Bioconjugate Chem. 1995, 6,673-682
673
Fluorescent Labeling of Cysteine 39 on Escherichia coli Primase Places the Dye Near an Active Site? Mark A. Griep” a n d Teresa N. Mesman Department of Chemistry and the Center for Biotechnology, University of Nebraska, Lincoln, Nebraska 68588-0304. Received February 3, 1995@
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) a t this cysteine. Modification with FM at this residue does not lead to any activity loss in a coupled RNMDNA 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 pK,$ of the fluoresceins from both FMand 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 a t 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.
Primase plays the central role a t the replication fork during DNA synthesis (Kornberg and Baker, 1992; Marians, 1992). It interacts with DnaB helicase which unwinds the duplex DNA to create the single-stranded DNA (ssDNAl) template that is used by both primase and DNA polymerase I11 holoenzyme. Primase initiates primer synthesis once every approximately 1500 nucleotides on the lagging strand (Okazaki et al., 1968; Wu et al., 1992a,b; Zechner et al., 1992). Primase activity is limited to the replication fork by its strong interaction with DnaB helicase (McMacken et al.,1977) and its weak interaction with ssDNA (Swart and Griep, 1993). When the lagging strand DNA polymerase completes the previous Okazaki fragment, primase binds to the one of the next d(CTG) trinucleotides on the lagging strand ssDNA template and begins synthesizing a n 11 & 1 nucleotide RNA primer (Yoda et al., 1988; Yoda and Okazaki, 1991; Swart and Griep, 1993). The lagging strand DNA polymerase displaces primase from the RNA primer and elongates processively for about 1500 nucleotides from it to create the Okazaki fragment. Far from the replication fork, DNA polymerase I, RNase H, and DNA ligase
* Author to whom correspondence should be addressed. Tel.: (402) 472-3429. E-mail:
[email protected]. + This work was supported by funds from the UNL Center for Biotechnology and NIH Grant GM 47490 (to M.A.G.) and by funds from the NSF Research Experience for Undergraduates (to T.N.M.). Abstract published in Advance ACS Abstracts, October 1, 1995. Abbreviations: DMSO, dimethyl sulfoxide; D’M’,dithiothreitol; FM, 5-fluorescein maleimide; FMl.o-primase, primase modified with a single FM (the maleimide moiety of the dye reacts by Michael addition with a primase sulfhydryl to create a succinimide linkage); IAF,6-(iodoacetamido)fluorescein;I A F 0 . e primase, primase modified with 0.48 IAF/polypeptide (the iodide moiety of the dye is displaced upon reaction with primase to create acetamidefluoresceinylated primase); MALDI, matrixassisted laser desorption ionization; PMPS, p-hydroxymercuriophenyl sulfonate. @
are all thought to be involved in removing the RNA primer and ligating the lagging strand into a high molecular weight DNA (Westergaard et al.., 1973; Lehman, 1974; McMacken and Kornberg, 1978; Ogawa and Okazaki, 1980; Funnel et al., 1986). To participate in lagging strand DNA synthesis, the proteins with which primase must minimally interact are DnaB helicase and DNA polymerase I11 holoenzyme. DnaB helicase is a hexamer of 52 300-Da protomers that migrates along the 3’75’ ssDNA strand (the lagging strand) in the direction of the replication fork (LeBowitz and McMacken, 1986). In the absence of duplex DNA to unwind, helicase is a single-stranded DNA-specific ATPase that is capable of enhancing the ability of primase to bind to the template. In the presence of -36 nM hexameric DnaB helicase, 150 nM primase provides maximal primer synthesis (Arai and Kornberg, 19811, whereas, in the absence of DnaB helicase, much higher concentrations of primase are required for primer synthesis on single-stranded DNA (Swart and Griep, 1993). Thus, to initiate primer synthesis primase binds to helicase and then to the single-stranded DNA template. It is in this way that DnaB helicase acts as a “mobile promoter’’ for primer RNA synthesis (McMacken et al., 1977). Recent functional evidence indicates that helicase binds to the carboxyl-terminal16-kDa portion of primase (Tougu et al., 1994). On the basis of sequence analysis (Ilyina et al., 19921, it can be predicted that it is the amino-terminal portion of DnaB helicase that binds to primase. The evidence for a primaselDNA polymerase I11 holoenzyme interaction is considerable. The DNA yield and synthesis rates when starting from DNA polymerase initiation complexes (template/primer/DNA polymerase) are influenced by whether primase is allowed to synthesize a primer in the presence of the DNA polymerase (Griep and McHenry, 1989). When DNA polymerase binds to the primer while primase is synthesizing it, then the complex is more active than if primer synthesis is
1043-1802/95/2906-0673$09.00/0 0 1995 American Chemical Society
674 Bioconjugate Chem., Vol. 6,No. 6,1995 Putative Function
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Griep and Mesman ?
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Figure 1. Seven cysteines of E. coli primase and their locations with respect to conserved sequences. Motifs 1-6 were established from amino acid sequence analysis and some of their possible functions taken from analogous motifs in other proteins (Ilyina et al., 1992). The “RNAF”’ and “bacterial primases”motifs were also identified by sequence analysis (Versalovic and Lupski, 1993). The location of the DnaB helicase binding domain was determined functionally by Tougu and co-workers (Tougu et al., 1994).
