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Protein-Protein and Protein-DNA Interactions at the Bacteriophage T4 DNA Replication Fork

Protein-Protein and Protein-DNA Interactions at the Bacteriophage T4 DNA Replication Fork THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 45, Issue of November 8, pp. 28045–28051, 1996 © 1996 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Protein-Protein and Protein-DNA Interactions at the Bacteriophage T4 DNA Replication Fork CHARACTERIZATION OF A FLUORESCENTLY LABELED DNA POLYMERASE SLIDING CLAMP* (Received for publication, May 28, 1996, and in revised form, August 3, 1996) Daniel J. Sexton, Theodore E. Carver, Anthony J. Berdis, and Stephen J. Benkovic‡ From the Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802 The T4 DNA polymerase holoenzyme is composed of ture is now known to resemble the “sliding clamp” ring struc- the polymerase enzyme complexed to the sliding clamp ture previously shown for the analogous proteins from E. coli (the 45 protein), which is loaded onto DNA by an ATP- (b-clamp) and eucaryotes (proliferating cell nuclear antigen) dependent clamp loader (the 44/62 complex). This paper (5, 6). The 45 protein is loaded onto DNA at a primer template describes a new method to directly investigate the junction through the ATP-dependent activity of the 44/62 pro- mechanism of holoenzyme assembly using a fluores- tein complex. The 44/62 complex has a functional and a limited cently labeled cysteine mutant of the 45 protein. This sequence homology (7) to the g complex clamp loader of E. coli protein possessed unaltered function yet produced sub- (8) and the RF-C complex of eucaryotes (9). The oligomeric stantial changes in probe fluorescence intensity upon stoichiometry of the 44/62 complex is four 44 subunits to one 62 interacting with other components of the holoenzyme. subunit with the ATPase activity residing on each of the 44 These fluorescence changes provide insight into the role subunits (10). Recently, it was quantitatively demonstrated of ATP hydrolysis in holoenzyme assembly. Using either that the 44/62 complex loads the 45 protein in a catalytic ATP or the non-hydrolyzable ATP analog, adenosine 5*- manner and also functions as a chaperone protein to ensure O-(3-thiophosphate), events in holoenzyme assembly productive holoenzyme complex (11, 12). The amount of ATP were assigned as either dependent or independent of consumed in the 44/62-dependent loading of the 45 protein onto ATP hydrolysis. A holoenzyme assembly mechanism is primed DNA has recently been shown to be consistent with the proposed in which the 44/62 complex mediates the asso- hydrolyis of ATP at all four ATPase sites in the 44/62 complex ciation of the 45 protein with DNA in an ATP-dependent (13). manner not requiring ATP hydrolysis. Upon ATP hy- Despite the wealth of information regarding the roles of the drolysis, the 44/62 complex triggers a conformational different T4 replication proteins in DNA synthesis, there re- change in the 45 protein that may be attributed to the clamp loading onto DNA. main a number of individual events or “microprocesses” for which mechanisms are undetermined. For example, by divid- ing the T4 replication activity into three processes, 1) the assembly of the holoenzyme, 2) the synthesis of DNA, and 3) In most biological systems, the replication of DNA involves the coordinated actions of a multitude of different proteins. In the disassembly of the holoenzyme, it is evident that there exist events within each process that may be considered micro- general, as the complexity of the organism increases so do the number of proteins participating in DNA replication. The bac- processes. One such microprocess within the assembly of the holoenzyme concerns the development of the protein-protein teriophage T4 DNA replication system is well suited for a mechanistic investigation of the protein-protein interactions interaction(s) between the 45 protein and the 44/62 complex that are required to load the 45 protein onto DNA. The mech- required for replication due to the relatively low number of proteins involved and the apparent functional similarities to anism by which the 44/62 complex loads the 45 protein onto DNA is not known. It is known that ATP hydrolysis is required other more complicated systems such as those found in Esche- richia coli and eucaryotes (reviewed in Refs. 1 and 2). for the stimulation of the T4 polymerize processivity by the accessory proteins (14). However, ATP hydrolysis appears not The bacteriophage T4 replication fork is comprised of the DNA polymerase enzyme (the product of gene 43), the DNA to be necessary for a 44/62 complex-mediated 45 protein-DNA interaction since this interaction was observed with the non- polymerase accessory proteins (the 44/62 complex and the 45 protein), as well as the ssDNA-binding protein (the 32 protein), hydrolyzable ATP analog, ATPgS, by both DNA footprinting (15) and protein-DNA cross-linking experiments (16). ATP hy- the helicase (the 41 protein), the primase (the 61 protein), and the helicase accessory protein (the 59 protein) (3, 4). The T4 drolysis was required to observe, by cyroelectron microscopy, structures (termed “hash marks”) on nicked DNA that corre- DNA polymerase holoenzyme consists of the T4 polymerase, which possesses both a 59–39 polymerase and a 39–59 exonucle- sponded to 45 protein trimers (17) that emphasizes the impor- tance of ATP hydrolysis in loading the 45 protein onto DNA. ase activity, and the 45 protein. The homotrimeric 45 protein is the processivity factor for the polymerase, and its x-ray struc- The investigation of the DNA loading mechanism of the 45 protein is made difficult by the fact that 45 protein does not * This work was supported by a National Institutes of Health Grant GM13306 (to S. J. B.). The costs of publication of this article were J. Kuriyan, personal communication. defrayed in part by the payment of page charges. This article must The abbreviations used are: ATPgS, adenosine 59-O-(3-thiophos- therefore be hereby marked “advertisement” in accordance with 18 phate); bio, biotin; DCIA, 7-diethylamino-3-((49-iodoacetyl)amino)phen- U.S.C. Section 1734 solely to indicate this fact. yl)-4-methylcoumarin; DTNB, 5,59-dithio-bis(2,29-nitrobenzoic acid); ‡ To whom correspondence should be addressed: Dept. of Chemistry, IAANS, 2-(49-(iodoacetamido)anilino)naphthalene-6-sulfonic acid, so- 152 Davey Laboratory, Pennsylvania State University, University dium salt; IAEDANS, 5-((((iodoacetyl)amino)ethyl)amino)naphthalene- Park, PA 16802. Tel.: 814-865-2882; Fax: 814-865-2973. E-mail: 1-sulfonic acid; IANBD, N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7- [email protected]. nitrobenz-2-oxa-1,3-diazole; IAF, 5-iodoacetamidofluorescein. This paper is available on line at http://www-jbc.stanford.edu/jbc/ 28045 This is an Open Access article under the CC BY license. 28046 Fluorescently Labeled DNA Polymerase Sliding Clamp on protein function was assessed by substitution for wild type, unla- possess a measurable intrinsic activity. It would therefore be beled 45 protein. Initial rates of ATP hydrolysis were obtained under advantageous to have a means of directly monitoring the 45 conditions where less than 10% of the limiting reactant was utilized protein interactions during complex formation. over the reaction time course. In this paper, we describe a fluorescently labeled cysteine Pre-steady State ATPase Assay—The pre-steady state ATPase rates mutant of the 45 protein. It is demonstrated that this fluores- were performed using a rapid quench instrument (21) as described cently labeled 45 protein provides a novel means for the direct previously (13). The ATPase rates were obtained from an assay mixture containing 250 nM 44/62 complex, 250 nM 45 protein (or labeled 45 investigation of certain 45 protein-protein interactions as well protein), 500 nM biotinylated 34/62/36-mer DNA, 1 mM streptavidin, 1 as 45 protein-DNA interactions. The observed fluorescence mM ATP, 50 nM [g- P]ATP in buffer C at 25 °C. Briefly, the reactions changes enabled the presentation of a partial mechanism for were quenched with 1 M HCl and neutralized with an appropriate T4 holoenzyme assembly. The change in the fluorescence of the amount of 3 M NaOH in 1 M Tris base followed by a thin layer chro- 32 32 labeled 45 protein that occurs upon holoenzyme formation has matographic separation of [g- P]ATP from g- P and then radiochem- also provided a new assay for the determination of the T4 ical analysis using a Molecular Dynamics Phosphorimager. Strand Displacement Assay—A strand displacement assay was uti- holoenzyme-DNA complex dissociation rate constant. lized to further characterize the effect of T7C-45 protein thiol modifi- EXPERIMENTAL PROCEDURES cation on the formation of active holoenzyme. This assay has been Materials—Oligonucleotides were synthesized with an Expedite described in detail elsewhere (12). Briefly, an assay mixture containing 8909 DNA synthesizer (Perceptive Biosystems) and purified according 50 nM P-59-end-labeled, biotinylated 34/62/36-mer DNA, 55 nM to Capson et al. (18). Biotin-labeled oligonucleotides were prepared streptavidin, 1 mM ATP, and 10 mM dCTP is preincubated 30 s with 10 using a BioTEG phosphoramidite as obtained from Glen Research. The nM T4 D219A polymerase, 55 nM 44/62 complex, 55 nM 45 protein before fluorescent probes DCIA, dibromobimane, IAEDANS, IANBD, IAF, addition of 10 mM remaining deoxynucleotides (dATP, dTTP, and dGTP) IAANS, 2-(49-(iodoacetamido)anilino)naphthalene-6-sulfonic acid, so- and 1 mg/ml salmon sperm single strand DNA trap. Following the dium salt were obtained from Molecular Probes, and pyrene maleimide addition of the remaining dNTPs and single strand DNA trap, aliquots was obtained from Acros. ATPgS was purchased from Boehringer were removed at different times and quenched in 2 M HCl and extracted Mannheim as a 98% pure solution and used without further purifica- with phenol:chloroform:isoamyl alcohol (25:24:1). The zero point was tion. DuPont NEN was the source of the [g- P]ATP used to 59-end label obtained by omitting the protein from the assay mixture. The omission oligonucleotides with T4 polynucleotide kinase (U. S. Biochemical of the remaining three nucleotides provided an experimental control to Corp.). All other biochemicals and chemicals were obtained from Sigma assess the incorporation extent of the first nucleotide. Prior to loading or Fisher and were of analytical grade or better. onto a 16% polyacrylamide, 8 M urea, sequencing gel, the samples were DNA encoding a threonine to cysteine mutation at position 7 in the neutralized with the addition of an appropriate amount of 3 M NaOH in 45 protein (T7C-45 protein) ligated into a pET 26b expression vector 1 M Tris-base. The sequencing gels were exposed to constant current was a generous gift of Dr. John Kuriyan (Rockefeller University). Fol- electrophoresis, and the distribution of the radioactivity was analyzed lowing transformation of the DNA into BL21-DE3 E. coli and IPTG- using a Molecular Dynamics Phosphorimager. induced expression, the T7C-45 protein was purified according to Nos- Steady State Fluorescence—Steady state fluorescence measurements sal (19). The wild type 45 protein and the 44/62 complex were purified were performed using an SLM Aminco 8000C photon counting spec- from overproducing strains obtained from Dr. William Konigsberg (Yale trofluorometer equipped with a thermostated cell compartment that University) as described previously (19). The T4 exonuclease-deficient was maintained at 25 °C. The experiments were performed in buffer C polymerase D219A mutant was purified according to Frey et al. (20). in the presence of an appropriate amount of T7C-45-ANBD protein to The D219A mutant of the T4 polymerase was used in these studies to provide a satisfactory fluorescence signal (usually 250 nM). The effects avoid complications arising from the presence of the exonuclease activ- of the other components of the T4 polymerase holoenzyme (i.e. proteins, ity. The polymerase activity of the T4 D219A polymerase is identical to DNA, and/or ATP) on the T7C-45-ANBD fluorescence were observed that of the wild type enzyme (20). upon their direct addition to the fluorescence cuvette. The fluorescence Primer Template Construction—The biotinylated 34/62/36-mer DNA spectra obtained were normalized for the effects of dilution. substrate was constructed as described previously (12). This DNA sub- Stopped-flow Fluorescence—Stopped-flow fluorescence measure- strate is composed of a 34-mer primer annealed to a 39-biotinylated ments were performed using an Applied Photophysics stopped-flow 62-mer with a 36-mer fork strand annealed to the 59-end of the template instrument at a constant temperature of 25 °C. The changes in fluores- with an 18-base overhang. cence were observed upon stopped-flow mixing of syringe A and syringe T7C-45 Protein Cysteine Modification—The modification of the cys- B. Syringe A contained 500 nM T7C-45-ANBD protein, 500 nM of the teine thiol of the T7C-45 protein was performed upon passage of the 44/62 complex, 1 mM ATP, 500 nM biotinylated 34/62/36-mer DNA, 500 protein solution over a G-25 column equilibrated with degassed buffer nM T4 D219A polymerase, and 550 nM streptavidin in buffer C. Syringe A (50 mM HEPES, 150 mM KOAc, 1 mM EDTA, 10% glycerol, pH 7.8) B contained 20 mM glucose and 20 units/ml glucose hexokinase in buffer followed by the addition of a 10-fold molar excess of thiol reagent over C. The concentration of protein and DNA in the two syringes of the protein monomer. The cysteine labeling reaction was allowed to proceed stopped-flow instrument was diluted by a factor of 2 upon stopped-flow approximately4hat4 °C. Excess thiol reagent was removed by exten- mixing. sive dialysis against buffer B (50 mM Tris, 50 mM NaCl, 5 mM EDTA, 1 Data Analysis—Data obtained from the pre-steady state rate of ATP mM b-mercaptoethanol, 10% glycerol, pH 8.0). The labeled protein so- hydrolysis by the 44/62 complex were fit to Equation 1: lution was then concentrated in a microconcentrator (Centricon-30) and 2Bt stored at 280 °C. The extent of cysteine modification was quantitated y 5 Ae 1 C (Eq. 1) by measuring the dye concentration from its molar extinction at a where A is the amplitude of the pre-steady state phase, B is the wavelength other than 280 nm. Protein concentration was determined pre-steady state rate constant, and C is a constant. Data obtained from using the Bradford assay with the 45 protein as the standard. Labeling the stopped-flow fluorescence determination of the kinetics of holoen- efficiencies are reported as mol of dye/mol of 45 trimer. zyme dissociation were best fit by Equation 2: Steady State ATPase Assay—Steady state ATP hydrolysis measure- ments were performed using a phosphoenolpyruvate kinase/lactate de- 2Bt y 5 Ae 1 Ct 1 D (Eq. 2) hydrogenase enzyme coupled system where the consumption of ATP was monitored spectrophotometrically (OLIS-Cary-14 spectrophotome- where A is the amplitude of the exponential decay, B is the exponential ter) upon oxidation of NADH (13). ATPase activity was observed upon decay rate constant, C is a linear decay rate, and D is a constant. addition of the 45 protein (250 nM) to a solution containing 250 nM 44/62 complex, 250 nM biotinylated 34/62/36-mer DNA, 1 mM streptavidin, 1 RESULTS mM ATP, 10 mM phosphoenolpyruvate, 200 mM NADH, 6 units of phos- phoenolpyruvate kinase/lactate dehydrogenase mix, and buffer C (150 Fluorescent Labeling of the 45 Protein—The wild type 45 mM potassium acetate, 10 mM magnesium acetate, 10 mM b-mercapto- protein is devoid of cysteine residues. The site-selective intro- ethanol, and 25 mM Tris, pH 7.5) at 25 °C. The T4 D219A polymerase duction of a cysteine residue into the sequence of the 45 protein protein was subsequently added to observe the inhibition of the ATP enables the covalent attachment of a thiol-reactive fluorescent hydrolysis upon stable complex formation. The effect of the mutation of molecule at a known position in the primary structure. A mu- the threonine at position 7 to a cysteine in the 45 protein (the T7C-45 protein) as well as the subsequent thiol derivatization of the T7C thiol tated 45 protein, in which a threonine residue at position 7 Fluorescently Labeled DNA Polymerase Sliding Clamp 28047 TABLE I 44/62 complex steady state ATPase activity (Table II). Thiol reagent labeling efficiency of the T7C-45 protein A rapid chemical quench method has been developed to Thiol reagent Probe:trimer measure the pre-steady state kinetics of ATP hydrolysis by the 44/62 complex (13). The ability of the T7C-45-ANBD protein to mol/mol substitute for the wild type 45 protein under pre-steady state DCIA 0.6 conditions was also assessed (Table II). No significant differ- Dibromobimane 2.3 DTNB 3 ence was observed between the T7C-45-ANBD protein and the IAANS 0.4 wild type 45 protein with respect to the measured pre-steady IAEDANS 1.3 state rates and the burst amplitudes (Table II). The pre-steady IAF 1.5 state ATPase rate is determined by the chemical rate of ATP IANBD 0.9 Pyrene maleimide 1.2 hydrolysis rather than product release (steady state rate). The burst amplitude relative to the 44/62 complex concentration The labeling efficiency was determined as described under “Exper- provides the stoichiometry of ATP consumption. At a concen- imental Procedures” where the concentration of each probe was deter- mined using the following molar extinction coefficients: DCIA, 33,000 tration of 250 nM the burst amplitude for both forms of the 45 21 21 liter z cm z mol at 382 nm; dibromobimane, 5,300 liter z cm z mol at protein was approximately 1 mM which corresponds to 4 mol 397 nm; DTNB 13,600 liter z cm z mol at 412 nm; IAANS, 26,000 21 21 of ATP hydrolyzed per mol of 44/62 complex as previously liter z cm z mol at 329 nm; IAEDANS, 5,700 liter z cm z mol at 336 reported (13). nm; IAF, 82,000 liter z cm z mol at 491 nm; IANBD, 23,000 liter z cm z mol at 472 nm and pyrenemaleimide, 36,000 liter z To further examine the effect of the T7C mutation and sub- cm z mol at 339 nm. sequent thiol derivatization of the 45 protein on holoenzyme function, a strand displacement assay was utilized. In this from the N terminus, was site-specifically mutated to a cys- 32 assay the fork strand (36-mer) of a 59- P-biotinylated 62/34/ teine residue producing the threonine 7 to cysteine 45 mutant 36-mer is capable of being displaced by the holoenzyme but not protein (T7C-45 protein). The details for the creation of this by the T4 polymerase alone (11, 12). Displacement of the fork cysteine mutant will be published elsewhere. strand gives rise to the production of a fully extended 62-mer The cysteine mutant, T7C-45 protein, was modified using primer strand that can be differentiated from the bio62-mer several different thiol-reactive probes (Table I). The thiol quan- template strand on a polyacrylamide sequencing gel. Quanti- titating reagent 5,59-dithiobis(2,29-nitrobenzoic acid) (DTNB; tation of the radioactivity of the 62-mer primer strand yields a Ref. 22) reacted slowly with three of the thiols in the trimer measure of the ability of the T4 replicative proteins to form indicating the presence of only the reduced form of the cys- productive holoenzyme complex. Fig. 1 demonstrates that the teines. No evidence of nonequivalent cysteine thiols in the T7C-45 mutant protein displays strand displacement activity T7C-45 protein trimer was provided by the DTNB reactivity comparable with the wild type 45 protein even when derivat- since the reaction was best approximated by a single exponen- ized with IANBD. tial (data not shown). However, thiol modification of the cys- Steady State Fluorescence of T7C-45-ANBD—The T7C-45- teine T7C-45 protein with the majority of the fluorescent ANBD protein was selected for further study based on two probes consistently led to a probe:trimer ratio less than 3:1, the important properties. First, the ability of the T7C-45-ANBD ratio expected if all thiols in the trimer were modified (Table I). protein to substitute for the wild type 45 protein in the assays It is evident that for most of the probes tested in this study the described above, without a measurable loss of activity (Table II probe:trimer ratio was close to 1:1 (omitting DTNB, the aver- and Fig. 1), makes this labeled 45 protein potentially suitable age probe:trimer ratio equals 1.2:1) which suggests that only for monitoring the interactions of the 45 protein at the T4 one out of three of the cysteines in the trimer is modified. Of the replication fork. Second, the environmentally sensitive nature fluorescent probes tested in Table I, dibromobimane incorpo- of T7C-45-ANBD has been shown to permit the detection of rated to the greatest extent (2.3:1) while the naphthalene de- certain 45 protein-44/62 complex and 45 protein-DNA interac- rivative (IAANS) was the least reactive (0.4:1). A titration of tions (Fig. 2). As shown in Fig. 2, the addition of the 44/62 the remaining thiols of the T7C-45-ANBD protein with DTNB complex to a solution of the T7C-45-ANBD protein resulted in yielded 2 thiols per trimer, which was again consistent with a no measurable change in the fluorescence emission spectrum. labeling efficiency for IANBD of only 1 probe:trimer. The subsequent addition of ATP to the above solution contain- Activity of Fluorescently Labeled T7C-45 Protein—The meas- ing T7C-45-ANBD and 44/62 complex caused a marked (ap- urement of the degree of stimulation of the 44/62 complex proximately 1.7-fold) increase in the fluorescence intensity. In steady state ATPase activity by the fluorescently labeled the absence of the 44/62 complex ATP had no effect on the T7C-45 proteins provides a convenient assay to assess the fluorescence of the T7C-45-ANBD protein (data not shown). effects of derivatization on 45 protein function (Table II). ATP That fluorescence increase was then attenuated by the addition is consumed as the 44/62 complex loads the 45 protein onto the of the bio34/62/36-mer to an intermediate level of fluorescent biotinylated 34/62/36 DNA substrate. In the absence of the T4 intensity approximately 1.3 times greater than that of the polymerase, the 45 protein dissociates from DNA allowing it to T7C-45-ANBD alone. The fluorescence of the T7C-45-ANBD be reloaded by the 44/62 complex at the expense of additional protein is insensitive to the addition of the T4 polymerase ATP hydrolysis. The addition of the T4 polymerase diminishes whether it is added to the mixture of T7C-45-ANBD protein, ATP consumption through the formation of holoenzyme, which 44/62 complex, and DNA (Fig. 2) or in any other order of is more stable on DNA than the 45 protein alone (13). As shown addition (data not shown). in Table II, the T7C-45 mutant displays essentially identical A demonstration