptide, it was found that the fluorescence quantum yield of the single tyrosine increased by 46% and the average lifetime by 34%. The binding stoichiometry was one zinc per peptide. Below pH 6.0 and above pH 8.5, the zinc-peptide binding affinity was less than 1 microM and could be accurately determined. Interpolation from those binding constants suggested that the affinity at pH 7.5 was between 10 and 100 nM. The absorption spectrum of the cobalt(II)-peptide complex was consistent with tetrahedral metal coordination by three sulfur and one imidazole nitrogen ligands. The peptide affinity for cobalt was less than for zinc, indicating metal specificity. Analysis of the fluorescence intensity pH profile, circular dichroism spectra, the effect of extrinsic quenchers indicated that at neutral pH (1) the free peptide up into a structure to place the tyrosine in an environment protected from solvent, (2) the peptide bound zinc via its three cysteines and one of its histidines resulting in little change to the polypeptide secondary structure or to the tyrosine solvent accessibility, and (3) when the peptide bound zinc, it bound directly to or caused the immobilization of the groups that had been intramolecularly collisionally quenching the tyrosine which resulted in the observed increases in tyrosine quantum yield and lifetime.

DNA Primase↗

PubMed1996

The role of zinc and the reactivity of cysteines in Escherichia coli primase.

Primase is the zinc metalloenzyme responsible for synthesizing RNA primers for use during DNA synthesis. To establish whether the zinc played a catalytic or structural role, the zinc was removed and the activity of the apoprimase determined. The zinc was removed with p-(hydroxymercuri)-benzenesulfonate (PMPS), which covalently reacts with cysteine sulfhydryls, EDTA was added to chelate the zinc, DTT was added to remove the PMPS from the apoprimase, and then the apoprimase was separated from the small molecules. The resulting apoprimase was fully active, indicating that the zinc played a structural role but not one involved in thermodynamic folding/unfolding. PMPS and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) cysteine reactivities indicated that the cysteines fell into three categories: one or two were fast-reacting, three were zinc-ligating, and two or three were slow or nonreacting. The major distinction between apoprimase and natural primase was that apoprimase became inactivated during storage at 4 degrees C for 10 days. Storage-induced inactivation correlated with disulfide bond formation and could be reversed by incubation with a mild reducing agent. Apoprimase oxidation also prevented zinc reconstitution which was only achieved with freshly-reduced enzyme, indicating that the zinc-ligating cysteines participated in the inactivating disulfide bonds. The conclusion was that, in natural primase, the zinc prevented disulfide bond formation which, in turn, prevented inactivation. The zinc reconstitution studies identified a strong and a weak zinc binding site. Zinc could be prevented from binding to the weak site by the presence of magnesium, indicating that the weak site may be the catalytic magnesium site in which two of the seven cysteines were located.

Amino Acid Sequence↗

PubMed1995

Magnesium acetate induces a conformational change in Escherichia coli primase.

Primase from Escherichia coli is a single-stranded DNA-dependent RNA polymerase. As such, it requires magnesium to carry out catalysis. Limited tryptic digestion was used to probe the conformations of primase as a function of magnesium acetate concentration. In the absence of magnesium, trypsin cleaved primase at three sites. Magnesium acetate induced a conformational change such that one of these sites became inaccessible to trypsin digestion and a new site became trypsin accessible. The conformational change was only induced by Mg(OAc)2 and not MnCl2, CaCl2, NaOAc or LiCl, indicating a clear magnesium acetate-dependent conformational change. The effect was slightly induced by MgSO4 and MgCl2. An allosteric binding model indicates that primase binds at least two magnesiums in a cooperative manner. The data were best fit to a two-state model in which one conformation had a high affinity for magnesium, KR = 83.4 M-1, and the other state had virtually no affinity.

Acetates↗

PubMed1995

Primer synthesis kinetics by Escherichia coli primase on single-stranded DNA templates.