complete prior to adding the DNA polymerase. Thus, primer synthesis influences the ability of DNA polymerase to bind to the primer. Conversely, there is evidence that primer synthesis is highly controlled by DNA polymerase during rolling circle synthesis. A primer is not synthesized until the lagging strand DNA polymerase completes the elongation of the previous Okazaki fragment (Zechner et al., 1992;Wu et al., 199213). Together with single-stranded DNA binding protein, the helicase/primase/DNApolymerase complex represent the minimal proteins needed to replicate lagging strand DNA (Mok and Marians, 1987). The three enzymes form a complex, however transient, that is in excess of 1 MDa. Fluorescence resonance energy transfer is one of the few effective techniques that could establish the solution structure of such a large dynamic complex. To determine its overall structure and orientation a t the replication fork will require establishing many different distances between protein and template sites. Likewise, specific reporter groups on each of the enzymes will be useful tools for establishing the static and dynamic effects that these enzymes have on one another. This paper describes the procedure for dye labeling primase a t its active site to create a site-specific probe suitable for use in primase/ helicase interaction studies. The most chemically reactive groups on proteins are their amines and thiols. There are many fluorescent dyes of various chemistries that target these groups (Brinkley, 1992). The cysteine thiols are better targets for sitespecific labeling than are the amines of the amino terminus and lysine side chain because there are usually fewer of them. For instance, primase from E. coli has seven cysteines and 22 lysines (Burton et al., 1983). Given the lower number of primase cysteines, these were first targeted by our laboratory as possible sites for specific fluorophore labeling. The number of cysteines in E. coli primase that can be expected to be chemically reactive is further limited because three of its cysteines (40,61,and 64)are proposed to be involved in binding its one zinc atom (Ilyina et al., 1992;Stamford et al., 1992). This leaves cysteines 39, 306, 307, and 492 available for dye labeling. The best reporter groups are near functional domains but do not interfere with their activity. Unfortunately for primase, its four available cysteines reside in three identified motifs (Figure 1). It is possible that labeling any one of them will inhibit some but not all activities of primase. Motif 1 is the putative zinc finger of primase, and cysteine 39 resides at its amino terminus (Ilyina et al., 1992). The role of the analogous zinc finger motif from bacteriophage T7 primasehelicase is to cause primer synthesis initiation to be template sequencespecific (Bernstein and Richardson, 1988). A fluorophore at this site may prevent primer synthesis because the
primase may not be able to specifically bind to ssDNA. However, it may not interfere with binding to DNA polymerase or to DnaB helicase. Cysteines 306 and 307 reside in conserved motif 5 , a putative magnesium binding site (Ilyina et al., 1992). This domain shows some similarity to the catalytically active magnesium binding sites of DNA and RNA polymerases (Argos, 1988). Again, labeling a t this site may inhibit primer synthesis activity but not binding to ssDNA, DNA polymerase, or DnaB helicase. Cysteine 492 resides within the helicase binding carboxyl terminus of primase (Tougu et al., 1994). A label a t this site should not interfere with primer synthesis activity but may interfere with helicase binding. This study shows that cysteine 39 of E. coli primase is the most chemically reactive cysteine. Labeling this residue with fluorescein maleimide is rapid (less than 10 min) and noninhibitory. In contrast, labeling this same residue with iodoacetamide fluorescein is slow (not complete in 4 h) and stoichiometricallyinhibiting. This indicates that even though cysteine 39 is near an active site, it can be safely labeled by fluorescein maleimide. The ability to place a noninhibitory maleimide derivative in this location will provide a sensitive probe of the action at this site. EXPERIMENTAL PROCEDURES
Proteins and Enzymes. Single-stranded DNA binding protein was isolated according to a published protocol (Lohman et al., 1986). DNA polymerase I11 holoenzyme and primase were isolated as described previously (Swart and Griep, 1993). Primase was isolated from a primaseoverproducer that was manufactured by Dr. Roger McMacken’s laboratory at of Johns Hopkins University. The concentration of native primase was determined using its extinction coefficient of 47 800 M-l cm-l a t 280 nm. The activity of primase was measured by the standard assay in which primer synthesis is coupled to DNA synthesis. One unit of primase was defined as the amount needed to incorporate 1 pmol of (total) nucleotide/ min into acid-precipitable DNA. The assay was performed at 30 “C for 5 min under conditions in which all other components were saturating. Primase from several preparations has had a specific activity of 2.1 unitslng (f0.3). The activities of primase and the labeled primases were determined using the coupled primer synthesisDNA synthesis assay (Bouchb et al., 1975; Wickner, 1977; Johanson and McHenry, 1980;Griep and McHenry, 1989; Swart and Griep, 1993). In this assay, bacteriophage G4 single-stranded DNA complementary strand origin is primed once by primase a t a known initiation sequence. The DNA polymerase I11 holoenzyme, also present in the assay, processively elongates a DNA polymer from the
Primase Cysteine 39 Modification
RNA primer. The assay is quantitated according to the amount of tritiated thymidine incorporated into the nascent DNA strand. Primase could lose any of a number of activities and not be active in this assay. It could lose its ability to do the following: bind single-stranded DNA, specifically recognize its trinucleotide initiation sequence CTG; bind ribonucleotides; bind magnesium; bind DNA polymerase I11 holoenzyme. Thus, any step that precedes processive DNA synthesis by the DNA polymerase will lead to a loss of activity in this assay. Fluorescent Labeling of Primase. 6-(Iodoacetamido)fluorescein (IAF) and 5-fluorescein maleimide (FM) were purchased from Molecular Probes (Eugene, OR) and prepared as 10-mM stock solutions in DMSO. In a typical 100 p L labeling reaction (Griep and McHenry, 19881, 50 p M primase and 600 pM dye were incubated in 50 mM HEPES, pH 7.5,50 mM NaC1,2.5% DMSO in the dark a t room temperature for 15 min. The sample was then gel filtered on a S-200 column (0.9 x 18 cm) to remove unreacted dye from the primase and to establish the aggregation state of primase. Typical recoveries of both labeled and unlabeled primase from this column were in the range of 90 to 95%. Fractions containing greater than 30% of the peak fraction absorbance were pooled. The concentration of the IAF fluorescein was determined using c495 of 75 000 M-l cm-l and the FM fluorescein using €495 of 83 000 M-l cm-I (Molecular Probes, Eugene, OR); these represent the extinction coefficients for the fully unprotonated species (Martin and Lindqvist, 1975). As demonstrated in the Results, the pK,'s of the fluoresceins attached to primase were shifted to such low values that they were fully deprotonated a t pH 7.5, allowing these extinction coefficients to be used. The primase concentration was determined by the Bradford assay (Bradford, 1976) using unlabeled primase as the standard. The concentration of the unlabeled primase was accurately determined using its extinction coefficient. Primase binds 2.03 times as much Coomassie Brilliant Blue as does immunoglobulin G, the usual Bradford assay standard. The protein concentration within the labeled protein can be estimated using Az~o', the corrected A280 of the labeled primase for the contribution from the dye (Brinkley, 19921, using the following equation: A,,w = A,,,(labeled primase) A,,,(free dye)
