the journal of biological vol. 267, no. 20. of pp. 1992 ... · processive proofreading is intrinsic...

Vol. 267, No. 20. Issue of July 15, pp. 14157-14166. 1992 Printed in U. S. A.

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

Processive Proofreading Is Intrinsic to T4 DNA Polymerase* (Received for publication, December 20,1991)

Michael K. Reddy, Stephen E. Weitzel, and Peter H. von Hippel$ From the Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, Oregon 97403

DNA replication occurs in vivo with very high processivity, meaning that the replication complex assem- bles at the origin(s) of replication and then performs template-directed synthesis of DNA over virtually the entire genome without dissociation. Such processivity also characterizes reconstituted replication holoenzyme complexes in vitro. However, the isolated DNA polymerases are much less processive, especially under physiological conditions. In this paper we monitor the degree of processivity displayed by the bacteriophage T4-coded DNA polymerase while in its proofreading mode by asking whether an isolated polymerase can “edit-out” the 3”terminal nucleotide from the primer (using the 3‘45‘-exonuclease activity of the polymerase) and then switch into the synthesis mode without dissociating from the DNA template. This “switch experiment’’ is accomplished by using mismatched primer/template substrates as an experimental tool to mimic the situation that T4 DNA polymerase encounters after a misincorporation event has occurred. By performing experiments under single-turnover conditions (obtained using a heparin trap), we demonstrate that T4 DNA polymerase, upon encountering a misincorporated base, neither synthesizes the next base nor dissociates into solution. Instead, with a greater than 80% probability, it removes the misincorporated base and then continues synthesis in a fully processive manner. We also show that the removal of a doubly mispaired sequence from the 3”terminus of the primer, followed by synthesis, is comparably processive. In contrast, the apparent processivity of removing a triply mispaired terminus is much reduced. Taken together, these observations are consistent with the notion that the “editing active site” of the T4 enzyme optimally accommodates only two unpaired nucleotide residues. Our results do not support the idea that the exonuclease activity of T4 DNA polymerase is highly selective for mismatched termini; they suggest instead that the dwell time at a misincorporated base deter- mines overall editing efficiency. The integrated results of this study provide additional insight into the structure of the T4 DNA polymerase, as well as into the interactions between the polymerase and the polymerase accessory proteins that are required to provide the holoenzyme complex with full processivity.

* These studies were supported in part by United States Public Health Service Research Grants GM-15792 and GM-29158 (to P. H. von H.) and by a grant from the Lucille P. Markey Charitable Trust to the Institute of Molecular Biology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ American Cancer Society Research Professor of Chemistry. To whom correspondence and reprint requests should be addressed. Tel.: 503-346-5151: Fax: 503-346-5891.

DNA replication complexes involved in the elongation phase of replication in prokaryotes and eukaryotes contain a number of subunits, ranging from two for the simplest Esch- erichia coli bacteriophage (T7) to over 20 for the entire functional E . coli complex (see Kornberg and Baker, 1992). During in vivo replication, the nascent DNA is elongated in a highly processive manner, suggesting that the multisubunit complexes are assembled initially at the origins of replication and then carry out continuous replication of the chromosome (or of a major part of the chromosome) without falling apart or dissociating from the template DNA.

The central functional entity carried within these replication complexes is the one or more polymerase molecules that actually catalyze the template-directed addition of single nucleotide residues to the growing DNA chain. In contrast to their behavior when integrated into the i n vivo complex, these polymerases tend to synthesize DNA with very low processivity (i.e. they polymerize very few nucleotides per binding- dissociation event) when functioning in isolation at physiological salt concentrations. Thus, in a typical in vitro experiment under such conditions, the polymerase adds one, or a very few, nucleotide residues to the growing DNA chain and then dissociates from the template (Das and Fujimura, 1980; Newport et al., 1980). Clearly, as has been demonstrated for the T7 system (Tabor et al., 1987), the T4 system (Newport et al., 1980; Sinha et al., 1980), and the E. coli system (Fay et al., 1981,1982), one of the major functions of a number of the additional subunits of the DNA replication complex is to make it possible for the polymerase (working as a part of the functional holoenzyme) to operate in a fully processive manner under physiological conditions.