The kinetics of primer RNA initiation and elongation by Escherichia coli primase were measured. The single-stranded DNA template that was used to develop the system, d(CAGA-(CA)5CTGCAAAGC), contained: (1) the preferred initiating trinucleotide d(CTG); (2) five residues 3' to the d(CTG), the minimum required for efficient primer synthesis; and (3) a single guanine placed near the 5'-end to facilitate study of cytidine triphosphate analog incorporation at a unique site. The assay monitored radiolabeled nucleotide incorporation into the RNA primers. The various primers were separated by length using denaturing polyacrylamide gel electrophoresis. Different types of primers were observed when synthesis was monitored using gamma- versus alpha-radiolabeled nucleotides as the probe. When [gamma-32P]-ATP incorporation was the probe, only primers initiated with ATP from the unique template thymine were observed. The sequences of these primers were complementary to that of the template. No primers shorter than a 12-mer accumulated, demonstrating that formation of the first phosphodiester bond was much slower than that of the next 10 phosphodiester bonds. The longest primer observed when monitoring [gamma-32P]ATP incorporation was 16 nucleotides long, the correct length for a primer completely template-directed and initiated at the unique thymine. Misinsertion of a noncognate nucleotide at the template's guanine indicated very poor nucleotide discrimination by this enzyme. When [alpha-32P]UTP was the probe for primer synthesis, all primers synthesized were observed whether or not they were initiated with ATP. Under these conditions, "overlong" primers and the above-described template length-dependent primers were observed. The template length-dependent primers accumulated faster than the overlong primers, but, at long incubation times, the overlong primers became the dominant species. The overlong primers were not fully related to the template length-dependent primers since they were not initiated complementary to the template d(CTG). Nevertheless, the overlong primers did appear to arise as a consequence of the template length-dependent species since their length was double and they arose in the time course after the length-dependent species.

Base Sequence↗

PubMed1995

Fluorescence recovery assay: a continuous assay for processive DNA polymerases applied specifically to DNA polymerase III holoenzyme.

A continuous assay was developed for processive DNA polymerases. The specific enzyme used to develop the assay was the most processive polymerase known, Escherichia coli DNA polymerase III holoenzyme. The assay was based upon the recovery of the intrinsic fluorescence of single-stranded DNA binding protein (SSB) as it was displaced from the DNA template during DNA synthesis. The intrinsic fluorescence of SSB was quenched by as much as 80% when it bound to single-stranded DNA. As the DNA was replicated, SSB was displaced and recovered its fluorescence. The amount of fluorescence recovered was directly proportional to the amount of DNA synthesized and was used to quantitate the rate of DNA synthesis. However, since 50 to 60 nucleotides must be replicated for every SSB tetramer released, the assay is expected to work best for processive DNA polymerases. The only requirement for using this assay with other DNA polymerases is that they be able to synthesize DNA on a template coated with SSB. The replication SSBs do not pose an obstacle to the assay because they all appear to have intrinsic fluorescence that is sensitive to their ssDNA-bound state.

Bacterial Proteins↗

PubMed1995

Primase structure and function.

Primase is the ssDNA-dependent RNA polymerase that synthesizes RNA primers during DNA replication. In common with all DNA and RNA polymerases, primase has structural and functional features involved in polymer elongation. As RNA polymerase, it has structural and functional features for initiating chain synthesis. As a primase, it has structural and functional features for initiating chain synthesis on ssDNA. Using amino acid sequence analysis the structure of Escherichia coli primase responsible for binding zinc, at least three magnesium, and DnaB helicase has been identified. One of the magnesium binding motifs resembles the ¿active magnesium¿ motif found in all DNA and RNA polymerases. This motif can be considered to be involved in phosphodiester bond formation. The region with the putatuve zinc binding motif is the most highly conserved portion, including more than 25% of identical residues among bacterial primases. The function of the zinc finger may be to bind ssDNA in a sequence-specific manner. Primase has ¿RNAP¿ motif, a sequence found in all RNA polymerases which may be involved in chain initiation. Many of the observations concerning primer synthesis initiation in vivo have been reproduced by several of the in vitro assay systems. Important among these is that Okazaki fragments are initiated in vivo from d(CTG) most of the time. This trinucleotide initiation specificity has been shown to be an intrinsic property of pure primase in vitro. Using artificial ssDNA templates, primase has been shown to be the slowest and most error-prone polymerase yet studied. The rate-determining step is the first phosphodiester bond formed. Any protein which can influence either the dinucleotide synthesis rate or primase-ssDNA binding affinity will also play a key role in the regulation of primer synthesis initiation.

Amino Acid Sequence↗

PubMed1993

Primase from Escherichia coli primes single-stranded templates in the absence of single-stranded DNA-binding protein or other auxiliary proteins. Template sequence requirements based on the bacteriophage G4 complementary strand origin and Okazaki fragment initiation sites.