The A28dA495 correction ratio a s taken from the spectra of the free IAF is 0.326 and free FM is 0.254. Using these values gives protein concentrations that are in disagreement with Bradford assay determinations. Possible sources of error in the equation are that it assumes (1) that the dye does not perturb the UV-absorbing properties of the tyrosines and tryptophans of the protein and (2) that the protein does not perturb the UV- or visibleabsorbing properties of the dye. The first assumption is reasonable for iodoacetamide and maleimide dyes since they do not react with the tyrosines or tryptophans. That there was no interference with the UV-absorbing properties of the protein was confirmed as indicated in the Results. Also, as indicated in the Results, the UVabsorbing properties of the dye are not altered when it is covalently attached to the protein. However, the visible absorption spectrum is significantly altered such that the dye's spectrum shifts by 4 nm to the red when covalently attached to the protein. The A2sdA495 correction ratio can be recalculated as -0.43 for IAF and -0.30
Bioconjugate Chem., Vol. 6,No. 6, 1995 675
for FM when proper account is taken of the known concentrations of protein from the Bradford assay. Protein concentrations determined with these correction ratios were consistent (&lo%) with the Bradford assay determinations. Fluorescence Measurements. The fluorescence measurements were made using an Aminco-Bowman 2 spectrofluorometer controlled by a n IBM running OS/2. The sample compartment was equipped with a stirring assembly and a temperature regulator; the compartment was maintained a t 30.0 f 0.1 "C. The excitation and emission band widths were each set at 4 nm except during the quantum yield measurements when the emission bandpass was set to 1nm. All measurements have been corrected for background fluorescence and dilution effects. Quantum yields were determined using the relationship Qx= (FAn&ref)/(FFefA.),where Q was quantum yield, A was absorption a t the excitation wavelength, F was area under the fluorescence emission curve, and the subscripts x and ref were the unknown and the reference samples (Parker and Rees, 1960). The concentrations of the unknown and reference were adjusted so that their absorbances were less than 0.05 to prevent any inner filter effects (Lakowicz, 1983). The reference quantum yield for disodium fluorescein in 100 mM NaOH was 0.92 (Weber and Teale, 1957). The pH dependence studies were performed in APHTC buffer (Griep and McHenry, 1990) containing 10 mM of each of the following: acetic acid (pK4.8 a t 25 "C), PIPES (pK 6.81, HEPES (pK 7.6), Tris (pK 8.31, and CHES (pK 9.3). KOH was used to adjust the pH of the buffer combinations to create solutions of known pH. Aliquots of IAF-primase were added to these buffer solutions and absorbance and fluorescence emission spectra taken. Tryptic Digestion, Fluorescent Peptide Isolation, and Peptide Identity. Tryptic digestion and fluorescent peptide isolation were performed essentially as described for the /3 subunit of DNA polymerase I11 holoenzyme (Griep and McHenry, 1990). Prior to tryptic digestion, fluorescently labeled primase (2 nmol, 130 pg) was dried to completion in a Savant SpeedVac and the residue dissolved in 50 pL of 7.0 M urea, 400 mM NH4HC03, pH 8.0, 5 mM DTT. This sample was incubated a t 60 "C for 15 min to denature the protein and maintain the cysteines in a reduced state. After the mixture was cooled to room temperature, a final concentration of 10 mM iodoacetic acid was added to carboxymethylate the other cysteines to prevent them from forming mixed disulfides during subsequent steps. After this mixture was incubated for 5 min a t ambient temperature, the volume was diluted 1:4 with water to lower the urea to 1.8 M. Trypsin was added at a 1:25 enzyme:substrate weight ratio and the mixture incubated for 20 h a t 37 "C. The tryptic peptides were resolved on an ISCO Spherisorb C18 column (100 x 4.6 mm). The modular HPLC setup consisted of a Milton Roy CM400 multiple solvent delivery system, a RheoDyne manual injector, a LDC analytical spectroMonitor 3100X for absorbance detection a t 215 nm, a Shimadzu RF-535 for fluorescence detection by excitation a t 440 nm and emission a t 520 nm, and a Gilson FC-80 fraction collector set to collect 1 min of eluent per fraction. Half of the above described tryptic digestion sample was loaded into the injector loop and then the column. The peptides were eluted from the column using a 0.1% trifluoroacetate (pH 2.2) solution and the following acetonitrile gradient a t a flow rate of 1mumin: 0-13% acetonitrile gradient over 5 min; 1327% over 60 min; 27-90% over 5 min; maintain 90% over
676 Bioconjugate Chem., Vol. 6, No. 6, 1995
5 min; 90-0% over 5 min. Fractions of 1 mL were collected. The fractions containing fluorescent substances were pooled, concentrated in a Speed-Vac, and rechromatographed on the C18 column with an even shallower central gradient. In the case of the main fluorescent peak, it was rechromatographed with a central gradient of 20-26% acetonitrile over a 60-min period. A spreadsheet program was written to predict the percent acetonitrile required to elute each of the cysteinecontaining peptides from the C18 column. The equation used in the prediction was based on the amino acid composition of the eluting peptides and the C18/0.1% trifluoroacetate partition coefficients for the amino acids in the peptide (Sasagawa et al., 1982). The equation for a linear 1%acetonitrile/min gradient was: % acetonitrile = A In(1 ZDj’nj) C , where A was an empirically derived slope constant (= 12.4); Dj’ was the nonweighted C18/0.1% trifluoroacetate partition coefficient for amino acid j (taken from Table I1 of Sasagawa et al. (1982)); n, was the number of residues of amino acid j in the peptide; and C‘ was an empirically derived intercept constant (= -30.3). The partition coefficient for cysteine was available for both the carboxymethyl-modified or aminoethylmodified forms but not for the fluoresceinylated form. In our calculations, the carboxymethylated cysteine partition coefficient was used for all cysteines. Peptide amino acid sequence was determined by Edman degradation in an Applied Biosystems 420H by Laurey Steinke of the University of Nebraska Medical Center Protein Structural Core Facility. Mass spectral analysis was determined by MALDI in a Bruker BenchTOF and by electrospray mass spectrometry by Ron Cerny of the University of Nebraska-Lincoln Midwest Center for Mass Spectrometry.