of whether or not ATP hydrolysis is re- activity to that of the wild type 45 protein. Labeling the T7C-45 quired for a particular biochemical event is often made possible protein with iodoacetamidofluorescein (IAF) led to a decrease by testing the ability of nonhydrolyzable ATP analogs to sub- in the observed stimulation of the ATPase activity of the 44/62 stitute for ATP. ATPgS has been shown to be a potent inhibitor complex (Table II). The modification of the T7C-45 protein with of the 44/62 complex ATPase activity, whereas the ATP analogs IANBD, however, did not affect its ability to stimulate the 4 AMP-PNP and AMP-PCP are much weaker inhibitors (14). The T7C-45 mutant protein was obtained from the Kuriyan Labo- ratory (Rockefeller University). A. J. Berdis, unpublished data. 28048 Fluorescently Labeled DNA Polymerase Sliding Clamp TABLE II Stimulation of the DNA-dependent 44/62 complex ATPase activity by fluorescently labeled 45 proteins Steady state Steady state Pre-steady state Pre-steady state 45 protein a a b b rate 1 rate 2 rate amplitude nM/s nM/s s mM WT 45 398 87 3.1 0.9 T7C-45 421 110 ND ND T7C-ANBD 428 115 3.9 1.2 T7C-AF 228 103 ND ND Steady state ATPase activity was performed as described under “Experimental Procedures.” Steady state rate 1 corresponds to the ATPase rate resulting from the stimulation by the 45 protein, and steady state rate 2 is the rate observed upon addition of the D219A T4 polymerase to the solution that contains the 45 protein. The difference between these values and those reported in Berdis and Benkovic (13) may be due to different preparations of the same DNA substrate. Pre-steady state ATPase measurements were performed as described under “Experimental Procedures.” ND, not determined. FIG.1. Strand displacement assay in the presence of the T7C- 45-ANBD protein. The above sequencing gel (16% acrylamide, 8 M urea) was used to separate the polymerase extension products using the P-59-end-labeled, biotinylated 34/62/36-mer DNA substrate. The lanes in A are the extension products observed in the presence of the polym- erase alone (i.e. no accessory proteins), at different time points (0, 10, FIG.2. Steady state fluorescence of the T7C-45-ANBD protein. 20, and 30 s) plus a control point. The control point represents the The T7C-45-ANBD steady state fluorescence was observed in buffer C incorporation of just the first nucleotide. B represents the results of the with 275 nM streptavidin at 25 °C at an excitation wavelength of 475 holoenzyme with wild type 45 protein, and C and D represent the nm and an emission wavelength of 530 nm. Spectrum 1 was obtained in observed results of the holoenzyme when the T7C-45 protein and the the presence of 250 nM T7C-45-ANBD protein, and spectrum 2 is the T7C-45-ANBD protein, respectively, were substituted for the wild type result of the addition of 250 nM of the 44/62 complex. Spectrum 3 45 protein. Strand displacement was observed according to the appear- contains 250 nM T7C-45-ANBD protein, 250 nM of the 44/62 complex, ance of the fully extended primer as a 62-mer (indicated by the arrow) and1mM ATP. Spectrum 4 contains 250 nM T7C-45-ANBD protein, 250 that was resolved from the template biotinylated 62-mer. The strand nM of the 44/62 complex, 1 mM ATP, and 250 nM biotinylated 34/62/36- displacement experiment was performed as described under “Experi- mer DNA, and spectrum 5 was recorded with 250 nM T7C-45-ANBD mental Procedures.” protein, 250 nM of the 44/62 complex, 1 mM ATP, 250 nM biotinylated 34/62/36-mer DNA, and 250 nM T4 D219A polymerase. The ATP analogs, AMP-PNP and AMP-PCP, did not produce any change in the fluorescence of the T7C-45-ANBD protein Holoenzyme-DNA Dissociation Rate Constant—Stopped-flow under the above experimental conditions and at a concentra- fluorescence rapid mixing experiments were performed to tion of up to 10 mM ATP analog (data not shown). measure the dissociation rate of the holoenzyme from the At a concentration of 1 mM, ATPgS was not able to elicit all bio34/62/36-mer. In order to measure the holoenzyme dissoci- of the fluorescence changes mediated by ATP in Fig. 2. How- ation rate, the holoenzyme was assembled onto the DNA sub- ever, there is a fluorescence increase upon the addition of DNA strate in one syringe and pushed against another syringe con- to a mixture containing T7C-45-ANBD and the 44/62 complex taining excess glucose and hexokinase that rapidly consume in the presence of ATPgS (Fig. 3). That fluorescence increase is the ATP. Under the experimental conditions (10 units/ml hex- not affected by the presence or absence of T4 polymerase or the okinase) the ATP (1 mM) should be consumed in approximately order of addition (data not shown). Magnesium ion (10 mM) was 6 s. The approximate dead time of the experiment is therefore required to observe the ATPgS-dependent fluorescence change. 6 s. The data obtained are shown in Fig. 5 where it can be seen The Stoichiometry of the T7C-45-ANBD:44/62 Interaction— that after stopped-flow mixing of the assembled holoenzyme- The stoichiometry of the ATP-dependent interaction between DNA complex with glucose and hexokinase there is a decrease the T7C-45-ANBD protein and the 44/62 complex was deter- in fluorescence that can be approximated by a single exponen- mined by varying the concentration of 44/62 complex. As shown ) followed by a linear decrease in Fig. 4, the increase in fluorescence reaches a plateau at tial decay (0.011 6 0.002 s 25 26 21 (7.8 3 10 6 2 3 10 s ). The single exponential decay rate approximately a 1:1 molar ratio of 44/62 complex to T7C-45- ANBD protein. represents the dissociation rate constant of the holoenzyme- Fluorescently Labeled DNA Polymerase Sliding Clamp 28049 FIG.5. Determination of the T4 polymerase holoenzyme-DNA dissociation rate constant. Stopped-flow fluorescence was used to measure the T4 polymerase holoenzyme-DNA dissociation rate con- stant where syringe A contained 500 nM T7C-45-ANBD protein, 500 nM of the 44/62 complex, 1 mM ATP, 500 nM biotinylated 34/62/36-mer DNA, 500 nM T4 D219A polymerase, and 550 nM streptavidin in buffer C. Syringe B contained 20 mM glucose and 20 units/ml glucose hexoki- FIG.3. Effect of ATPgS on the changes in the steady state nase in buffer C. Stopped-flow mixing of syringe A and B resulted in the fluorescence of the T7C-45-ANBD protein. The T7C-45-ANBD rapid depletion of ATP as well the dilution of the above concentrations steady state fluorescence was observed in buffer C with 275 nM strepta- by a factor of 2. The thick line represents a fit of the data to a single vidin at 25 °C at an excitation wavelength of 475 nm and an emission exponential followed by a steady state as described under “Experimen- wavelength of 530 nm. Spectrum 1 was obtained in the presence of 250 tal Procedures.” nM T7C-45-ANBD protein, and spectrum 2 is the result of the addition of 250 nM of the 44/62 complex. Spectrum 3 contains 250 nM T7C-45- ANBD protein, 250 nM of the 44/62 complex, and 1 mM ATPgS. Spec- trum 4 contains 250 nM T7C-45-ANBD protein, 250 nM of the 44/62 cently labeled 45 protein was then demonstrated to be useful in complex, 1 mM ATPgS, and 250 nM D219A T4 polymerase, and spectrum monitoring certain 45 protein-protein and 45 protein-DNA in- 5 was recorded with 250 nM T7C-45-ANBD protein, 250 nM of the 44/62 teractions associated with the T4 replication fork. complex, 1 mM ATPgS, 250 nM T4 polymerase, and biotinylated 34/62/ Fluorescent Labeling of the 45 Protein—Since the wild type 36-mer DNA. 45 protein is devoid of cysteine residues, it was expected that the introduction of a single cysteine residue by site-directed mutagenesis would result in the incorporation of a single thiol- reactive fluorescent probe per monomer. However, the extent of T7C 45 protein thiol modification was consistently less than that expected for a 45 protein trimer with one cysteine residue per monomer (Table I). The probe to protein trimer ratio for many of the thiol-reactive probes was approximately 1:1 which may indicate that the 45 protein occupies an asymmetrical conformation. Asymmetry may be introduced in the 45 protein by the interconversion between open and closed conformations (Fig. 6). In this model, the reactivity of the T7C thiol is influenced by whether the 45 protein is in the putative open or closed con- formation. The modification of the first cysteine thiol may weaken subunit interactions at one interface so that intercon- version between the open and closed conformation favors sub- unit opening at that weakened interface. Assuming that the cysteine thiols of the T7C-45 protein are derivatized only when they are near an open subunit interface, the remaining two FIG.4. The stoichiometry of the T7C-45-ANBD protein:44/62 complex interaction. The T7C-45-ANBD steady state fluorescence cysteines would remain unmodified. This hypothesis suggests was observed in buffer C at 25 °C at an excitation wavelength of 475 nm that thiol derivatization alters the structure of 45 protein. It and an emission wavelength of 530 nm. The fluorescence intensity of was therefore necessary to thoroughly determine the effects of 500 nM T7C-45-ANBD was monitored as a function of the 44/62 complex thiol modification on the 45 protein function. concentration in the absence of DNA. Fluorescence measurements were recorded immediately following addition of the 44/62 complex that It should be noted that the existence of a mixed population of ensured no significant ATP depletion. multiply labeled and unlabeled 45 proteins that combine to yield an average probe to protein trimer ratio of 1:1 cannot be completely disregarded at this time. However, under conditions DNA complex, while the linear rate can be explained by a small of up to 50-fold molar excess of probe to protein thiol and at amount of protein precipitation in the absence of ATP. prolonged reaction times (24 h) additional equivalent thiols in DISCUSSION the T7C-45 protein should be completely reacted. Moreover, the In this paper, we have succeeded in labeling a cysteine mu- apparent similarity with respect to the extent of T7C-45 pro- tant of the sliding clamp (the 45 protein) with an environmen- tein thiol modification with several of the different thiol re- tally sensitive, thiol-reactive fluorescent probe. This fluores- agents listed in Table I suggests that the observed ratio of 28050 Fluorescently Labeled DNA Polymerase Sliding Clamp There were two fluorescence changes induced in the T7C-45- ANBD protein by the addition of the 44/62 complex and ATP. One change was DNA-independent and the other was DNA-de- pendent. The DNA-independent change was triggered by the addition of the 44/62 complex and ATP and had an apparent stoichiometry of 1:1. The 1:1 stoichiometry of the T7C-45- ANBDz44/62 complex indicates that the observed increase in fluorescence is likely due to complex formation rather than a catalytic event such as the 44/62 complex-catalyzed opening or closing of the 45 protein trimeric ring. The ATP dependence and the inability of the nonhydrolyz- able ATP analogs AMP-PNP, AMP-PCP, or ATPgS to substi- tute in the formation of this DNA-independent fluorescence change suggests two possible scenarios. One possibility is that the fluorescence change is the result of a conformational change induced by binding ATP that cannot be induced by the FIG.6. Model for the observed stoichiometry of T7C-45 protein ATP analogs. A more likely explanation is that this