We find that neither the hairpins nor bound single-stranded DNA-binding protein (SSB) are required for in vitro primase activity at the G4 complementary strand origin. Primase has the ability to prime synthetic DNA templates in the absence of any auxiliary proteins, including SSB, and in the absence of any DNA secondary structure. primase does, however, initiate primer synthesis starting at a specific nucleotide sequence present in both the G4 origin system and at sites of Okazaki fragment initiation. We tested a series of single-stranded DNA templates that consisted of various portions of the G4 complementary strand origin for the ability to support primer synthesis. Primer synthesis activity was determined using an assay in which primer length and quantity are monitored by the incorporation of [gamma-32P]ATP once per primer and the products analyzed on a denaturing polyacrylamide gel. We find that primase requires only a short DNA template, 5'-(AC)7CTG CAA AGC-3', to synthesize a 17-nucleotide primer starting at the thymidine residue. We also report primase's ability to incorporate dideoxyribonucleotide triphosphates into primers, which has allowed us to determine directly both length and sequence of primers synthesized.

Base Sequence↗

PubMed1992

Fluorescence energy transfer between the primer and the beta subunit of the DNA polymerase III holoenzyme.

We report here our initial success in using fluorescence energy transfer to map the position of the subunits of the DNA polymerase III holoenzyme within initiation complexes formed on primed DNA. Using primers containing a fluorescent derivative 3 nucleotides from the 3'-terminus and acceptors of fluorescence energy transfer located on Cys333 of the beta subunit, a donor-acceptor distance of 65 A was measured. Coupling this distance with other information enabled us to propose a model for the positioning of beta within initiation complexes. Examination of the fluorescence properties of a labeled primer with the unlabeled beta subunit and other assemblies of DNA polymerase III holoenzyme subunits allowed us to distinguish all of the known intermediates of the holoenzyme-catalyzed reaction. Specific fluorescence changes could be assigned for primer annealing, Escherichia coli single-stranded DNA-binding protein binding, 3'----5' exonucleolytic hydrolysis of the primer, DNA polymerase III* binding, initiation complex formation upon the addition of beta in the presence of ATP, and DNA elongation. These fluorescence changes are sufficiently large to support future detailed kinetic studies. Particularly interesting was the difference in fluorescence changes accompanying initiation complex formation as compared to binding of DNA polymerase III holoenzyme subunit assemblies. Initiation complex formation resulted in a strong fluorescence enhancement. Binding of DNA polymerase III* led to a fluorescence quenching, and transfer of beta to primed DNA by the gamma delta complex did not change the fluorescence. This demonstrates a rearrangement of subunits accompanying initiation complex formation. Monitoring fluorescence changes with labeled beta, we have determined that beta binds with a stoichiometry of one monomer/primer terminus.

DNA Polymerase III↗

PubMed1991

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.

Autoradiography↗

PubMed1990

Dissociation of the DNA polymerase III holoenzyme beta 2 subunits is accompanied by conformational change at distal cysteines 333.

The beta subunit of DNA polymerase III holoenzyme is in a dimer-monomer equilibrium at physiological beta concentrations. Dissociation is accompanied by the fluorescence enhancement of a fluorophore attached to a unique sulfhydryl group of beta (Griep, M. A., and McHenry, C. S. (1988) Biochemistry 27, 5210-5215). Sequencing of the isolated tryptic peptides of beta revealed that the fluorescent maleimide group was attached to cysteine 333. The 2 residues, lysine 332 and glutamate 334, that flank this residue are hydrophilic and may place cysteine 333 on the surface of beta, explaining its high reactivity. Fluorescence energy transfer permitted us to locate the uniquely labeled cysteines 333 of beta at the distal ends of the beta dimer. When the beta dimer was dissociated to monomers, the accompanying alteration of the conformational state was reported by the fluorescein-5-maleimide (fluorescein)-labeled cysteines which were located far from the dimer interface. The carboxyl of fluorescein had a fluorescence pKa of 6.9 when beta was in its dimeric state. The pKa decreased by 0.3 pH unit upon dissociation to monomers and resulted in the fluorescence enhancement that was observed when the signal was monitored at constant pH. The adjacent glutamate 334 apparently increased the pKa of the attached fluorescein when beta was in its dimeric state. Movement of either the adjacent lysine 332 amino side chain to a closer position or glutamate 334 to a position further away could lower the pKa upon beta monomerization. Thus, beta undergoes a conformational change concomitant with dimer dissociation that was transmitted to the opposite ends of the beta dimer. The pKa of fluorescein attached to the distal cysteines was shifted, leading to greater ionization and enhanced fluorescence.

Chromatography, Gel↗

PubMed1990

Reduction of the potent DNA polymerase III holoenzyme 3'----5' exonuclease activity by template-primer analogues.