+
+
RESULTS
To monitor the interactions between the enzymes a t a replication fork will require a way to monitor each of the enzymes independently of the others. One way to do this would be to attach different fluorescent probes to each of the participating enzymes. In this way, each enzyme could be monitored for the effects that the other enzymes have on it. The p subunit of DNA polymerase I11 holoenzyme has already been site-specifically labeled in such a way that it retains full activity (Griep and McHenry, 1988, 1990, 1992). As the next step for achieving the above goal, primase was targeted for fluorescent labeling. Labeling Primase with Fluorescein Dyes. In the early trials, which were with IAF,primase was incubated with a 16-fold molar excess IAF dye for 15 min a t pH 7.5. The same excess of dye was added to the solution three more times, each time incubating for 15 min. The solution was gel filtered on a n S-200 column to remove unreacted dye and assess the Stokes’radius of the labeled primase. The fractions containing labeled primase were pooled and then quantitated for fluorescein using its extinciton coefficient of 7 5 000 M-l cm-l and for primase using the Bradford assay. Under the conditions described above, primase eluted as a monomer that was labeled with 0.48 IAF/primase and had a specific activity of 1.32 unitdng. This was about half of full activity (Figure 2). Unlabeled primase did not lose activity when incubated a t room temperature for up to 2 days, indicating that it was the label that caused primase to lose activity. To modify primase to a greater extent with IAF requires incubations up to 4.5 h, although only involving three or four additions of excess IAF.The plot of specific activity versus modification demonstrated that primase
Griep and Mesman
FluoresceinPrimase (molimol)
Figure 2. Activity plot of modified primase. Following modification of the primase with either IAF or FM, the protein was gel filtered on an S-200 column and then characterized for both the extent of modification and its specific activity in the coupled RNA/DNA synthesis assay. The line through the IAF modification data was drawn assuming that first site labeling caused proportional loss of activity. The lines through the FM modification data was drawn assuming that the first site labeled caused no loss of activity while the second site labeled caused proportional loss of activity.
was inhibited in direct proportion to modification. Therefore, the modified cysteine had either been an essential residue or the fluorescein was interfering with the function of an adjacent region. FM reacted with primase much more rapidly than did IAF,especially the first residue modified. Within 5 s of adding a 16-fold molar excess of FM dye to a primase solution, the solution became orange rather than the yellow it would have been if added to buffer alone. After only 10 min of incubation, 1.0 cysteine was modified per primase, and it retained full activity (Figure 2). For FM, an extinction coefficient of 83 000 M-’ cm-l was used. Merely 90 min was required to obtain the highest amount of FM modification presented (1.56 FWprimase and a specific activity of 0.36 unitdng). In contrast to IAF modification, the activity/modification plot of FM demonstrated that the first FM-modified residue did not inhibit primase activity (Figure 2), while the second FMmodified cysteine caused proportional inhibition. The extinction coefficients for the fully unprotonated fluorescein were used in the above trials to quantitate the moles fluorescein. It will be shown below that the fluorescein pK’s were low enough to justify this use with the pH 7.5 buffers used here. However, it should be noted that the IAF modification plot (Figure 2) would have a better proportional fit if an extinction coefficient of 92 000 M-l cm-I had been used and the FM modification data plotted would be better fit if an extinction coefficient of 75 000 M-l cm-l had been used. Absorbance Spectra. A comparison of the FMl,oprimase spectrum with that of the unreacted dye revealed that there was a 3.5-nm red shift from 492.5 to 496.0 nm of the fluorescein’s absorption maximum when the dye was attached to primase (Figure 3). Similarly, the IAFo.4g-primase absorbance maximum red-shifted from 493.0 to 497.0 nm (data not shown). The visible difference spectrum between FMl.o-primase and FM in pH 8.5 buffer also revealed the large spectral shift. Above 350 nm, the area above and below zero absorbance was nearly equal, indicating a simple environmental change rather than a change in the electronic structure of the moiety. When the FMl,o-primase/FM difference spectrum below 300 nm was compared to the absorption spectrum of unlabeled primase, they were found to be nearly superimposable (data not shown). Replacing the maleimide double bond with a thio-carbon bond in the labeled primase did not lead to a change in the dye’s W
Bioconjugate Chem., Vol. 6,No. 6,1995 677
Primase Cysteine 39 Modification L""'"'
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absorbing properties. Below 300 nm, the UV difference spectrum between FMl,o-primase and FM can be fully accounted for by the UV absorbing properties of primase alone. Thus, the dye did not alter the tyrosine and tryptophan spectral properties and none of the primase residues altered the dye's UV absorbing properties. Quantum Yield and pKa of Labeled Primase. The environment surrounding the reactive cysteine was further explored by determining the fluorescence properties of the labeled primases. The fluorescence emission maximum wavelength for IAF0,4~-primasein a moderate ionic strength pH 7.5 buffer was typical at 520 nm, and its quantum yield was 0.50. The emission maximum was also 520 nm for FMl,o-primase but its quantum yield at pH 7.5 was 0.85. Fluorescein fluorescence is sensitive to its protonated state (Martin and Lindqvist, 1975) because deprotonation alters its electronic structure. As a result, both the extinction coefficient and the quantum yield are pH dependent. Buffers of known pH were prepared, and the same amount of labeled primase was added to each solution. The same solution was used for both absorbance and fluorescence emission measurements. The fluorescence emission scans were collected while exciting, a t 470 nm, the absorption isosbestic point to eliminate absorption differences from having an effect on the quantum yield determination. The quantum yield for IAFo.48-primase was again determined to be 0.50 a t pH 7.5. Hill plots of the A495and quantum yield data (Figure 4) indicated that the pKa for fluorescein on LAFo.48-primase were 5.19 and 5.45, respectively (the data for FMl,o-primase were similar and are summarized in Table 1). These were negatively shifted pK:s for a fluoresceinylated compound. For IAF0,4s-primase, the Hill coefficients were 1.09 and 0.98, respectively, confirming that there was one protonatable group involved. Since the pK,)s were more than two pH units lower than pH 7.5, the fluoresceins will be more than 99% unprotonated a t pH 7.5 and a t their full quantum yields. Determining the Cysteines to Which the FM and IAF' Were Attached. There are seven cysteines in primase, and they reside on four tryptic peptides (Table 2). Three ofthe tryptic peptides have two cysteines. Even though this leads to a reduction in the number of possible peptides to which the fluorescein may be attached, it also leads to ambiguity as to which of the two cysteines were labeled on those tryptic peptides. Some evidence relating to this issue was obtained during Edman degradation. FMl.o-primase (and separately IAFo~-primase)was denatured, labeled with iodoacetate, and tryptically digested, and the tryptic peptides were resolved by a
5
6
7
8
PH Figure 4. Hydrogen ion Hill plots of absorbance and quantum yields of IAFo.4s-primase. The absorbance and quantum yield were measured in the APHTC buffers described in the Experimental Procedures. The ratio was the A495 nm or quantum yield at the indicated pH divided by the difference from the total signal change observed at the highest pH. Least-squares analysis to the absorbance data yielded the equation Ratio = 101.086(pH-5.19) with R2 = 0.982 and for the quantum yield data Ratio = 100.9WH-5.44)with R2 = 0.992.