Processivity is an essential feature of a functional replication complex. Aside from the obvious logistical problems associated with reassembling the complex at each nucleotide insertion step, simple calculations for the T4 DNA replication system have shown that the time that would be required for diffusion to bring just the polymerase itself back to the primer- template (P/T)’ junction under in vivo polymerase concentrations after a nonprocessive nucleotide insertion event is likely to be of the order of seconds.’ Since replication in vivo at T4 DNA replication forks proceeds at rates approaching 500 nucleotide residues per polymerase molecule per s (McCarthy et al., 1976), the ability to conduct replication in a processive manner is obviously a central functional requirement for any DNA replication complex. Therefore, in studying the stepwise assembly of the functional T4 DNA replication complex from its subunit constituents i n vitro, we have monitored the onset of processivity to determine the components and interactions that are necessary to establish this activity under physiological conditions.

’ The abbreviations used are: P/T, primer/template; HEPES, 4-

M. C. Young, M. K. Reddy, and P. H. von Hippel, submitted for (2-hydroxyethy1)-1-piperazineethanesulfonic acid.

publication.

14157

14158 Processive Proofreading with T4 DNA Polymerase

Most DNA polymerases manifest a 3’45‘-exonuclease activity, which is thought to serve in an editing (or proofreading) capacity to improve the overall fidelity of the DNA synthesis process by preferentially removing misincorporated 3’-terminal residues from the growing DNA chain prior to further synthesis (Brutlag and Kornberg, 1972). This activity may be intrinsic to the polymerase, or it may be carried on a separate polypeptide that interacts functionally with the polymerase within the holoenzyme complex (see Kornberg and Baker, 1992). In uiuo it seems clear that this editing process occurs while the replication complex moves processively along the chromosomal template, since the complex as a whole clearly does not dissociate in order to remove a misincorporated nucleotide from the nascent DNA.

On the other hand, it is not clear whether this ability of the polymerase, functioning within the integrated replication complex, to switch from the synthesis to the editing mode and then back again without dissociating into solution represents a function of the complex as a whole, or whether it is “built-in” to the polymerase itself. A definitive answer to this question is important in attempting to understand the roles played by the various holoenzyme subunits in setting up the functional replication complex, because if this ability is (at least latently) present in the polymerase alone this signifi- cantly changes one’s views of what is required of the other components of the integrated complex. In this paper we have therefore attempted to determine whether, at least under some conditions, the switch between the synthesis and misincorporation modes can be carried out processively by the polymerase alone at a primer-template junction free in solution, or whether (in the absence of the other subunits of the complex) the polymerase is required to dissociate and then to return to the functional “substrate” by a free diffusion process in mov- ing from one of these modes to the other.

A processive ability to switch between these two modes at the free polymerase level is rendered more mechanistically demanding, at least for E. coli DNA polymerase I (Pol I) for which a detailed molecular structure has been determined by x-ray crystallography, by the demonstration that the enzymic sites involved in synthesis and exonuclease editing are 25 8, to 30 A apart (Ollis et al., 1985). Experiments to establish the amino acid residues that are directly concerned with these two enzymic activities in Pol I (Derbyshire et al., 1988), as well as in T4 DNA polymerase (Reha-Krantz, 1988) and T7 DNA polymerase (Tabor and Richardson, 1989), are also consistent with a significant spatial separation of these two functions.

Experiments designed in part to determine which of these editing pathways is followed by polymerase alone have been attempted with various prokaryotic polymerases. Although referred by different terms; i.e. “intramolecular switching” for T5 DNA polymerase (Das and Fujimura, 1980), “DNA shut- tling” for the Klenow fragment of Pol I (Joyce, 1989), and “bidirectional channeling” for T7 DNA polymerase (Donlin et al., 1991), the protocols used in these studies were all structured to attempt to determine what happens to a bound DNA polymerase molecule when it must “relocate” the 3‘- hydroxyl terminus of the nascent DNA from the active site for synthesis to the physically and chemically distinct exonuclease active site. Preliminary experiments of this sort have also been carried out by Dolejsi (1988) in this laboratory (see also Jarvis et al., 1990b). In the present study, we use a new approach to demonstrate definitively that the T4 DNA polymerase follows a processive “switching” pathway; we have been able to use this approach to define a number of the molecular details of this process. As suggested above, the results have

important implications for future studies of the structural and functional integration of the polymerase into the T4 DNA holoenzyme complex and ultimately into the entire replication apparatus.