The DNA polymerase III holoenzyme of Escherichia coli contains a potent 3'----5' exonuclease that removes the terminal nucleotide from a synthetic deoxyoligonucleotide primer with a half-life of approximately 2 s. Degradation of primers could not be effectively prevented by permitting the holoenzyme to "idle" at the primer terminus in the presence of limited deoxynucleoside triphosphates. To further characterize this exonuclease and to develop stable primers to facilitate experimental manipulations, we synthesized a series of twelve 25-mer oligonucleotides that differed only in the two 3'-terminal residues. The penultimate position contained either a CMP or a dCMP residue, while at the terminal position either AMP, dAMP, 2',3'-dideoxyAMP, cordycepin (3'-dAMP), dAMP alpha S, or 2',3'-dideoxyAMP alpha S was incorporated. No single change at either the 3'-penultimate or 3'-terminal positions resulted in a decrease in the exonuclease rate greater than 10-fold; however, combined changes at these two sites resulted in a strong synergistic effect. Placing a ribonucleotide at the penultimate position coupled by a phosphorothioate linkage to a terminal 2',3'-dideoxynucleotide reduced the rate of exonucleolytic activity almost 30,000-fold (half-life approximately 16 h). If only the ribonucleotide and phosphorothioate substitutions were made, a primer capable of being efficiently elongated was generated that exhibited a 500-fold increase in stability (half-life = 40 min). The elemental effect observed by substituting a nonbridging oxygen in the terminal phosphodiester bond for sulfur increased from 1.5 to 200 as other substitutions were made that decreased the exonuclease rate. This was consistent with a change in the rate-limiting step of the exonuclease reaction from a conformational change to the chemical step where the covalent bond is cleaved. At least part of this effect appears to be due to perturbations within the enzyme's active site and not solely due to changes in electrophilicity.

DNA Polymerase III↗

PubMed1989

Glutamate overcomes the salt inhibition of DNA polymerase III holoenzyme.

Even though Escherichia coli can grow in media containing up to 1 M NaCl, one-fifth that amount of NaCl will completely inhibit the in vitro activity of DNA polymerase III holoenzyme. It has been established that the major intracellular ionic osmolytes are potassium and glutamate (Richey, B., Cayley, D. S., Mossing, M. C., Kolka, C., Anderson, C. F., Farrar, T. C., and Record, M. T., Jr. (1987) J. Biol. Chem. 262, 7157-7164). We have found that holoenzyme catalyzes replication efficiently in vitro in up to 1 M potassium glutamate. Two salt effects on the replication of single-stranded DNA were observed. At low salt replicative activity was enhanced and at high salt there was anion-specific inhibition. We have found that DNA polymerase III holoenzyme tolerated 10-fold higher concentrations of glutamate than chloride. The ability of various anions to extend the useful range of salt concentrations followed the order: phosphate less than chloride less than N-Ac-glutamate less than acetate less than glycine less than aspartate less than glutamate. With the exception of phosphate, this order followed the Hofmeister series indicating that the anion-specific effects were due to anions interacting at the protein-water interface at weak anion binding sites. Glutamate did not reverse the inhibition by chloride. The low salt enhancement and high salt inhibition effects were additive for the two anions indicating that they competed for common anion binding sites. The major salt-sensitive step was holoenzyme binding to template rather than the subsequent elongation reaction.

Anions↗

PubMed1988

The dimer of the beta subunit of Escherichia coli DNA polymerase III holoenzyme is dissociated into monomers upon binding magnesium(II).

The beta subunit of Escherichia coli DNA polymerase III holoenzyme binds Mg2+. Reacting beta with fluoresceinmaleimide (FM) resulted in one label per beta monomer with full retention of activity. Titration of FM-beta with Mg2+ resulted in a saturable 11% fluorescence enhancement. Analysis indicated that there was one noncooperative magnesium binding site per beta monomer with a dissociation constant of 1.7 mM. Saturable fluorescence enhancement was also observed when titration was with Ca2+ or spermidine(3+) but not with the monovalent cations Na+ and K+. The Mg2+-induced fluorescence enhancement was specific for FM-beta and was not observed with FM-glutathione, dimethoxystilbenemaleimide-beta, or pyrenylmaleimide-beta. Gel filtration studies indicated that the beta dimer-monomer dissociation occurred at physiologically significant beta concentrations and that the presence of 10 mM Mg2+ shifted the dimer-monomer equilibrium to favor monomers. Both the gel-filtered dimers and the gel-filtered monomers were active in the replication assay. These and other results suggested that the fluorescence increase which accompanies beta dissociation is due to a relief from homoquenching of FM when the beta dimer dissociates into monomers.