conforma- thiol modification. Structure 1 represents the 45 protein in the closed tional change is triggered by ATP. This apparent requirement conformation that may be in equilibrium with an open conformation (structure 2). Structure 1 is shown with all cysteine thiols solvent for ATP hydrolysis suggests that the T7C-45-ANBDz44/62 com- inaccessible. Upon opening of the 45 protein at a single subunit inter- plex has undergone an ATP hydrolysis-driven conformational face, the environment of one cysteine thiol may change making one change, such as the opening of the 45 protein. Currently, the cysteine thiol more reactive. The reaction of this thiol with a thiol identity of this ATP hydrolysis-dependent conformational reagent (RX) may produce a 45 protein (structures 3 and 4) that is preferentially “opened” at the interface closest to the modified cysteine change in the 45 protein has not been assigned. thiol. T7C-45-ANBD Protein-DNA Interactions—The DNA- dependent change in T7C-45-ANBD fluorescence in the pres- probe:trimer is not due to the thiol reagent structure but rather ence of ATP was attributable to the 44/62 complex catalyzed some aspect of the protein itself. The ability of DTNB to modify loading of the T7C-45-ANBD protein onto DNA. The assign- all three cysteine thiols with equivalent kinetics may be attrib- ment of this DNA-dependent fluorescence change was made by uted to its relatively small size. measuring its rate of dissociation upon ATP consumption. The Activity of Fluorescently Labeled T7C-45 Protein—The 45 observed rate of fluorescence decay (0.011 s ) was similar to protein does not possess a measurable catalytic activity by that obtained for the holoenzyme-DNA dissociation constant itself. Instead its function is assessed through either its stim- using a strand displacement assay method (0.002 s , 11). The ulation of the DNA-dependent 44/62 ATPase activity or its difference in the dissociation rate constants may be attributed ability to enhance the processivity of the T4 DNA polymerase. to the fact that the substantial decrease in the fluorescence A steady state DNA-dependent ATPase assay provided a con- signal was best approximated by a single exponential decay, venient assay for screening the effects of thiol modification. The whereas the data obtained from the strand displacement assay T4 polymerase-45 protein interaction can be observed by the were better approximated by a double exponential decay reduction in the ATP hydrolysis rate upon holoenzyme assem- where, in addition to the 0.002 s rate, there was a population bly. These measurements demonstrate that the 45 protein can- (30%) that decayed at a faster rate (0.028 s , 11). The not tolerate large thiol reagents, such as the fluorescein deriv- weighted average of these rates (0.0097 s ) is very similar to ative, at the T7C position, but it can tolerate small thiol that obtained here (0.011 s ) which suggests that the fluores- reagents such as IANBD. This observation is supported by the cence assay is not sensitive to the presence of the two distinct x-ray crystal structure of the 45 protein that demonstrates that holoenzyme species observable by the strand displacement the T7 position is buried on the interior surface of the protein 1 method. ring. Interestingly, T7C-45 protein modification with the rel- Interestingly, the DNA-dependent change in T7C-45-ANBD atively large fluorescein derivative did not affect the T4 poly- fluorescence was also induced when ATPgS was substituted for merase-induced ATPase shut down rate. This lack of an appar- ATP. Other ATP analogs that were examined (AMP-PNP and ent effect on the holoenzyme ATPase rate by some large thiol AMP-PCP) did not induce this fluorescence change. In this reagents suggests that the T4 polymerase interacts at a site case, ATPgS appears to provide a clue toward determining the removed from the T7C position in the 45 protein. role of ATP hydrolysis in holoenzyme assembly. This effect of Proper functioning of the T7C-45 protein labeled with ATPgS may suggest that ATP binding rather than hydrolysis is IANBD (T7C-45-ANBD) was also demonstrated by a pre-steady required for the 44/62 complex to chaperone the 45 protein onto state kinetic analysis of the stimulation of the DNA-dependent DNA. This may also suggest that the T7C-45-ANBD fluores- ATPase activity of the 44/62 complex and by a strand displace- cence change is independent of whether the 45 protein is cor- ment assay. The pre-steady state ATPase measurements pro- rectly loaded onto DNA (i.e. with ATP) or merely associated vided the ATPase burst rate and burst amplitude. It appears with DNA in some manner. It was previously shown by DNA that it is the ATP hydrolysis burst rate that limits holoenzyme footprinting (15) and protein-DNA cross-linking (16) that the assembly (13, 11). The strand displacement assay demon- 45 protein is complexed with DNA in the presence of the 44/62 strated that the T7C-45 protein retains another function of the complex and ATPgS. Since ATPgS is not able to support func- wild type 45 protein, namely the polymerase processivity tional holoenzyme assembly (14), the 45 protein is likely in- enhancement. completely loaded onto DNA in its presence. T7C-45-ANBD Protein-Protein Interactions—The evaluation of several thiol-directed fluorescent probes led to the discovery Partial Mechanism for Holoenzyme Assembly—Based on the ATP hydrolysis dependence of the observed fluorescence that the T7C-45 protein modified with the environmentally sensitive probe, IANBD, produced substantial changes in fluo- changes in the presence and absence of DNA, a putative partial mechanism is proposed (Fig. 7). The proposed mechanism is rescence intensity upon interactions with the other components of T4 replication. incomplete due to the fact that the loading of the polymerase Fluorescently Labeled DNA Polymerase Sliding Clamp 28051 more stable ring closed form on DNA concomitant with 44/62 complex release. Comparison with the DNA Polymerases of Other Systems— The E. coli replication system sliding clamp (the b clamp) is loaded by a five protein complex termed either the DnaX com- plex or the g complex. The five proteins that assemble to form the E. coli clamp loader are the d, the d9, the x, the c, and either the g or the t subunit. DnaX complexes formed with either the g or the t subunit differ in their ability to hydrolyze ATPgS (23) and are thought to possess different roles in holoenzyme for- mation (23, 24, 25). The existence of a clamp loader-clamp intermediate has been shown in this system using surface plasmon resonance (24). This intermediate displayed similar ATP requirements to that of the 44/62–45 complex observed here in that it was dependent upon ATP hydrolysis for its function (24). In conclusion, the fluorescence data presented here are highly suggestive of a mechanism in which the 44/62 complex mediates the association of the 45 protein with DNA in an ATP-dependent manner but without ATP hydrolysis. Upon ATP hydrolysis a conformational change is invoked in the 45 protein by the 44/62 complex that results in the functionally loaded 45 protein on DNA. Acknowledgments—We are grateful to Ismail Moarefi and John Kuriyan for providing us with the cysteine mutant of the 45 protein. REFERENCES FIG.7. Partial mechanism for holoenzyme assembly. The 44/62 1. Kornberg, A., and Baker, T. (1992) DNA Replication, 2nd Ed., pp. 113–217, complex is shown by four spheres that represent the 44 protein and one W. H. Freeman and Co., New York rectangle (the 62 protein). Upon binding ATP a conformational change 2. Stillman, B. (1994) Cell 78, 725–728 is induced in the 44/62 complex that enables interaction with the 45 3. Young, M. C., Reddy, M. K., and von Hippel, P. H. (1992) Biochemisty 31, protein (trimeric ring). This 45 protein:44/62:ATP bound complex inter- 8675–8690 acts with DNA without ATP hydrolysis to yield a noncatenated form of 4. Nossal, N. G. (1992) FASEB J. 6, 871–878 the 45 protein on DNA. The model has ring opening on DNA coupled to 5. Kong, X.-P., Onrust, R., O’Donnell, M., and Kuriyan, J. (1992) Cell 69, ATP hydrolysis. The opened form of the ring then decays to the closed 425–437 ring on DNA with the release of the 44/62 complex. 6. Krishna, T. S. R., Kong, X.-P., Gary, S., Burgers, P. M., and Kuriyan, J. (1994) Cell 79, 1233–1243 7. O’Donnell, M., Onrust, R., Dean, F. B., Chen, M., and Hurwitz, J. (1993) Nucleic Acids Res. 21, 1–3 does not cause a fluorescence change. The popular conception is 8. Onrust, R., Stukenberg, P. T., and O’Donnell, M. (1991) J. Biol. Chem. 266, that ATP causes a conformational change in the 44/62 complex 21681–21686 such that the 62 protein is better positioned for interaction 9. Yoder, B. L., and Burgers, P. M. J. (1991) J. Biol. Chem. 266, 22689–22697 10. Rush, J., Lin, T.-C., Quinones, M., Spicer, E. K., Douglas, I., Williams, K. R., with the 45 protein (3). The mechanism in Fig. 7 uses ATP and Konigsberg, W. H. (1989) J. Biol. Chem. 264, 10943–10953 binding to the 44/62 complex to cause the conformational 11. Kaboord, B. F., and Benkovic, S. J. (1995) Curr. Biol. 5, 149–157 change that exposes the 62 protein for interaction with the 45 12. Kaboord, B. F., and Benkovic, S. J. (1996) Biochemistry 35, 1084–1092 13. Berdis, A. J., and Benkovic, S. J. (1996) Biochemistry, 35, 9253–9265 protein. ATP hydrolysis within the 45–44/62 complex causes 14. Piperno, J. R., and Alberts, B. M. (1978) J. Biol. Chem. 253, 5174–5179 an observable fluorescence change that may represent the 15. Munn, M. M., and Alberts, B. M. (1991) J. Biol. Chem. 266, 20024–20033 opening of the 45 protein. As shown in Fig. 7 our results 16. Capson, T. L., Benkovic, S. J., and Nossal, N. G. (1991) Cell 65, 249–258 17. Gogel, E. P., Young, M. C., Kubasek, W. L., Jarvis, T. C., and von Hippel, P. H. suggest that the ATP-dependent 45–44/62 complex can be as- (1992) J. Mol. Biol. 224, 395–412 sociated with DNA without ATP hydrolysis. In the model (Fig. 18. Capson, T. L., Peliska, J. A., Kaboord, B. F., Frey, M. W., Lively, C., Dahlberg, M., and Benkovic, S. J. (1992) Biochemistry 31, 10984–10994 7), this ATP hydrolysis independent DNA-45–44/62 complex 19. Nossal, N. G. (1979) J. Biol. Chem. 254, 6026–6031 represents a form of the 45 protein that does not have DNA 20. Frey, M. W., Nossal, N. G., Capson, T. L., and Benkovic, S. J. (1993) Proc. Natl. through the center of the protein ring (nonconcatenated form). Acad. Sci. U. S. A. 90, 2579–2583 21. Johnson, K. A. (1986) Methods Enzymol. 134, 677–705 Since ATP binding must occur prior to ATP hydrolysis, it is 22. Ellman, G. L. (1958) Arch. Biochem. Biophys. 82, 70–77 plausible that the 44/62 complex chaperones the 45 protein to 23. Dallmann, H. G., Thimmig, R. L., and McHenry, C. S. (1995) J. Biol. Chem. the primer template before the 45 protein ring is opened. The 270, 29555–29562 24. Naktinis, V., Onrust, R., Fang, L., and O’Donnell, M. (1995) J. Biol. Chem. 270, opening of the protein ring on DNA would enable the DNA to 13358–13365 enter the center of the 45 protein. The ring opened 45 protein- 25. Xiao, H., Naktinis, V., and O’Donnell, M. (1995) J. Biol. Chem. 270, 44/62 complex on DNA may decay to a thermodynamically 13378–13383 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Protein-Protein and Protein-DNA Interactions at the Bacteriophage T4 DNA Replication Fork

Sexton, Daniel J.; Carver, Theodore E.; Berdis, Anthony J.; Benkovic, Stephen J.