MATERIALS AND METHODS

Preparation of T4 DNA Polymerase“T4 DNA polymerase (gene 43 protein or gp43) was purified from an overproducer strain (gift of T. C. Lin from the laboratory of W. Konigsberg) as described by Rush and Konigsberg (1989; see also Jarvis et al., 1991). The polymerase was judged to be >98% pure, based on sodium dodecyl sulfate- polyacrylamide gel electrophoresis analysis of the preparation followed by silver staining, and was shown to be free of contaminating endonuclease by the absence of chain breaks after incubation with supercoiled pBR322 DNA or single-stranded M13 DNA. Protein concentrations were determined spectrophotometrically using a calculated (Gill and von Hippel, 1989) molar extinction coefficient at 280 nm (cM, 280) of 1.28 X lo5 M” cm”, based on the published amino acid sequence of gp43 (Spicer et al., 1988). This value differs by -15% from a value recently reported by Reha-Krantz et aL(1991). T4 DNA polymerase (gp43) exists in solution, and presumably also bound to our DNA primer-template constructs, as protein monomers (Goulian et al., 1968; Nossal and Hershfield, 1971) with a calculated molecular mass of 103,572 daltons (Spicer et al., 1988). In this paper, we report the concentrations of gp43 in nanomolar units.

Preparation of Synthetic Oligonucleotides-The oligonucleotides used in this study (see Table I) were synthesized and purified as described elsewhere (Jarvis et al., 1989, 1990b). Concentrations of oligonucleotides were estimated by UV absorbance at 260 nm, using the following molar extinction coefficients (eM, 2 ~ ) ) for the various primers and templates (see Table I for the primer and template designations) that were calculated by nearest-neighbor analysis (War- shaw and Cantor, 1970). For the 25-mer template, tM, 260 = 23,800; for the 17-mer primer, t ~ , 2~ = 17,200; for the 16A-mer primer, EM, 260 = 17,800; for the 16C-mer primer, tM, 2M = 17,000; for the 16G- mer primer, C M , ~ M ) = 17,400; for the 15CC-mer primer, eM, 2~ = 16,700; and for the l4CCC-mer primer, tM, 2 6 ~ = 16,900.

Construction of the Primer-Template (P/T) Substrates-The 17- mer primer (60 pmol) was labeled at its 5’-end with [Y-~’P]ATP (Du Pont-New England Nuclear), using 6 units of T4 polynucleotide kinase (U. S. Biochemical Corp.) in a sample containing reaction buffer consisting of 50 mM Tris-OAc (pH 9.0), 100 mM KOAc, 10 mM dithiothreitol, and 10 mM Mg(OAc)2. We have optimized these conditions for end-labeling single-stranded DNA oligonucleotides using polynucleotide kinase (see also van de Sande et al., 1973). The reaction was allowed to proceed for 10 min in a 37 “C water bath, and the kinase was then inactivated by heating the sample at 70 “C for 10 min.

Hybridization of the primer and the template was accomplished by adding the complimentary 25-mer template oligonucleotide (72 pmol) directly to the kinase reaction mixture. The resulting solution was held at 70 “C for an additional 5 min and then cooled to room temperature over approximately 2 h. The hybridized primer-template constructs were separated from free ATP and M F on Bio-Spin 6 columns (Bio-Rad), pre-equilibrated, and eluted in 10 mM HEPES (pH 7.8), 50 mM KOAc, and 1 mM EDTA by centrifugation in a TH- 4 rotor at 1,200 X g for 4 min in a TJ-6 centrifuge (Beckman).