DNA Polymerase III↗

PubMed1986

Possible basis for the apparent surface selectivity of the contact activation of human blood coagulation factor XII.

The activation of factor XII by the proteases factor XIIa and kallikrein is known to be greatly enhanced by certain negatively charged surfaces. Studies that compared factor XII surface binding to factor XII activation found that binding alone was insufficient to account for surface enhancement of the activation rate. The temperature dependence of the reaction showed unusual behavior that may be related to the conformational change of factor XII following binding; the rate of factor XII activation had a relatively low temperature optimum (0-47 degrees C) that was sensitive to choice of surface and salt concentration. In temperature studies, below 47 degrees C, the decrease in the activation rate was not related to the thermal denaturation of enzyme or substrate, nor to the choice of activator enzyme (factor XIIa or kallikrein), nor to the species of factor XII (human or bovine) but to a behavior, designated a thermal transition, associated with the surface or the protein-surface interaction. The previously reported surface selectivity of contact activation is possible due to the temperature characteristics and other properties of the thermal transition; a surface that has a low-temperature thermal transition and that is highly sensitive to salt will be a "poor" contact surface under the usual choice of reaction conditions (approximately 150 mM ionic strength and 37 degrees C). However, solution conditions were identified that allowed the following negatively charged surfaces to function, in nearly equal potency, in the activation of factor XII: phosphatidylserine, phosphatidylglycerol, phosphatidic acid, phosphatidylinositol 4-phosphate, heparin, and 5-kDa dextran sulfate, as well as the previously characterized sulfatide and 500-kDa dextran sulfate.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzyme Activation↗

PubMed1985

Binding and activation properties of human factor XII, prekallikrein, and derived peptides with acidic lipid vesicles.

The binding of human factor XII and prekallikrein to vesicles of various compositions and the relationship to activation of factor XII were studied. Factor XII, factor XIIa, and the 40-kilodalton binding fragment of factor XII bound tightly to all of the negatively charged lipids investigated, including sulfatide, phosphatidylserine, and phosphatidylethanolamine, but not to the neutral lipid phosphatidylcholine. Binding could be reversed by high salt, and the dissociation constant for binding to sulfatide vesicles was in the nanomolar range at an ionic strength of 0.15 M. Prekallikrein did not bind significantly to either sulfatide or phosphatidylethanolamine vesicles under the conditions used. Stopped-flow studies showed that the association rate for the factor XII-sulfatide interaction was biphasic and very rapid; the faster rate corresponded to about 30% collisional efficiency. The kinetics of activation of factor XII was investigated and was in agreement with previous studies; sulfatide promoted activation but phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine did not. Autoactivation rates correlated closely with the packing density of factor XII and factor XIIa on the vesicle surface. In contrast, kallikrein activation of factor XII correlated with the amount of sulfatide-bound factor XII and was relatively insensitive to the density of factor XII on the vesicle surface. When the concentration of factor XII was reduced to only several molecules per vesicle, the autoactivation rate dropped very low whereas kallikrein activation held relatively constant. These results indicated that the autoactivation and the kallikrein activation of factor XII were dependent on different properties of the surface component.

Enzyme Activation↗

PubMedDate not supplied

Fluorescent labeling of cysteine 39 on Escherichia coli primase places the dye near an active site.

Cysteine 39 of Escherichia coli primase is the most chemically reactive cysteine. Its high chemical reactivity is likely due to its proximity to primase's zinc, which is probably ligated to the adjacent residues 40-62. The zinc may stabilize the deprotonated form of cysteine 39 to make it chemically reactive. Primase is rapidly, site-specifically modified by fluorescein maleimide (FM) at this cysteine. Modification with FM at this residue does not lead to any activity loss in a coupled RNA/DNA synthesis assay, indicating that it is not a catalytically essential residue. In contrast, iodoacetamidefluorescein (IAF) modifies cysteine 39 more slowly and stoichiometrically inhibits activity. It was not clear why these two similar fluorescent dyes should have such different inhibitory effects when attached to the same cysteine. The IAF inhibition must be due to some property of the link between the fluorescein and the cysteine because that is how it differs from FM. The pKa's of the fluoresceins from both FM- and IAF-modified primase are strongly shifted to lower values (approximately 5.4) compared to free fluorescein. These results strongly suggest that the deprotonated form of the fluoresceins are stabilized on primase by a strong interaction with the adjacent zinc in the zinc finger motif. The ability to place a noninhibitory FM at this site will be of great assistance in fluorescence energy transfer studies since the distances established to cysteine 39 will reflect the distance to the essential zinc finger motif.

Amino Acid Sequence↗