Journal of Biological Chemistry – Nov 1, 1996
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0021-9258
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 45, Issue of November 8, pp. 28045–28051, 1996 © 1996 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Protein-Protein and Protein-DNA Interactions at the Bacteriophage T4 DNA Replication Fork CHARACTERIZATION OF A FLUORESCENTLY LABELED DNA POLYMERASE SLIDING CLAMP* (Received for publication, May 28, 1996, and in revised form, August 3, 1996) Daniel J. Sexton, Theodore E. Carver, Anthony J. Berdis, and Stephen J. Benkovic‡ From the Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802 The T4 DNA polymerase holoenzyme is composed of ture is now known to resemble the “sliding clamp” ring struc- the polymerase enzyme complexed to the sliding clamp ture previously shown for the analogous proteins from E. coli (the 45 protein), which is loaded onto DNA by an ATP- (b-clamp) and eucaryotes (proliferating cell nuclear antigen) dependent clamp loader (the 44/62 complex). This paper (5, 6). The 45 protein is loaded onto DNA at a primer template describes a new method to directly investigate the junction through the ATP-dependent activity of the 44/62 pro- mechanism of holoenzyme assembly using a fluores- tein complex. The 44/62 complex has a functional and a limited cently labeled cysteine mutant of the 45 protein. This sequence homology (7) to the g complex clamp loader of E. coli protein possessed unaltered function yet produced sub- (8) and the RF-C complex of eucaryotes (9). The oligomeric stantial changes in probe fluorescence intensity upon stoichiometry of the 44/62 complex is four 44 subunits to one 62 interacting with other components of the holoenzyme. subunit with the ATPase activity residing on each of the 44 These fluorescence changes provide insight into the role subunits (10). Recently, it was quantitatively demonstrated of ATP hydrolysis in holoenzyme assembly. Using either that the 44/62 complex loads the 45 protein in a catalytic ATP or the non-hydrolyzable ATP analog, adenosine 5*- manner and also functions as a chaperone protein to ensure O-(3-thiophosphate), events in holoenzyme assembly productive holoenzyme complex (11, 12). The amount of ATP were assigned as either dependent or independent of consumed in the 44/62-dependent loading of the 45 protein onto ATP hydrolysis. A holoenzyme assembly mechanism is primed DNA has recently been shown to be consistent with the proposed in which the 44/62 complex mediates the asso- hydrolyis of ATP at all four ATPase sites in the 44/62 complex ciation of the 45 protein with DNA in an ATP-dependent (13). manner not requiring ATP hydrolysis. Upon ATP hy- Despite the wealth of information regarding the roles of the drolysis, the 44/62 complex triggers a conformational different T4 replication proteins in DNA synthesis, there re- change in the 45 protein that may be attributed to the clamp loading onto DNA. main a number of individual events or “microprocesses” for which mechanisms are undetermined. For example, by divid- ing the T4 replication activity into three processes, 1) the assembly of the holoenzyme, 2) the synthesis of DNA, and 3) In most biological systems, the replication of DNA involves the coordinated actions of a multitude of different proteins. In the disassembly of the holoenzyme, it is evident that there exist events within each process that may be considered micro- general, as the complexity of the organism increases so do the number of proteins participating in DNA replication. The bac- processes. One such microprocess within the assembly of the holoenzyme concerns the development of the protein-protein teriophage T4 DNA replication system is well suited for a mechanistic investigation of the protein-protein interactions interaction(s) between the 45 protein and the 44/62 complex that are required to load the 45 protein onto DNA. The mech- required for replication due to the relatively low number of proteins involved and the apparent functional similarities to anism by which the 44/62 complex loads the 45 protein onto DNA is not known. It is known that ATP hydrolysis is required other more complicated systems such as those found in Esche- richia coli and eucaryotes (reviewed in Refs. 1 and 2). for the stimulation of the T4 polymerize processivity by the accessory proteins (14). However, ATP hydrolysis appears not The bacteriophage T4 replication fork is comprised of the DNA polymerase enzyme (the product of gene 43), the DNA to be necessary for a 44/62 complex-mediated 45 protein-DNA interaction since this interaction was observed with the non- polymerase accessory proteins (the 44/62 complex and the 45 protein), as well as the ssDNA-binding protein (the 32 protein), hydrolyzable ATP analog, ATPgS, by both DNA footprinting (15) and protein-DNA cross-linking experiments (16). ATP hy- the helicase (the 41 protein), the primase (the 61 protein), and the helicase accessory protein (the 59 protein) (3, 4). The T4 drolysis was required to observe, by cyroelectron microscopy, structures (termed “hash marks”) on nicked DNA that corre- DNA polymerase holoenzyme consists of the T4 polymerase, which possesses both a 59–39 polymerase and a 39–59 exonucle- sponded to 45 protein trimers (17) that emphasizes the impor- tance of ATP hydrolysis in loading the 45 protein onto DNA. ase activity, and the 45 protein. The homotrimeric 45 protein is the processivity factor for the polymerase, and its x-ray struc- The investigation of the DNA loading mechanism of the 45 protein is made difficult by the fact that 45 protein does not * This work was supported by a National Institutes of Health Grant GM13306 (to S. J. B.). The costs of publication of this article were J. Kuriyan, personal communication. defrayed in part by the payment of page charges. This article must The abbreviations used are: ATPgS, adenosine 59-O-(3-thiophos- therefore be hereby marked “advertisement” in accordance with 18 phate); bio, biotin; DCIA, 7-diethylamino-3-((49-iodoacetyl)amino)phen- U.S.C. Section 1734 solely to indicate this fact. yl)-4-methylcoumarin; DTNB, 5,59-dithio-bis(2,29-nitrobenzoic acid); ‡ To whom correspondence should be addressed: Dept. of Chemistry, IAANS, 2-(49-(iodoacetamido)anilino)naphthalene-6-sulfonic acid, so- 152 Davey Laboratory, Pennsylvania State University, University dium salt; IAEDANS, 5-((((iodoacetyl)amino)ethyl)amino)naphthalene- Park, PA 16802. Tel.: 814-865-2882; Fax: 814-865-2973. E-mail: 1-sulfonic acid; IANBD, N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7- [email protected]. nitrobenz-2-oxa-1,3-diazole; IAF, 5-iodoacetamidofluorescein. This paper is available on line at http://www-jbc.stanford.edu/jbc/ 28045 This is an Open Access article under the CC BY license. 28046 Fluorescently Labeled DNA Polymerase Sliding Clamp on protein function was assessed by substitution for wild type, unla- possess a measurable intrinsic activity. It would therefore be beled 45 protein. Initial rates of ATP hydrolysis were obtained under advantageous to have a means of directly monitoring the 45 conditions where less than 10% of the limiting reactant was utilized protein interactions during complex formation. over the reaction time course. In this paper, we describe a fluorescently labeled cysteine Pre-steady State ATPase Assay—The pre-steady state ATPase rates mutant of the 45 protein. It is demonstrated that this fluores- were performed using a rapid quench instrument (21) as described cently labeled 45 protein provides a novel means for the direct previously (13). The ATPase rates were obtained from an assay mixture containing 250 nM 44/62 complex, 250 nM 45 protein (or labeled 45 investigation of certain 45 protein-protein interactions as well protein), 500 nM biotinylated 34/62/36-mer DNA, 1 mM streptavidin, 1 as 45 protein-DNA interactions. The observed fluorescence mM ATP, 50 nM [g- P]ATP in buffer C at 25 °C. Briefly, the reactions changes enabled the presentation of a partial mechanism for were quenched with 1 M HCl and neutralized with an appropriate T4 holoenzyme assembly. The change in the fluorescence of the amount of 3 M NaOH in 1 M Tris base followed by a thin layer chro- 32 32 labeled 45 protein that occurs upon holoenzyme formation has matographic separation of [g- P]ATP from g- P and then radiochem- also provided a new assay for the determination of the T4 ical analysis using a Molecular Dynamics Phosphorimager. Strand Displacement Assay—A strand displacement assay was uti- holoenzyme-DNA complex dissociation rate constant. lized to further characterize the effect of T7C-45 protein thiol modifi- EXPERIMENTAL PROCEDURES cation on the formation of active holoenzyme. This assay has been Materials—Oligonucleotides were synthesized with an Expedite described in detail elsewhere (12). Briefly, an assay mixture containing 8909 DNA synthesizer (Perceptive Biosystems) and purified according 50 nM P-59-end-labeled, biotinylated 34/62/36-mer DNA, 55 nM to Capson et al. (18). Biotin-labeled oligonucleotides were prepared streptavidin, 1 mM ATP, and 10 mM dCTP is preincubated 30 s with 10 using a BioTEG phosphoramidite as obtained from Glen Research. The nM T4 D219A polymerase, 55 nM 44/62 complex, 55 nM 45 protein before fluorescent probes DCIA, dibromobimane, IAEDANS, IANBD, IAF, addition of 10 mM remaining deoxynucleotides (dATP, dTTP, and dGTP) IAANS, 2-(49-(iodoacetamido)anilino)naphthalene-6-sulfonic acid, so- and 1 mg/ml salmon sperm single strand DNA trap. Following the dium salt were obtained from Molecular Probes, and pyrene maleimide addition of the remaining dNTPs and single strand DNA trap, aliquots was obtained from Acros. ATPgS was purchased from Boehringer were removed at different times and quenched in 2 M HCl and extracted Mannheim as a 98% pure solution and used without further purifica- with phenol:chloroform:isoamyl alcohol (25:24:1). The zero point was tion. DuPont NEN was the source of the [g- P]ATP used to 59-end label obtained by omitting the protein from the assay mixture. The omission oligonucleotides with T4 polynucleotide kinase (U. S. Biochemical of the remaining three nucleotides provided an experimental control to Corp.). All other biochemicals and chemicals were obtained from Sigma assess the incorporation extent of the first nucleotide. Prior to loading or Fisher and were of analytical grade or better. onto a 16% polyacrylamide, 8 M urea, sequencing gel, the samples were DNA encoding a threonine to cysteine mutation at position 7 in the neutralized with the addition of an appropriate amount of 3 M NaOH in 45 protein (T7C-45 protein) ligated into a pET 26b expression vector 1 M Tris-base. The sequencing gels were exposed to constant current was a generous gift of Dr. John Kuriyan (Rockefeller University). Fol- electrophoresis, and the distribution of the radioactivity was analyzed lowing transformation of the DNA into BL21-DE3 E. coli and IPTG- using a Molecular Dynamics Phosphorimager. induced expression, the T7C-45 protein was purified according to Nos- Steady State Fluorescence—Steady state fluorescence measurements sal (19). The wild type 45 protein and the 44/62 complex were purified were performed using an SLM Aminco 8000C photon counting spec- from overproducing strains obtained from Dr. William Konigsberg (Yale trofluorometer equipped with a thermostated cell compartment that University) as