The amounts of radiolabeled primer-templates present in various experiments were measured by running aliquots of the sample on polyethyleneimine-cellulose F thin layer chromatography plates (EM Science), which were then developed in 0.3 M potassium phosphate (pH 7.0) and quantitated on an AMBIS radioanalytic scanner (AM- BIS Inc., San Diego). The extent of hybridization of the primer- template structures was routinely determined to be greater than 95% by electrophoresis on 16% nondenaturing polyacrylamide gels in a buffer system containing 6 mM Mg(OAc)? and 1 X TBE (89 mM Tris- HCI (pH 8.3), 89 mM boric acid, 2.5 mM EDTA). Radioactive bands on dried polyacrylamide gels were also quantitated using the AMBIS radioanalytic scanner.

Polymerase Assays-Except where otherwise noted, the following standard assay protocol was used. T4 DNA polymerase was diluted in a buffer containing 25 mM HEPES (pH 7.5; diluted from a 1 M stock of pH 7.8 at 20 “C), 2 mg/ml acetylated bovine serum albumin (LJ. S. Biochemical Corp.), 5 mM dithiothreitol, and 20% (v/v) glycerol. The diluted polymerase was preincubated at 37 “C (except where otherwise noted) for 2 min with the indicated primer-template in a

Processive Proofreading with T4 DNA Polymerase 14159

buffer containing all four deoxynucleoside triphosphates (Pharmacia LKB Biotechnology Inc.). Preincubations were always performed in the absence of M$+. Enzymic reactions on the preformedpolymerase- P/T complexes were initiated by the addition of M e for multiple turnover reactions and M$+/heparin for single-turnover reactions. The complete mixture was incubated for 15 s at 37 “C (unless noted otherwise), and reactions were quenched with the addition of 2 volumes (20 pl) of a formamide-dye mixture (95% deionized formamide, 0.25% xylene cylanol, 0.25% bromphenol blue, and 20 mM EDTA). After reaction, the samples were boiled briefly, chilled rapidly, and then subjected to denaturing electrophoresis on 20% polyacrylamide (20:l acry1amide:bis) gels (0.4-mm-thick) containing 8 M urea (Maxam and Gilbert, 1980). The gels were prerun to a temperature of 50 “C, and the samples were then electrophoresed for 2 h at 30 watts (constant power). Gels were dried on Whatman No. 3MM paper and autoradiographed on x-ray film (Fuji Aif or Kodak X- Omat). In addition, products from DNA polymerase reactions were quantitated on the AMBIS scanner.

The final reactions in the DNA synthesis and “switching” experiments were run in 10-pl volumes and contained (unless otherwise noted) 25 mM HEPES (pH 7.5), 60 mM KOAc, 5 mM dithiothreitol, 0.5 mM EDTA, 125 p~ each of up to four deoxyribonucleoside triphosphates (dNTPs), 0.2 mg/ml bovine serum albumin, 6 mM Mg(OAc),, 2% glycerol, 1 mg/ml heparin, 70 nM P/T substrate (expressed in extendable 3’-primer ends), and 128 nM polymerase

under the same buffer conditions, except that the dNTPs were (expressed as protein monomers). The exonuclease assays were run

omitted. Heparin was omitted when single-turnover conditions were not desired.

Although the original characterization (Goulian et al., 1968) of T4 DNA polymerase reported a pH optimum of 8.8 for the purified enzyme, we perform our in vitro reactions in a HEPES buffer at pH 7.5 for two reasons. The first is the physiological relevance of this pH value. The other is the observation, made originally by Huang and Lehman in 1972, that in a HEPES buffer (as opposed to a Tris-HC1 buffer a t this same pH) activity of the polymerase remains maximal at pH 7.5.

RESULTS

Use of Heparin to Obtain Single-turnover Conditions-The processivity of T4 DNA polymerase or holoenzyme has previously been measured in our laboratory using “single-hit” (Newport et al., 1980; Dolejsi, 1988; Jarvis et al., 1990a, 1991) as well as “single-turnover” (Jarvis et al., 1990a, 1991) kinetic conditions. The relative merits of each of these approaches has been discussed by Jarvis et al. (1991). The central point in such experiments is that, using either the single-hit or the single-turnover protocol, any given primer on a primer/template (P/T) substrate will have an effectively zero probability of being extended by more than one polymerase molecule during a single reaction.