6-IAF labeled cysteine
5-FM labeled cysteine
Figure 5. Structures of the fluorescein dyes attached to primase. The dyes were 6-(iodoacetamido)fluorescein and 5-fluorescein maleimide, and their structures as attached to primase are shown. The succinimide is shown in one of its two possible open ring forms. The numbering system is for the lactone form of the fluorescein rings, even though that was not the form studied here. Table 1. Summary of Fluorescein-LabeledPrimase modified primase IAFo.48-primasea FM1.o-primase labeling condns four consecutive 15-fold dye excess aliquots 15-fold during less than dye excess for 10 min 15 min each dyelprimase 0.48 1.0 specific activityb 1.32 (-63%) 2.06 (-100%) (unitslng) absorbance max (nm) 497 496 fluorescein pKa 5.19 5.38 Hill coefficient 1.09 0.68 fluorescence emission 520 520 max (nm) quantum yield (%) 0.50 0.85 fluorescein pK, 5.45 5.52 Hill coefficient 0.98 0.98 a The primase could be labeled with IAF to greater extents by incubating for longer periods of time. Unlabeled primase from several preparations has a specific activity of 2.1 f 0.3 unitslng.
shallow acetonitrile gradient on an analytical C18 column. The major fluorescent peak (80% of the total fluorescent species) eluted a t about 25%acetonitrile and consisted of a primary peak with a trailing shoulder peak. There was a minor fluorescent peak (10% of the total) which eluted a t about 13% acetonitrile. This peak was composed of several compounds with masses in the range of 400-500 amu as observed by MALDI and probably represents free fluorescein generated during workup. Another minor peak (10% of the total) eluted a t the
Griep and Mesman
678 Bioconjugate Chem., Vol. 6,No. 6, 1995 Table 2. Predicted Cysteine-Containing Tryptic Peptides from Primase
mass* amino acid sequence NFHACCPFHNEK QFYHmGCGAHG NAIDFLMNYDK ATNNVICCYDGDR ELVNTcmQPGLTT GQLLEHYR
residue nos. 35-46 57-79 300-312 487-508
no. of residues 12 23 13 22
% acetonitrile"
24.6 33.9 22.5 32.0
+IAF+CM 1892.1 3097.5 1889.0 2860.3
+FM+CM 1932.1 3137.5 1929.0 2900.3
=The predicted percent acetonitrile to elute the peptide from a C18 reversed-phase column was calculated as described in the Experimental hocedures. The prediction was for peptides containing all S-carboxymethylated cysteines. * Masses of the uncharged peptides were calculated as the sum of their residue masses. Acetamidofluorescein (IAF) will add 387.4 amu to the IAF-peptide, succinimidofluorescein (FM) will add 427.4 amu to the FM-peptide, and S-carboxymethyl (CM) will add 58.0 amu to those peptides containing two cysteines.
location expected from full length primase. The major peak was pooled, concentrated, and rechromatographed on an even shallower gradient to remove closely eluting contaminants. Again, a n early eluting species was observed (about 10% of the total fluorescent species) which had a mass in the range of 400-500 amu. The first evidence as to the identity of the labeled peptide was the percent acetonitrile a t which it eluted. The percent acetonitrile a t which the four cysteinecontaining peptides were predicted to elute was calculated using their amino acid compositions and the amino acid C18/0.1% trifluoroacetate partition coefficients (Sasagawa et al., 1982) (Table 2). The calculation assumed that the cysteines were labeled with iodoacetate, not fluorescein. Fluoresceinylated peptides would be expected to elute a t slightly higher acetonitrile than the S-carboxymethylated peptides. Because the major peak eluted a t about 25% acetonitrile, the calculations predicted that the labeled cysteine on either peptide 35-46 or peptide 300-312. Both of these peptides have two adjacent cysteines that could be labeled by a fluorophore. The FM-peptide (estimated to be 220 pmol by fluorescence intensity comparison to FM-primase standards) was subjected to Edman degradation and a low yield of peptide (average 25 pmol) with the sequence NFHA?CPFHN?K was obtained. This corresponded to peptide 3546 in which the fifth residue should be a cysteine and the eleventh residue should be a glutamate (Burton et al., 1983). During cycle 6 but not 5, it was possible to establish that a cysteine was present by the appearance of a phenylthiohydantoin derivative of S-carboxymethylated cysteine which eluted near the position for a serine derivative. Only the zinc-ligated cysteine would have been free following denaturation and susceptible to iodoacetate labeling. Thus, by inference, cysteine 39 was the FM-labeled cysteine. No residue was positively identified during eleventh Edman degradation cycle due to a nonproteinaceous contaminant which eluted during every cycle where glutamate would. The nonproteinaceous material may have also reduced the Edman degradation yield. These data effectively ruled out peptide 300-312 as the site of fluorescent labeling. The major fluorescent peak was characterized by electrospray mass spectrometry. Electrospray is precise to within about 0.01% (or 0.2 amu in 2000). The FMpeptide mass (MI was 1951.0 amu. This mass was 19 amu more than that predicted for a succinimide linkage t o peptide 35-46 (Table 2). The less precise measurement by MALDI (about 1.0 amu in 2000) indicated that the mass (M) was 1947 which was 15 amu more that predicted for a succinimide linkage (Table 2). There were a t least two possibilities to account for an increase in peptide mass of this magnitude. Ring opening of the succinimide to its succinamic acid form would increase the mass by 18 amu (due to HzO). Succinimide ring opening has been reported by other workers (Wu et al., 1976; Lux and GBrard, 1981; Ishii and Lehrer, 1986).