described previously (19). The T4 exonuclease-deficient was maintained at 25 °C. The experiments were performed in buffer C polymerase D219A mutant was purified according to Frey et al. (20). in the presence of an appropriate amount of T7C-45-ANBD protein to The D219A mutant of the T4 polymerase was used in these studies to provide a satisfactory fluorescence signal (usually 250 nM). The effects avoid complications arising from the presence of the exonuclease activ- of the other components of the T4 polymerase holoenzyme (i.e. proteins, ity. The polymerase activity of the T4 D219A polymerase is identical to DNA, and/or ATP) on the T7C-45-ANBD fluorescence were observed that of the wild type enzyme (20). upon their direct addition to the fluorescence cuvette. The fluorescence Primer Template Construction—The biotinylated 34/62/36-mer DNA spectra obtained were normalized for the effects of dilution. substrate was constructed as described previously (12). This DNA sub- Stopped-flow Fluorescence—Stopped-flow fluorescence measure- strate is composed of a 34-mer primer annealed to a 39-biotinylated ments were performed using an Applied Photophysics stopped-flow 62-mer with a 36-mer fork strand annealed to the 59-end of the template instrument at a constant temperature of 25 °C. The changes in fluores- with an 18-base overhang. cence were observed upon stopped-flow mixing of syringe A and syringe T7C-45 Protein Cysteine Modification—The modification of the cys- B. Syringe A contained 500 nM T7C-45-ANBD protein, 500 nM of the teine thiol of the T7C-45 protein was performed upon passage of the 44/62 complex, 1 mM ATP, 500 nM biotinylated 34/62/36-mer DNA, 500 protein solution over a G-25 column equilibrated with degassed buffer nM T4 D219A polymerase, and 550 nM streptavidin in buffer C. Syringe A (50 mM HEPES, 150 mM KOAc, 1 mM EDTA, 10% glycerol, pH 7.8) B contained 20 mM glucose and 20 units/ml glucose hexokinase in buffer followed by the addition of a 10-fold molar excess of thiol reagent over C. The concentration of protein and DNA in the two syringes of the protein monomer. The cysteine labeling reaction was allowed to proceed stopped-flow instrument was diluted by a factor of 2 upon stopped-flow approximately4hat4 °C. Excess thiol reagent was removed by exten- mixing. sive dialysis against buffer B (50 mM Tris, 50 mM NaCl, 5 mM EDTA, 1 Data Analysis—Data obtained from the pre-steady state rate of ATP mM b-mercaptoethanol, 10% glycerol, pH 8.0). The labeled protein so- hydrolysis by the 44/62 complex were fit to Equation 1: lution was then concentrated in a microconcentrator (Centricon-30) and 2Bt stored at 280 °C. The extent of cysteine modification was quantitated y 5 Ae 1 C (Eq. 1) by measuring the dye concentration from its molar extinction at a where A is the amplitude of the pre-steady state phase, B is the wavelength other than 280 nm. Protein concentration was determined pre-steady state rate constant, and C is a constant. Data obtained from using the Bradford assay with the 45 protein as the standard. Labeling the stopped-flow fluorescence determination of the kinetics of holoen- efficiencies are reported as mol of dye/mol of 45 trimer. zyme dissociation were best fit by Equation 2: Steady State ATPase Assay—Steady state ATP hydrolysis measure- ments were performed using a phosphoenolpyruvate kinase/lactate de- 2Bt y 5 Ae 1 Ct 1 D (Eq. 2) hydrogenase enzyme coupled system where the consumption of ATP was monitored spectrophotometrically (OLIS-Cary-14 spectrophotome- where A is the amplitude of the exponential decay, B is the exponential ter) upon oxidation of NADH (13). ATPase activity was observed upon decay rate constant, C is a linear decay rate, and D is a constant. addition of the 45 protein (250 nM) to a solution containing 250 nM 44/62 complex, 250 nM biotinylated 34/62/36-mer DNA, 1 mM streptavidin, 1 RESULTS mM ATP, 10 mM phosphoenolpyruvate, 200 mM NADH, 6 units of phos- phoenolpyruvate kinase/lactate dehydrogenase mix, and buffer C (150 Fluorescent Labeling of the 45 Protein—The wild type 45 mM potassium acetate, 10 mM magnesium acetate, 10 mM b-mercapto- protein is devoid of cysteine residues. The site-selective intro- ethanol, and 25 mM Tris, pH 7.5) at 25 °C. The T4 D219A polymerase duction of a cysteine residue into the sequence of the 45 protein protein was subsequently added to observe the inhibition of the ATP enables the covalent attachment of a thiol-reactive fluorescent hydrolysis upon stable complex formation. The effect of the mutation of molecule at a known position in the primary structure. A mu- the threonine at position 7 to a cysteine in the 45 protein (the T7C-45 protein) as well as the subsequent thiol derivatization of the T7C thiol tated 45 protein, in which a threonine residue at position 7 Fluorescently Labeled DNA Polymerase Sliding Clamp 28047 TABLE I 44/62 complex steady state ATPase activity (Table II). Thiol reagent labeling efficiency of the T7C-45 protein A rapid chemical quench method has been developed to Thiol reagent Probe:trimer measure the pre-steady state kinetics of ATP hydrolysis by the 44/62 complex (13). The ability of the T7C-45-ANBD protein to mol/mol substitute for the wild type 45 protein under pre-steady state DCIA 0.6 conditions was also assessed (Table II). No significant differ- Dibromobimane 2.3 DTNB 3 ence was observed between the T7C-45-ANBD protein and the IAANS 0.4 wild type 45 protein with respect to the measured pre-steady IAEDANS 1.3 state rates and the burst amplitudes (Table II). The pre-steady IAF 1.5 state ATPase rate is determined by the chemical rate of ATP IANBD 0.9 Pyrene maleimide 1.2 hydrolysis rather than product release (steady state rate). The burst amplitude relative to the 44/62 complex concentration The labeling efficiency was determined as described under “Exper- provides the stoichiometry of ATP consumption. At a concen- imental Procedures” where the concentration of each probe was deter- mined using the following molar extinction coefficients: DCIA, 33,000 tration of 250 nM the burst amplitude for both forms of the 45 21 21 liter z cm z mol at 382 nm; dibromobimane, 5,300 liter z cm z mol at protein was approximately 1 mM which corresponds to 4 mol 397 nm; DTNB 13,600 liter z cm z mol at 412 nm; IAANS, 26,000 21 21 of ATP hydrolyzed per mol of 44/62 complex as previously liter z cm z mol at 329 nm; IAEDANS, 5,700 liter z cm z mol at 336 reported (13). nm; IAF, 82,000 liter z cm z mol at 491 nm; IANBD, 23,000 liter z cm z mol at 472 nm and pyrenemaleimide, 36,000 liter z To further examine the effect of the T7C mutation and sub- cm z mol at 339 nm. sequent thiol derivatization of the 45 protein on holoenzyme function, a strand displacement assay was utilized. In this from the N terminus, was site-specifically mutated to a cys- 32 assay the fork strand (36-mer) of a 59- P-biotinylated 62/34/ teine residue producing the threonine 7 to cysteine 45 mutant 36-mer is capable of being displaced by the holoenzyme but not protein (T7C-45 protein). The details for the creation of this by the T4 polymerase alone (11, 12). Displacement of the fork cysteine mutant will be published elsewhere. strand gives rise to the production of a fully extended 62-mer The cysteine mutant, T7C-45 protein, was modified using primer strand that can be differentiated from the bio62-mer several different thiol-reactive probes (Table I). The thiol quan- template strand on a polyacrylamide sequencing gel. Quanti- titating reagent 5,59-dithiobis(2,29-nitrobenzoic acid) (DTNB; tation of the radioactivity of the 62-mer primer strand yields a Ref. 22) reacted slowly with three of the thiols in the trimer measure of the ability of the T4 replicative proteins to form indicating the presence of only the reduced form of the cys- productive holoenzyme complex. Fig. 1 demonstrates that the teines. No evidence of nonequivalent cysteine thiols in the T7C-45 mutant protein displays strand displacement activity T7C-45 protein trimer was provided by the DTNB reactivity comparable with the wild type 45 protein even when derivat- since the reaction was best approximated by a single exponen- ized with IANBD. tial (data not shown). However, thiol modification of the cys- Steady State Fluorescence of T7C-45-ANBD—The T7C-45- teine T7C-45 protein with the majority of the fluorescent ANBD protein was selected for further study based on two probes consistently led to a probe:trimer ratio less than 3:1, the important properties. First, the ability of the T7C-45-ANBD ratio expected if all thiols in the trimer were modified (Table I). protein to substitute for the wild type 45 protein in the assays It is evident that for most of the probes tested in this study the described above, without a measurable loss of activity (Table II probe:trimer ratio was close to 1:1 (omitting DTNB, the aver- and Fig. 1), makes this labeled 45 protein potentially suitable age probe:trimer ratio equals 1.2:1) which suggests that only for monitoring the interactions of the 45 protein at the T4 one out of three of the cysteines in the trimer is modified. Of the replication fork. Second, the environmentally sensitive nature fluorescent probes tested in Table I, dibromobimane incorpo- of T7C-45-ANBD has been shown to permit the detection of rated to the greatest extent (2.3:1) while the naphthalene de- certain 45 protein-44/62 complex and 45 protein-DNA interac- rivative (IAANS) was the least reactive (0.4:1). A titration of tions (Fig. 2). As shown in Fig. 2, the addition of the 44/62 the remaining thiols of the T7C-45-ANBD protein with DTNB complex to a solution of the T7C-45-ANBD protein resulted in yielded 2 thiols per trimer, which was again consistent with a no measurable change in the fluorescence emission spectrum. labeling efficiency for IANBD of only 1 probe:trimer. The subsequent addition of ATP to the above solution contain- Activity of Fluorescently Labeled T7C-45 Protein—The meas- ing T7C-45-ANBD and 44/62 complex caused a marked (ap- urement of the degree of stimulation of the 44/62 complex proximately 1.7-fold) increase in the fluorescence intensity. In steady state ATPase activity by the fluorescently labeled the absence of the 44/62 complex ATP had no effect on the T7C-45 proteins provides a convenient assay to assess the fluorescence of the T7C-45-ANBD protein (data not shown). effects of derivatization on 45 protein function (Table II). ATP That fluorescence increase was then attenuated by the addition is consumed as the 44/62 complex loads the 45 protein onto the of the bio34/62/36-mer to an intermediate level of fluorescent biotinylated 34/62/36 DNA substrate. In the absence of the T4 intensity approximately 1.3 times greater than that of the polymerase, the 45 protein dissociates from DNA allowing it to T7C-45-ANBD alone. The fluorescence of the T7C-45-ANBD be reloaded by the 44/62 complex at the expense of additional protein is insensitive to the addition of the T4 polymerase ATP hydrolysis. The addition of the T4 polymerase diminishes whether it is added to the mixture of T7C-45-ANBD protein, ATP consumption through the formation of holoenzyme, which 44/62 complex, and DNA (Fig. 2) or in any other order of is more stable on DNA than the 45 protein alone (13). As shown addition (data not shown). in Table II, the T7C-45 mutant displays essentially identical A demonstration of whether or not ATP hydrolysis is