In the present study, we have investigated the processive editing by T4 DNA polymerase of various P/T constructs using a single-turnover protocol. This condition is attained by the strategic use of a reagent to “trap” the polymerase molecules that dissociate from the P/T substrate after a reaction has been initiated, thereby preventing polymerase rebinding and “multiple-hit” extension of the DNA primer.

Several past protocols have employed nonspecific DNA as the trapping agent of choice for studying processive events in DNA replication under single-turnover conditions (Huber et al., 1987; Joyce, 1989; Jarvis et al., 1990a, 1991). However, we have found heparin to be superior to a DNA trap in our mechanistic and functional dissection of T4 DNA polymerase. One problem with a DNA trap is that, in order to be effective, it must be present in vast molar excess (>ZOO fold) over the extendable primer ends of interest (see Joyce, 1989; Jarvis et al., 1991).3 Such quantities of DNA are tedious to obtain and to purify and require empirical re-evaluation for each new batch.

’’ M. K. Reddy, unpublished observations.

In addition, unlike heparin, a DNA trap is “used up” during the course of the reaction by virtue of the fact that it serves as a substrate for the polymerase. Thus, synthesis reactions involving the DNA trap convert it from a primer-template structure to a fully double-stranded structure for which the polymerase has a much lower affinity (Dolejsi, 19881, thereby decreasing the potency of the trap. It may also be necessary to use experimental protocols that require the incorporation of a-labeled dNTPs into the nascent DNA. Such an experimental procedure would result in the labeling of the DNA trap and therefore obscure the results with the P/T of interest (heparin, of course, is not labeled). Finally, the DNA trap may not be “passive,” in that it may bind to the polymerase- P/T complex and prematurely remove the polymerase from the template by a direct transfer mechanism (von Hippel et al., 1975). Such a process has been previously observed in single-turnover processivity reactions in which nonspecific DNA was used as a trap for T7 DNA polymerase (Huber et al., 1987).

The heparin trap may also present problems under some conditions. Addition of heparin to higher levels (>2 mg/ml) than used in this study (1 mg/ml) results in an artifactual reduction of primer usage by the p~lymerase.~ Since heparin is a polyanion, one plausible explanation for this observation is that it may “titrate” M g + ions out of the solution (presumably via ion-pairing), resulting in a decrease in polymerase activity. Another possibility is that at sufficiently high concentrations heparin might also actively displace polymerase molecules from P/T complexes by direct transfer mechanisms; to any extent that this may occur, the observed processivity would represent a lower limit of the actual processivity. Finally we have noticed, as also recently observed by Herendeen et al. (1990) in studies involving RNA polymerase in which heparin was used as a trap, that the effectiveness of the heparin trap decreases as the K+ ion concentration is raised. Again, this is most likely a result of direct ion-pair formation between the anionic groups of the heparin and K+ ions.

A display of the utility of heparin as a trap in single- turnover reactions involving T4 DNA polymerase is demonstrated under several different reaction conditions in Fig. 1. As in all the reactions presented in this paper, the DNA substrate was formed by annealing a 17-nucleotide primer oligonucleotide to a 25-nucleotide-long template strand (see Table I for the sequences of all the P/T constructs used in this study). As a control experiment to illustrate the effectiveness of heparin as a trap, the 17/25 PIT was preincubated with heparin and M$+ (the presence of this divalent cation in adequate concentrations is essential for the catalytic activities of the polymerase), and the reaction was initiated by the addition of polymerase. The experiment showed that preaddition of heparin prevents polymerase from binding to the 17/25 P/T construct and therefore absolutely no exonucleolytic degradation of the 5’-end-labeled primer strand occurs under such conditions (lane 1, Fig. 1).

Using heparin, we have also monitored the processivity of the 3’4’-exonuclease activity of T4 DNA polymerase on P/ T substrates. Products reflecting this exonuclease activity in a multihit (untrapped) regime are obtained when the polymerase is preincubated with (e.g.1 the 17/25 P/T construct and the reaction is then initiated by the addition of M$+ alone ( l a n e 2, Fig. 1). Here the T4 DNA polymerase effectively digests the fully base-paired primer strand by removing at least 14 nucleotide residues in less than 15 s.