!ban
It is interesting to note that the flexibility of the succinamic acid linkage of the FM-peptide would be similar to the acetamide linkage of IAF-peptide. Alternatively, sulfoxide formation a t one of the thioethers would increase the mass by 16 amu (due to 0).Oxidation could have occurred during peptide isolation. IAFo.48-primase was tryptically digested and chromatographed and its major fluorescent peptide collected (accounting for about 85% of the total fluorescent peptides). The IAF-peptide (estimated to be 430 pmol) was subjected to Edman degradation, and a low yield (average 23 pmol per residue) with the sequence NFHA?CPFHN?K was obtained. This sequence corresponded to peptide 35-46. The M mass of the IAF-labeled peptide was 1890 amu according to MALDI. This was 2 amu less than that predicted for peptide 35-46 (Table 2). Since the MALDI instrument was precise to within about 0.05% (or 1.0 amu in 2000), this indicated close agreement. The Effect of Magnesium, Nucleotides, SingleStranded DNA and PMPS upon Fluorescence. At pH 7.5, the fluorescence intensity of FMl,o-primase was not altered by the addition of up to 150 mM magnesium, up to 200 pM ATP or a mixture of nucleoside triphosphates, up to 100 pM nucleotides of M13Gori singlestranded DNA, or any combination thereof. This indicated that these components with which primase interacts either did not bind near cysteine 39 or their binding did not alter the fluorescein environment. The environment of fluorescein leads to changes in its fluorescence intensity changes only when the pK of its carboxyl is shifted higher or lower. The lack of fluorescence change when FM-primase binds to its substrates reflected a lack of pK shift to higher values for the fluorescein carboxylic acid. The only way in which the fluorescein fluorescence was observed to change was in a PMPS titration (Figure 6). The FM-primase and IAF-primase fluorescence was quenched to the same extent when 3 equiv of PMPS had been added. Given that the fluorescein was attached to cysteine 39, it was located immediately adjacent to the "zinc finger" motif of primase (Figure 1) (Ilyina et al., 1992). Other studies in our laboratory have confirmed that the zinc in primase is ligated by three cysteines (Cook and M. A. Griep, unpublished results) such that PMPS can release the bound zinc from primase. PMPS is a cysteine-specific reagent used to characterize zincchelating cysteines (Boyer, 1954; Hunt et al., 1984; Giedroc et al., 1986; Wu et al., 1992~).The fluorescence quenching by PMPS could be due to either static quenching by the mercury atoms now in proximity to the fluoresceins or by a positive shift in the pK of the fluorescein carboxylate upon release of the zinc such that there was a n apparent quenching a t pH 7.5. These two possibilities were tested by determining the pK of the fluorescein before and after the addition of 3 equiv of PMPS to FM-primase. The quantum yield of
Primase Cysteine 39 Modification
0.08' 0
'
'
"
Bioconjugate Chem., Vol. 6, No. 6,1995 679
'
"
"
'
5 IO PMPSPrimase (molimol)
'
'
'
15
Figure 6. Fluorescence quenching of dye-labeled primase by PMPS. Dye-labeled primase (400 yL, 80 nMj was titrated with 1yL aliquots of 50 yM PMPS in a buffer of 50 mM HEPES, pH 7.5, 50 mM KC1 at 30 "C. The fluorescence intensity was monitored with excitation at 496 nm and emission at 520 nm. Fluorescence quenching was calculated after correction for background and dilution using the equation (F, - FYF, (= AFI Foj,where F, was the fluorescence intensity of the sample before adding PMPS and F was the fluorescence in the presence of PMPS.
fluorescein did not change, indicating that the PMPS had not caused static quenching. However, the fluorescein pK's for absorbance and quantum yield were shifted, respectively, to 6.1 and 6.4. These pK's after the addition of PMPS were in the middle of the range expected of a fluorescein. This result confirmed that the presence of zinc in primase had shifted the pK's to the observed low values (Table 1)and that zinc removal (by PMPS addition) had allowed them to return to less perturbed values. DISCUSSION
It is possible to attach a noninhibiting fluorescent probe to a specific residue of primase that can be used in structural studies. Primase cysteine 39 is rapidly labeled by the fluorescent dye FM and retains full primer synthesis activity. The absorption and fluorescence properties of the FM are not significantly perturbed by its environment except that its pKa is shifted to about 5.4. A probe a t this site is even more useful since we also demonstrated that cysteine 39 is near a n active site. The iodoacetamide functional group of IAF reacts with cysteine 39 slower than does the maleimide of FM. This probably reflects the chemical reactivity of the dyes rather than the cysteine. The attached IAF inhibits primase activity completely in direct contrast to a n attached FM. Since cysteine 39 can be labeled by FM without causing inhibition, it indicates that cysteine 39 is not an essential residue. The structural cause of the inhibition must lie in the linkage between the fluorescein moiety and the sulfur of cysteine 39. The attached IAF and FM may have different flexibilities due to their linkages. The different flexibilities may allow IAF to interfere with an adjacent region (or function) or prevent FM from doing so. The most obvious site to interfere with is the immediately adjacent zinc finger motif (Motif 1in Figure 1)(Ilyina et al., 1992). Although it is also possible that the fluorophores interact with other sites within the tertiary structure. Differential inhibition by IAF and FM has been observed before with another protein (Griep and McHenry, 1988). What was not observed with this other protein was the large pKa shift that is common to both types of fluorophores when attached to primase. If the succinimide ring is hydrolyzed after attachment of FM to primase, then a carboxylate is created that is not present in the acetamide species (Figure 5). Perhaps the additional negative charge is beneficial to maintenance of activity? Succinimide ring opening has been
observed or postulated to occur in many other systems (Wu et al., 1976; Lux and Gerard, 1981; Farley et al., 1984; Ishii and Lehrer, 1986; Filoteo et al., 1987; Griep and McHenry, 1990). The pKa of either fluorescein attached to cysteine 39 is low at -5.4 and possibly due to a strong interaction with the nearby zinc. The zinc would be expected to strongly stabilize the enolate form of fluorescein and thereby reduce its pKa. Cysteine 39 may be the most chemically reactive cysteine among the seven of primase for the same reason. The zinc may lower the pKa of cysteine 39's thiol to make it especially reactive to sulfhydryl-specific reagents. The pKa shift indicates that the dye is strongly influenced by its environment, which includes the adjacent zinc-binding residues cysteine 40, 60, 64, and histidine 43. The pKa for free or proteinbound fluorescein is typically near 6.2-6.9. Deprotonation of free fluorescein's xanthenone enol (the position 6' oxygen in Figure 5) to create dianionic fluorescein leads to the high absorbing (€492 nm = 88 000 M-I cm-l) and fluorescing (quantum yield = 0.92) species (Martin and Lindqvist, 1975). The monoanionic monoprotonated fluorescein absorbs (€437 nm = 30 000 M-l cm-l and €475 nm = 31 000 M-l cm-l) and fluoresces (quantum yield = 0.25-0.35) a t lower wavelengths and with less intensity. The pKa for the final deprotonation of fluoresceinylated biomolecules in moderate ionic strength buffers are typically only slightly shifted upward or downward relative to the free dye: the pKa was 6.20 on ribonuclease A (Garel, 1976); the pKa was 6.9 on dimeric and 6.6 on monomeric DNA polymerase I11 holoenzyme /3 subunit (Griep and McHenry, 1990); and the pK, was from 6.57.2 on various transfer RNAs (Friedrich et al., 1988). A particularly illustrative example of a strongly downward shifted fluorescein pKa is that which occurs when free fluorescein binds to anti-fluorescein antibody 4-4-20 (Omelyanenko et al., 1993). In this case, the crystal structure of the complex is known (Herron et al., 1989) and it is possible to establish that a n adjacent arginine, L34, stabilized the enolate form of fluorescein which lowered the pKa to 5.2. In contrast, antibody which was mutated to have a histidine at that position was hypothesized to stabilize the protonated form of fluorescein since the pKa was slightly shifted upward by 0.4 pH units relative to free fluorescein. A fluorescent probe a t position 39 places it near two conserved motifs. The two motifs within the aminoterminal 70 residues of E. coli primase are a cationic cluster (Stamford et al., 1992) and a "zinc finger" motif (Ilyina et al., 1992). Residues 26-34 in bacterial primases consist of a cluster of five conserved lysines and arginines. This region is not present in bacteriophage primases. The importance of this region for function was demonstrated using E. coli primase. Mutant primase lacking the amino-terminal 27 residues is not functional in the coupled primerDNA synthesis assay (Stamford et al., 1992). This demonstrates the importance of the first 27 amino acids for function. Even though position 27 truncation would delete only one of the residues within the cationic cluster, it may be a critical residue. Stamford and co-workers (Stamford et al., 1992) have hypothesized that this mutant lacks function because without the amino-terminal 27 residues the adjacent zinc finger motif cannot fold properly. The motif that is even closer in the linear sequence to the fluorescent probe is the zinc finger motif. The zinc finger motif contains the highest percentage of conserved and identical residues among primases (Ilyina et al., 1992; Versalovic and Lupski, 1993). The sequence of this motif in bacterial primases is Cys-XZ-His-Xl7-Cys-Xd2ys