re- activity to that of the wild type 45 protein. Labeling the T7C-45 quired for a particular biochemical event is often made possible protein with iodoacetamidofluorescein (IAF) led to a decrease by testing the ability of nonhydrolyzable ATP analogs to sub- in the observed stimulation of the ATPase activity of the 44/62 stitute for ATP. ATPgS has been shown to be a potent inhibitor complex (Table II). The modification of the T7C-45 protein with of the 44/62 complex ATPase activity, whereas the ATP analogs IANBD, however, did not affect its ability to stimulate the 4 AMP-PNP and AMP-PCP are much weaker inhibitors (14). The T7C-45 mutant protein was obtained from the Kuriyan Labo- ratory (Rockefeller University). A. J. Berdis, unpublished data. 28048 Fluorescently Labeled DNA Polymerase Sliding Clamp TABLE II Stimulation of the DNA-dependent 44/62 complex ATPase activity by fluorescently labeled 45 proteins Steady state Steady state Pre-steady state Pre-steady state 45 protein a a b b rate 1 rate 2 rate amplitude nM/s nM/s s mM WT 45 398 87 3.1 0.9 T7C-45 421 110 ND ND T7C-ANBD 428 115 3.9 1.2 T7C-AF 228 103 ND ND Steady state ATPase activity was performed as described under “Experimental Procedures.” Steady state rate 1 corresponds to the ATPase rate resulting from the stimulation by the 45 protein, and steady state rate 2 is the rate observed upon addition of the D219A T4 polymerase to the solution that contains the 45 protein. The difference between these values and those reported in Berdis and Benkovic (13) may be due to different preparations of the same DNA substrate. Pre-steady state ATPase measurements were performed as described under “Experimental Procedures.” ND, not determined. FIG.1. Strand displacement assay in the presence of the T7C- 45-ANBD protein. The above sequencing gel (16% acrylamide, 8 M urea) was used to separate the polymerase extension products using the P-59-end-labeled, biotinylated 34/62/36-mer DNA substrate. The lanes in A are the extension products observed in the presence of the polym- erase alone (i.e. no accessory proteins), at different time points (0, 10, FIG.2. Steady state fluorescence of the T7C-45-ANBD protein. 20, and 30 s) plus a control point. The control point represents the The T7C-45-ANBD steady state fluorescence was observed in buffer C incorporation of just the first nucleotide. B represents the results of the with 275 nM streptavidin at 25 °C at an excitation wavelength of 475 holoenzyme with wild type 45 protein, and C and D represent the nm and an emission wavelength of 530 nm. Spectrum 1 was obtained in observed results of the holoenzyme when the T7C-45 protein and the the presence of 250 nM T7C-45-ANBD protein, and spectrum 2 is the T7C-45-ANBD protein, respectively, were substituted for the wild type result of the addition of 250 nM of the 44/62 complex. Spectrum 3 45 protein. Strand displacement was observed according to the appear- contains 250 nM T7C-45-ANBD protein, 250 nM of the 44/62 complex, ance of the fully extended primer as a 62-mer (indicated by the arrow) and1mM ATP. Spectrum 4 contains 250 nM T7C-45-ANBD protein, 250 that was resolved from the template biotinylated 62-mer. The strand nM of the 44/62 complex, 1 mM ATP, and 250 nM biotinylated 34/62/36- displacement experiment was performed as described under “Experi- mer DNA, and spectrum 5 was recorded with 250 nM T7C-45-ANBD mental Procedures.” protein, 250 nM of the 44/62 complex, 1 mM ATP, 250 nM biotinylated 34/62/36-mer DNA, and 250 nM T4 D219A polymerase. The ATP analogs, AMP-PNP and AMP-PCP, did not produce any change in the fluorescence of the T7C-45-ANBD protein Holoenzyme-DNA Dissociation Rate Constant—Stopped-flow under the above experimental conditions and at a concentra- fluorescence rapid mixing experiments were performed to tion of up to 10 mM ATP analog (data not shown). measure the dissociation rate of the holoenzyme from the At a concentration of 1 mM, ATPgS was not able to elicit all bio34/62/36-mer. In order to measure the holoenzyme dissoci- of the fluorescence changes mediated by ATP in Fig. 2. How- ation rate, the holoenzyme was assembled onto the DNA sub- ever, there is a fluorescence increase upon the addition of DNA strate in one syringe and pushed against another syringe con- to a mixture containing T7C-45-ANBD and the 44/62 complex taining excess glucose and hexokinase that rapidly consume in the presence of ATPgS (Fig. 3). That fluorescence increase is the ATP. Under the experimental conditions (10 units/ml hex- not affected by the presence or absence of T4 polymerase or the okinase) the ATP (1 mM) should be consumed in approximately order of addition (data not shown). Magnesium ion (10 mM) was 6 s. The approximate dead time of the experiment is therefore required to observe the ATPgS-dependent fluorescence change. 6 s. The data obtained are shown in Fig. 5 where it can be seen The Stoichiometry of the T7C-45-ANBD:44/62 Interaction— that after stopped-flow mixing of the assembled holoenzyme- The stoichiometry of the ATP-dependent interaction between DNA complex with glucose and hexokinase there is a decrease the T7C-45-ANBD protein and the 44/62 complex was deter- in fluorescence that can be approximated by a single exponen- mined by varying the concentration of 44/62 complex. As shown ) followed by a linear decrease in Fig. 4, the increase in fluorescence reaches a plateau at tial decay (0.011 6 0.002 s 25 26 21 (7.8 3 10 6 2 3 10 s ). The single exponential decay rate approximately a 1:1 molar ratio of 44/62 complex to T7C-45- ANBD protein. represents the dissociation rate constant of the holoenzyme- Fluorescently Labeled DNA Polymerase Sliding Clamp 28049 FIG.5. Determination of the T4 polymerase holoenzyme-DNA dissociation rate constant. Stopped-flow fluorescence was used to measure the T4 polymerase holoenzyme-DNA dissociation rate con- stant where syringe A contained 500 nM T7C-45-ANBD protein, 500 nM of the 44/62 complex, 1 mM ATP, 500 nM biotinylated 34/62/36-mer DNA, 500 nM T4 D219A polymerase, and 550 nM streptavidin in buffer C. Syringe B contained 20 mM glucose and 20 units/ml glucose hexoki- FIG.3. Effect of ATPgS on the changes in the steady state nase in buffer C. Stopped-flow mixing of syringe A and B resulted in the fluorescence of the T7C-45-ANBD protein. The T7C-45-ANBD rapid depletion of ATP as well the dilution of the above concentrations steady state fluorescence was observed in buffer C with 275 nM strepta- by a factor of 2. The thick line represents a fit of the data to a single vidin at 25 °C at an excitation wavelength of 475 nm and an emission exponential followed by a steady state as described under “Experimen- wavelength of 530 nm. Spectrum 1 was obtained in the presence of 250 tal Procedures.” nM T7C-45-ANBD protein, and spectrum 2 is the result of the addition of 250 nM of the 44/62 complex. Spectrum 3 contains 250 nM T7C-45- ANBD protein, 250 nM of the 44/62 complex, and 1 mM ATPgS. Spec- trum 4 contains 250 nM T7C-45-ANBD protein, 250 nM of the 44/62 cently labeled 45 protein was then demonstrated to be useful in complex, 1 mM ATPgS, and 250 nM D219A T4 polymerase, and spectrum monitoring certain 45 protein-protein and 45 protein-DNA in- 5 was recorded with 250 nM T7C-45-ANBD protein, 250 nM of the 44/62 teractions associated with the T4 replication fork. complex, 1 mM ATPgS, 250 nM T4 polymerase, and biotinylated 34/62/ Fluorescent Labeling of the 45 Protein—Since the wild type 36-mer DNA. 45 protein is devoid of cysteine residues, it was expected that the introduction of a single cysteine residue by site-directed mutagenesis would result in the incorporation of a single thiol- reactive fluorescent probe per monomer. However, the extent of T7C 45 protein thiol modification was consistently less than that expected for a 45 protein trimer with one cysteine residue per monomer (Table I). The probe to protein trimer ratio for many of the thiol-reactive probes was approximately 1:1 which may indicate that the 45 protein occupies an asymmetrical conformation. Asymmetry may be introduced in the 45 protein by the interconversion between open and closed conformations (Fig. 6). In this model, the reactivity of the T7C thiol is influenced by whether the 45 protein is in the putative open or closed con- formation. The modification of the first cysteine thiol may weaken subunit interactions at one interface so that intercon- version between the open and closed conformation favors sub- unit opening at that weakened interface. Assuming that the cysteine thiols of the T7C-45 protein are derivatized only when they are near an open subunit interface, the remaining two FIG.4. The stoichiometry of the T7C-45-ANBD protein:44/62 complex interaction. The T7C-45-ANBD steady state fluorescence cysteines would remain unmodified. This hypothesis suggests was observed in buffer C at 25 °C at an excitation wavelength of 475 nm that thiol derivatization alters the structure of 45 protein. It and an emission wavelength of 530 nm. The fluorescence intensity of was therefore necessary to thoroughly determine the effects of 500 nM T7C-45-ANBD was monitored as a function of the 44/62 complex thiol modification on the 45 protein function. concentration in the absence of DNA. Fluorescence measurements were recorded immediately following addition of the 44/62 complex that It should be noted that the existence of a mixed population of ensured no significant ATP depletion. multiply labeled and unlabeled 45 proteins that combine to yield an average probe to protein trimer ratio of 1:1 cannot be completely disregarded at this time. However, under conditions DNA complex, while the linear rate can be explained by a small of up to 50-fold molar excess of probe to protein thiol and at amount of protein precipitation in the absence of ATP. prolonged reaction times (24 h) additional equivalent thiols in DISCUSSION the T7C-45 protein should be completely reacted. Moreover, the In this paper, we have succeeded in labeling a cysteine mu- apparent similarity with respect to the extent of T7C-45 pro- tant of the sliding clamp (the 45 protein) with an environmen- tein thiol modification with several of the different thiol re- tally sensitive, thiol-reactive fluorescent probe. This fluores- agents listed in Table I suggests that the observed ratio of 28050 Fluorescently Labeled DNA Polymerase Sliding Clamp There were two fluorescence changes induced in the T7C-45- ANBD protein by the addition of the 44/62 complex and ATP. One change was DNA-independent and the other was DNA-de- pendent. The DNA-independent change was triggered by the addition of the 44/62 complex and ATP and had an apparent stoichiometry of 1:1. The 1:1 stoichiometry of the T7C-45- ANBDz44/62 complex indicates that the observed increase in fluorescence is likely due to complex formation rather than a catalytic event such as the 44/62 complex-catalyzed opening or closing of the 45 protein trimeric ring. The ATP dependence and the inability of the nonhydrolyz- able ATP analogs AMP-PNP, AMP-PCP, or ATPgS to substi- tute in the formation of this DNA-independent fluorescence change suggests two possible scenarios. One possibility is that the fluorescence change is the result of a conformational change induced by binding ATP that cannot be induced by the FIG.6. Model for the observed stoichiometry of T7C-45 protein ATP analogs. A more likely explanation is that