However, a dramatically different result is obtained when the same reaction is initiated by the simultaneous addition of

14160

25-

17- 16- 15-

14-

3- 2- 1-

Processive Proofreading with T4 DNA Polymerase

1 2 3 4 5

FIG. 1. Heparin prevents dissociated T4 DNA polymerase molecules from rebinding to the primer-template substrate. Exonuclease and synthesis assays were performed with the end- labeled 17/25 P/T construct under standard conditions (see "Mate- rials and Methods"). Lane 1 shows the results of an exonuclease reaction in which heparin (at 1 mg/ml) was preincubated with the DNA substrate before the addition of T4 DNA polymerase. The difference in product sizes obtained from multiple rounds of exonuclease activity (reactions initiated by the addition of Mg2f only to a preformed polymerase-P/T complex) compared with product sizes obtained from a single round of exonuclease activity (reactions initiated with Mg2f plus heparin) are shown in lanes 2 and 3, respectively. The products of a single round of DNA synthesis (+heparin) are also compared to products obtained from multiple rounds (-heparin) in lanes 4 and 5, respectively. In these synthesis experiments, all four dNTPs were present during the preincubation of the polymerase with the P/T construct.

M e and heparin ( l a n e 3, Fig. 1). Here the product distribution reflects the fact that the exonuclease reaction displays a very low processivity on this fully base-paired substrate. This experiment shows that, under the conditions used, no more than 3 nucleotide residues are removed from the 3'-hydroxyl end of the fully base-paired primer by T4 DNA polymerase before the enzyme dissociates from the P/T construct. In this experiment, of course, the polymerase is trapped by heparin and thus is prevented from rebinding.

Heparin can also be used to monitor the processivity of DNA synthesis. The initiation of a synthesis reaction by the addition of Mg2+ and all four dNTPs to a preformed polymerase-P/T complex in the absence of a trap results in multiple rounds of DNA synthesis, and all the primers are extended to full length products in much less than 15 s ( l u n e 5, Fig. 1). Identical reactions initiated by the simultaneous addition of M e and dNTPs and heparin also result in full-

TABLE I DNA sequences of the primerftemplate substrates used in this study

Each substrate is formed by the annealing of a 17-nucleotide residue primer strand to a %-residue template strand (see "Materials and Methods"). The designation for each primer/template (P/T) is shown in the left column and represents the number of matched base pairs followed by the sequence of the mismatched primer residue(s). The melting temperature ("C) of each P/T is given in the right column. T, values were determined in the identical buffer used for performing exonuclease reactions (see "Materials and Methods") at a strand concentration of 1 p ~ . Each value represents the average of two determinations to an accuracy of fl "C.

I- I 65

64

60

59

ea

64

length (25-mer) products, showing that the T4 DNA polymerase conducts synthesis reactions with complete processivity on this P/T construct ( l a n e 4, Fig. 1).

Extension of 3"Mispaired Primers-The central question we are concerned with is whether the T4 DNA polymerase can remove a misincorporated nucleotide residue from the 3'- end of the nascent DNA strand and then resume synthesis without undergoing dissociation from the DNA template. Two key requirements are central to the successful design of this experiment. The first, of course, is to use an effective trap to ensure single-turnover conditions. This requirement is met, as described above, by heparin. The second requisite is for a DNA substrate that "forces" the polymerase to perform an exonucleolytic event prior to a subsequent synthesis step. As the results of Fig. 2 show, this requirement is met for T4 DNA polymerase by using a P/T construct that features a mismatched 3'-OH primer terminus.

This fact was established by demonstrating that, although the T4 DNA polymerase can processively add the next single nucleotide to the 3'-end of the primer within the fully base- paired 17/25 P/T construct (a dC residue for this template sequence), it is unable to elongate the mismatched primer terminus of the 16C/25 P/T construct (compare lanes 3-5 with lanes 8-10 in Fig. 2). This observation is the core of the switch experiment that we describe in this paper. That is, under our experimental regime, any product longer than the initial (17-mer) primer must have been synthesized as the result of a preceding exonuclease event.