680 Bioconjugate Chem., Vol. 6,No. 6,1995
(CHCC class of zinc fingers) and in E. coli includes residues 40-64 (Figure 1). In fact, it has been determined that primase from E. coli has one zinc cofactor per polypeptide chain (Stamford et al., 1992) (Cook and M. A. Griep, unpublished results). Likewise, a peptide based on the E. coli zinc finger sequence is capable of binding zinc (M. A. Griep and Hromas, unpublished results), indicating that this portion of primase is responsible for the zinc binding. In the present study, we found that three PMPS equivalents caused fluorescence quenching that was the result of a shift in the fluorescein pK. The simplest explanation is that PMPS was reacting with the three zinc-chelating cysteines, a t residues 40, 61, and 64, to release the zinc. In the absence of zinc, the enolate form of the fluorescein would not be stabilized and the pK could return to more normal values. In fact, we have found that PMPS binds to the zinc-chelating cysteines of primase and releases its single zinc (Cook and M. A. Griep, unpublished results). The binding of PMPS to the remaining three cysteines of primase did not lead to additional fluorescein quenching. This was expected if the remaining three cysteines, residues 306, 307, and 492, were far removed from the modified cysteine 39. The role of the zinc finger in primase has been determined for the primase from bacteriophage T7. Even though the bacterial and bacteriophage primases have considerable amino acid sequence differences, they do appear to share many conserved residues (Ilyina et al., 1992). Perhaps an example of a minor difference is that the zinc finger motif in bacteriophage differs from that in bacteria in that the putative ligating residues are all cysteine, Cys-Xz-Cys-X1~-24-Cys-Xz-Cys (CCCC class of zinc fingers). One important difference is that bacteriophage T7 and P4 primases are a t the amino terminus of a polypeptide chain that also includes helicase (Ilyina et al., 1992; Ziegelin et al., 1993). In T7, it has been established that there is one zinc bound to the primase portion of the chain (Mendelman et al., 1994). It was established by site-directed mutagenesis that the aboveindicated four cysteines play a n important role with regard to protein function. A mutant primasehelicase in which the third cysteine was mutated to serine was found to bind less zinc than wild-type and did not catalyze template-dependent primer synthesis. On the basis of these observations, it is likely that all primases bind a t least one zinc and that the zinc finger plays an important role in template-directed primer synthesis. However, the mechanism by which the zinc finger carries out its role in template-directed synthesis has not been proven. To explain the role of the zinc finger in templatedirected mutagenesis, Bernstein and co-workers (Bernstein and Richardson, 1988) hypothesized that the zinc finger domain of T7 primasehelicase was responsible for the specific trinucleotide recognition that is characteristic of most primases. For instance, E. coli primase initiates primer synthesis from d(CTG)and bacteriophage T7 from d(GTC) (Mendelman and Richardson, 1991). This hypothesis was made in analogy to the CCHH class of zinc fingers which are found in a particular class of eukaryotic transcription factors. The CCHH class of zinc fingers fold into a common type of secondary structure (Lee et al., 1992) which is coded for by its sequence. Two of these transcription factors bind specific sequences of duplex DNA via particular residues in their single a helix according to their crystal structures (Pavletich and Pabo, 1991, 1993). One major difference between the CCHH class and the primase class of zinc fingers is that the CCHH class specifically binds duplex DNA while pri-
Griep and Mesman
mases specifically bind single-stranded DNA. It is also not clear whether all zinc fingers are capable of binding DNA or RNA with specificity. In addition, the secondary structure of CCHH zinc fingers may differ from the primase zinc fingers (Qian et al., 1993; Mendelman et al., 1994). However, this remains a very good hypothesis to explain the mechanism by which sequence-specific primer initiation is achieved using the zinc finger. A possible related mechanism for the function of the primase zinc finger is selective binding of the initiating nucleotides ATP and GTP. This is taken in analogy to the studies of E. coli RNA polymerase. Besides the obvious functional similarities between transcription RNA polymerases and primase, there are underlying structural and functional similarities especially with regard to one of the zincs. The multisubunit E. coli RNA polymerase has two zinc prosthetic groups. The zinc from the ,L? subunit of RNA polymerase (B-site Zn(I1)) is removable by PMPS treatment (Giedroc and Coleman, 1986) as is the zinc from primase (Cook and M. A. Griep, unpublished results). Since the role of the removable RNA polymerase zinc is to bind the initiating nucleotide ATP (Chatterji and Wu, 1982; Chatterji et al., 19841,this may be the role that it plays in primase. To disrupt this function should disrupt ATP-directed initiation which can be interpreted as ssDNA sequence-specific initiation disruption since complementary base pair formation is also required for initiation. Studies of the replication fork in action will require fluorescent probes for the various enzymes and proteins involved and once created can be used as sensitive probes of structure. For instance, the fluorescently labeled ,&subunit of DNA polymerase I11 holoenzyme has already been shown to be sensitive to four different DNA-bound states of the polymerase (Griep and McHenry, 1992). To study the interactions between primase and the DNA polymerase during replication will require a site-specific label on primase that does not alter its ability to synthesize a primer or interfere with its ability to bind to the DNA polymerase. The present study has shown that IAF-labeled primase cannot be used for this purpose since it has reduced activity in the coupled RNA/DNA synthesis assay and must be deficient in one of those properties. However, an inhibitory label near the zinc finger domain may prove useful in some studies. The zinc finger domain is very far removed in primary sequence from the helicase-interacting carboxyl-terminal domain of primase and will probably not interfere with primasehelicase binding. A lack of primase/ssDNA binding may prove desirable when studying the primase-helicase interaction. Nevertheless, FM-primase is labeled a t the same site, does not inhibit primer synthesis activity, and can certainly be used in further studies of the interaction of primase a t a replication fork. ACKNOWLEDGMENT
We would like to thank Jennifer Miller for her pioneering efforts toward the fluorescent labeling of primase in our lab. We would like to thank Elsbeth Cook for purifying the primase and the lab members for valuable critiquing of the manuscript while it was in preparation. LITERATURE CITED Arai, K.-i., and Kornberg, A. (1981) Mechanism of dnaB Protein Action. IV. General Priming of DNA Replication by dnaB Protein and Primase Compared with RNA Polymerase. J . Biol. Chem. 256, 5267-5272. h g o s , P. (1988)A Sequence Motif in Many Polymerases. Nucleic Acids Res. 16, 9909-9916.