this conforma- thiol modification. Structure 1 represents the 45 protein in the closed tional change is triggered by ATP. This apparent requirement conformation that may be in equilibrium with an open conformation (structure 2). Structure 1 is shown with all cysteine thiols solvent for ATP hydrolysis suggests that the T7C-45-ANBDz44/62 com- inaccessible. Upon opening of the 45 protein at a single subunit inter- plex has undergone an ATP hydrolysis-driven conformational face, the environment of one cysteine thiol may change making one change, such as the opening of the 45 protein. Currently, the cysteine thiol more reactive. The reaction of this thiol with a thiol identity of this ATP hydrolysis-dependent conformational reagent (RX) may produce a 45 protein (structures 3 and 4) that is preferentially “opened” at the interface closest to the modified cysteine change in the 45 protein has not been assigned. thiol. T7C-45-ANBD Protein-DNA Interactions—The DNA- dependent change in T7C-45-ANBD fluorescence in the pres- probe:trimer is not due to the thiol reagent structure but rather ence of ATP was attributable to the 44/62 complex catalyzed some aspect of the protein itself. The ability of DTNB to modify loading of the T7C-45-ANBD protein onto DNA. The assign- all three cysteine thiols with equivalent kinetics may be attrib- ment of this DNA-dependent fluorescence change was made by uted to its relatively small size. measuring its rate of dissociation upon ATP consumption. The Activity of Fluorescently Labeled T7C-45 Protein—The 45 observed rate of fluorescence decay (0.011 s ) was similar to protein does not possess a measurable catalytic activity by that obtained for the holoenzyme-DNA dissociation constant itself. Instead its function is assessed through either its stim- using a strand displacement assay method (0.002 s , 11). The ulation of the DNA-dependent 44/62 ATPase activity or its difference in the dissociation rate constants may be attributed ability to enhance the processivity of the T4 DNA polymerase. to the fact that the substantial decrease in the fluorescence A steady state DNA-dependent ATPase assay provided a con- signal was best approximated by a single exponential decay, venient assay for screening the effects of thiol modification. The whereas the data obtained from the strand displacement assay T4 polymerase-45 protein interaction can be observed by the were better approximated by a double exponential decay reduction in the ATP hydrolysis rate upon holoenzyme assem- where, in addition to the 0.002 s rate, there was a population bly. These measurements demonstrate that the 45 protein can- (30%) that decayed at a faster rate (0.028 s , 11). The not tolerate large thiol reagents, such as the fluorescein deriv- weighted average of these rates (0.0097 s ) is very similar to ative, at the T7C position, but it can tolerate small thiol that obtained here (0.011 s ) which suggests that the fluores- reagents such as IANBD. This observation is supported by the cence assay is not sensitive to the presence of the two distinct x-ray crystal structure of the 45 protein that demonstrates that holoenzyme species observable by the strand displacement the T7 position is buried on the interior surface of the protein 1 method. ring. Interestingly, T7C-45 protein modification with the rel- Interestingly, the DNA-dependent change in T7C-45-ANBD atively large fluorescein derivative did not affect the T4 poly- fluorescence was also induced when ATPgS was substituted for merase-induced ATPase shut down rate. This lack of an appar- ATP. Other ATP analogs that were examined (AMP-PNP and ent effect on the holoenzyme ATPase rate by some large thiol AMP-PCP) did not induce this fluorescence change. In this reagents suggests that the T4 polymerase interacts at a site case, ATPgS appears to provide a clue toward determining the removed from the T7C position in the 45 protein. role of ATP hydrolysis in holoenzyme assembly. This effect of Proper functioning of the T7C-45 protein labeled with ATPgS may suggest that ATP binding rather than hydrolysis is IANBD (T7C-45-ANBD) was also demonstrated by a pre-steady required for the 44/62 complex to chaperone the 45 protein onto state kinetic analysis of the stimulation of the DNA-dependent DNA. This may also suggest that the T7C-45-ANBD fluores- ATPase activity of the 44/62 complex and by a strand displace- cence change is independent of whether the 45 protein is cor- ment assay. The pre-steady state ATPase measurements pro- rectly loaded onto DNA (i.e. with ATP) or merely associated vided the ATPase burst rate and burst amplitude. It appears with DNA in some manner. It was previously shown by DNA that it is the ATP hydrolysis burst rate that limits holoenzyme footprinting (15) and protein-DNA cross-linking (16) that the assembly (13, 11). The strand displacement assay demon- 45 protein is complexed with DNA in the presence of the 44/62 strated that the T7C-45 protein retains another function of the complex and ATPgS. Since ATPgS is not able to support func- wild type 45 protein, namely the polymerase processivity tional holoenzyme assembly (14), the 45 protein is likely in- enhancement. completely loaded onto DNA in its presence. T7C-45-ANBD Protein-Protein Interactions—The evaluation of several thiol-directed fluorescent probes led to the discovery Partial Mechanism for Holoenzyme Assembly—Based on the ATP hydrolysis dependence of the observed fluorescence that the T7C-45 protein modified with the environmentally sensitive probe, IANBD, produced substantial changes in fluo- changes in the presence and absence of DNA, a putative partial mechanism is proposed (Fig. 7). The proposed mechanism is rescence intensity upon interactions with the other components of T4 replication. incomplete due to the fact that the loading of the polymerase Fluorescently Labeled DNA Polymerase Sliding Clamp 28051 more stable ring closed form on DNA concomitant with 44/62 complex release. Comparison with the DNA Polymerases of Other Systems— The E. coli replication system sliding clamp (the b clamp) is loaded by a five protein complex termed either the DnaX com- plex or the g complex. The five proteins that assemble to form the E. coli clamp loader are the d, the d9, the x, the c, and either the g or the t subunit. DnaX complexes formed with either the g or the t subunit differ in their ability to hydrolyze ATPgS (23) and are thought to possess different roles in holoenzyme for- mation (23, 24, 25). The existence of a clamp loader-clamp intermediate has been shown in this system using surface plasmon resonance (24). This intermediate displayed similar ATP requirements to that of the 44/62–45 complex observed here in that it was dependent upon ATP hydrolysis for its function (24). In conclusion, the fluorescence data presented here are highly suggestive of a mechanism in which the 44/62 complex mediates the association of the 45 protein with DNA in an ATP-dependent manner but without ATP hydrolysis. Upon ATP hydrolysis a conformational change is invoked in the 45 protein by the 44/62 complex that results in the functionally loaded 45 protein on DNA. Acknowledgments—We are grateful to Ismail Moarefi and John Kuriyan for providing us with the cysteine mutant of the 45 protein. REFERENCES FIG.7. Partial mechanism for holoenzyme assembly. The 44/62 1. Kornberg, A., and Baker, T. (1992) DNA Replication, 2nd Ed., pp. 113–217, complex is shown by four spheres that represent the 44 protein and one W. H. Freeman and Co., New York rectangle (the 62 protein). Upon binding ATP a conformational change 2. Stillman, B. (1994) Cell 78, 725–728 is induced in the 44/62 complex that enables interaction with the 45 3. Young, M. C., Reddy, M. K., and von Hippel, P. H. (1992) Biochemisty 31, protein (trimeric ring). This 45 protein:44/62:ATP bound complex inter- 8675–8690 acts with DNA without ATP hydrolysis to yield a noncatenated form of 4. Nossal, N. G. (1992) FASEB J. 6, 871–878 the 45 protein on DNA. The model has ring opening on DNA coupled to 5. Kong, X.-P., Onrust, R., O’Donnell, M., and Kuriyan, J. (1992) Cell 69, ATP hydrolysis. The opened form of the ring then decays to the closed 425–437 ring on DNA with the release of the 44/62 complex. 6. Krishna, T. S. R., Kong, X.-P., Gary, S., Burgers, P. M., and Kuriyan, J. (1994) Cell 79, 1233–1243 7. O’Donnell, M., Onrust, R., Dean, F. B., Chen, M., and Hurwitz, J. (1993) Nucleic Acids Res. 21, 1–3 does not cause a fluorescence change. The popular conception is 8. Onrust, R., Stukenberg, P. T., and O’Donnell, M. (1991) J. Biol. Chem. 266, that ATP causes a conformational change in the 44/62 complex 21681–21686 such that the 62 protein is better positioned for interaction 9. Yoder, B. L., and Burgers, P. M. J. (1991) J. Biol. Chem. 266, 22689–22697 10. Rush, J., Lin, T.-C., Quinones, M., Spicer, E. K., Douglas, I., Williams, K. R., with the 45 protein (3). The mechanism in Fig. 7 uses ATP and Konigsberg, W. H. (1989) J. Biol. Chem. 264, 10943–10953 binding to the 44/62 complex to cause the conformational 11. Kaboord, B. F., and Benkovic, S. J. (1995) Curr. Biol. 5, 149–157 change that exposes the 62 protein for interaction with the 45 12. Kaboord, B. F., and Benkovic, S. J. (1996) Biochemistry 35, 1084–1092 13. Berdis, A. J., and Benkovic, S. J. (1996) Biochemistry, 35, 9253–9265 protein. ATP hydrolysis within the 45–44/62 complex causes 14. Piperno, J. R., and Alberts, B. M. (1978) J. Biol. Chem. 253, 5174–5179 an observable fluorescence change that may represent the 15. Munn, M. M., and Alberts, B. M. (1991) J. Biol. Chem. 266, 20024–20033 opening of the 45 protein. As shown in Fig. 7 our results 16. Capson, T. L., Benkovic, S. J., and Nossal, N. G. (1991) Cell 65, 249–258 17. Gogel, E. P., Young, M. C., Kubasek, W. L., Jarvis, T. C., and von Hippel, P. H. suggest that the ATP-dependent 45–44/62 complex can be as- (1992) J. Mol. Biol. 224, 395–412 sociated with DNA without ATP hydrolysis. In the model (Fig. 18. Capson, T. L., Peliska, J. A., Kaboord, B. F., Frey, M. W., Lively, C., Dahlberg, M., and Benkovic, S. J. (1992) Biochemistry 31, 10984–10994 7), this ATP hydrolysis independent DNA-45–44/62 complex 19. Nossal, N. G. (1979) J. Biol. Chem. 254, 6026–6031 represents a form of the 45 protein that does not have DNA 20. Frey, M. W., Nossal, N. G., Capson, T. L., and Benkovic, S. J. (1993) Proc. Natl. through the center of the protein ring (nonconcatenated form). Acad. Sci. U. S. A. 90, 2579–2583 21. Johnson, K. A. (1986) Methods Enzymol. 134, 677–705 Since ATP binding must occur prior to ATP hydrolysis, it is 22. Ellman, G. L. (1958) Arch. Biochem. Biophys. 82, 70–77 plausible that the 44/62 complex chaperones the 45 protein to 23. Dallmann, H. G., Thimmig, R. L., and McHenry, C. S. (1995) J. Biol. Chem. the primer template before the 45 protein ring is opened. The 270, 29555–29562 24. Naktinis, V., Onrust, R., Fang, L., and O’Donnell, M. (1995) J. Biol. Chem. 270, opening of the protein ring on DNA would enable the DNA to 13358–13365 enter the center of the 45 protein. The ring opened 45 protein- 25. Xiao, H., Naktinis, V., and O’Donnell, M. (1995) J. Biol. Chem. 270, 44/62 complex on DNA may decay to a thermodynamically 13378–13383

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Journal of Biological Chemistry – Unpaywall

Published: Nov 1, 1996

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