Primase Cysteine 39 Modification Bernstein, J. A,, and Richardson, C. C. (1988) A 7-kDa Region of the Bacteriophage T7 Gene 4 Protein Is Required for Primase but not for Helicase Activity. Proc. Natl. Acad. Sci. U.S.A. 85, 396-400. Bouch6, J.-P., Zechel, K., and Kornberg, A. (1975) dnaG Gene Product, a Rifampicin-resistant RNA Polymerase, Initiates the Conversion of a Single-stranded Coliphage DNA to Its Duplex Replicative Form. J . Biol. Chem. 250, 5995-6001. Boyer, P. D. (1954) Spectrophotometric Study of the Reaction of Protein Sulfhydryl Groups with Organic Mercurials. J . Am. Chem. SOC. 76, 4331-4337. Bradford, M. M. (1976) A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Anal. Biochem. 72, 248254. Brinkley, M. (1992) A Brief Survey of Methods for Preparing Protein Conjugates with Dyes, Haptens, and Cross-Linking Reagents. Bioconjugate Chem. 3, 2-13. Burton, Z. F., Gross, C. A., Watanabe, K. K., and Burgess, R. R. (1983) The Operon that Encodes the Sigma Subunit of RNA Polymerase also Encodes Ribosomal Protein S21 and DNA Primase in Escherichia coli K12. Cell 32, 335-349. Chatterji, D., and Wu, F. Y.-H. (1982) Direct Coordination of Nucleotide with the Intrinsic Metal in Escherichia coli RNA Polymerase: A Nuclear Magnetic Resonance Study with Cobalt-Substituted Enzyme. Biochemistry 21, 4657-4664. Chatterji, D., Wu, C.-W., and Wu, F. Y.-H. (1984) Nuclear Magnetic Resonance Studies on the Role of Intrinsic Metals in Escherichia coli RNA Polymerase: Effect of DNA Template on the Nucleotide-Enzyme Interaction. J . Biol. Chem. 259, 284-289. Farley, R. A., R a n , C. M., Carilli, C. T., Hawke, D., and Shively, J. E. (1984) The Amino Acid Sequence of a FluoresceinLabeled Peptide from the Active Site of (Na,K)-ATPase. J . Biol. Chem. 259, 9532-9535. Filoteo, A. G., Gorski, J. P., and Penniston, J. T. (1987) The ATP-Binding Site of the Erythrocyte Membrane Ca2+Pump: Amino Acid Sequence of the Fluorescein IsothiocyanateReactive Region. J . Biol. Chem. 262, 6526-6530. Friedrich, K., Woolley, P., and Steinhauser, K. G. (1988) Electrostatic Potential of Macromolecules Measured by pKa Shift of a Fluorophore. 2. Transfer RNA. Eur. J . Biochem. 173, 233-239. Funnel, B. E., Baker, T. A., and Kornberg, A. (1986) Complete Enzymatic Replication of Plasmids Containing the Origin of the Escherichia coli Chromosome. J . Biol. Chem. 261, 56165624. Garel, J.-R. (1976) pK Changes of Ionizable Reporter Groups as an Index of Conformational Changes in Proteins: A Study of Fluorescein-Labeled Ribonuclease A. Eur. J. Biochem. 70, 179-189. Giedroc, D. P., and Coleman, J. E. (1986) Structural and Functional Differences between the Two Intrinsic Zinc Ions of Escherichia coli RNA Polymerase. Biochemistry 25,49694978. Giedroc, D. P., Keating, K. M., Williams, K. R., Konigsberg, W. H., and Coleman, J. E. (1986) Gene 32 Protein, the SingleStranded DNA Binding Protein from Bacteriophage T4, Is a Zinc Metalloprotein. Proc. Natl. Acad. Sci. U.S.A. 83, 84528456. Griep, M. A., and McHenry, C. S. (1988) The Dimer of the p Subunit of Escherichia coli DNA Polymerase I11 Holoenzyme Is Dissociated into Monomers upon Binding Magnesium(I1). Biochemistry 27, 5210-5215. Griep, M. A., and McHenry, C. S. (1989) Glutamate Overcomes the Salt Inhibition of DNA Polymerase 111 Holoenzyme. J . Biol. Chem. 264, 11294-11301. Griep, M. A., and McHenry, C. S. (1990) Dissociation of the DNA Polymerase I11 Holoenzyme pz Subunits Is Accompanied by Conformational Change a t Distal Cysteines333. J. Biol. Chem. 265, 20356-20363. Griep, M. A., and McHenry, C. S. (1992) Fluorescence Energy Transfer Between the Primer and the /3 Subunit of Escherichia coli DNA Polymerase I11 Holoenzyme. J. Biol. Chem. 267, 3052-3059.
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