We have tried to determine whether we can achieve processive extension of each of the different mismatched P/T constructs used in this study (see Table I for structures), without nucleolytic removal of the mismatched primer terminus, by increasing the initial concentration of dCTP up to 10 mM. We find that at dCTP concentrations of 2 mM and above, T4 DNA polymerase can be forced to synthesize directly from a

Processive Proofreading with T4 DNA Polymerase

16C/25 17/25 mismatched base - paired

‘1 2 3 4 5“6 7 8 9 10

+primer +1 -primer

FIG. 2. T4 DNA polymerase does not extend a DNA chain directly from a terminally mismatched primer-template construct under single-turnover conditions. Incorporation of the next correct nucleotide from the 3”terminus of the 17-base primer (corresponding to a dC residue opposite the template G ) is compared for a mismatched P/T construct (lanes 1-5) and a base-paired P/T construct (lanes 6-10) as a function of the concentration of dCTP added to the reaction. The following concentrations of dCTP were included in the preincubation protocol: 0 pM (lanes 2 and 7), 100 pM (lanes 3 and 8), 500 p~ (lanes 4 and 9) , and 1,000 p~ (lanes 5 and 10). Reactions were initiated by the simultaneous addition of Mg2‘ and heparin. Markers showing the respective end-labeled primer strands are in lanes I and 6.

3”terminally mismatched P/T construct. A somewhat similar observation has been reported in a previous study on the fidelity of T4 DNA polymerase (see Table V of Kunkel et al., 1984). Also, consistent with previous conclusions (Fersht and Knill-Jones, 1981), we observe a substantial decrease in exonuclease (i.e. proofreading) activity a t these extremely high (>2 mM) dCTP concentrations (data not shown). The amount of “next nucleotide” synthesis that we observe reaches a maximum of -15% of the total products formed and appears to be independent of the sequence of the mismatch (data not shown). However, we stress again that at the concentration of dNTPs in our experiments (125 pM), absolutely m next nucleotide synthesis is observed with T4 polymerase with any of the mismatched constructs employed in this study.

The “Switch” Experiment-The experimental protocol and conceptual design we have used to determine whether T4 DNA polymerase can processively switch from one mode of activity (synthesis or exonuclease) to the other is diagrammed in Fig. 3. Processive exonuclease products, displayed in the left lane of the inset to Fig. 3, are represented as Species I. As described above, these products are obtained simply by the omission of dNTPs from the initiation reaction. Note that although primer usage is greater on the mismatched substrate, there is no dramatic change in exonuclease processivity on the mismatched P/T construct compared to the fully base- paired substrate (compare to lane 3 of Fig. 1).

The actual switch experiment is performed by preincubat- ing T4 DNA polymerase with a mismatched P/T (Reaction A of Fig. 3), followed by the simultaneous addition of Mg2‘ and all four dNTPs and heparin to initiate reaction under single- turnover conditions (Reaction B of Fig. 3). The initial event under these conditions is the processive exonucleolytic digestion of the primer by the polymerase. If, after the required removal of the mismatch, the polymerase dissociates into solution, then Species I11 will represent the end point of the reaction pathway (Reaction D) since the presence of heparin precludes rebinding of the polymerase to the P/T construct (i.e. Reaction E is blocked). However, if the primer terminus is transferred from the exonuclease to the polymerase active site (Reaction C), then a switch in catalytic modes occurs, and synthesis on the P/T construct gives rise to Species 11. The results of such a switch experiment on the 16C/25 P/T

1.

14161

\

\ / QUESCH

FIG. 3. T4 DNA polymerase can switch from an exonuclease to a polymerase mode without dissociating from the primer- template. The drawing depicts the conceptual as well as experimental pathway of interaction of T4 DNA polymerase with a mismatched P/T substrate under single-turnover conditions. The initial step of the pathway illustrates a preincubated polymerase-P/T complex in which the 3”terminus of the primer is prebound in the synthesis active site of the enzyme (S) . Step A represents the simultaneous addition of Mg2‘ and heparin and dNTPs to initiate the reaction. The next event is a switch in location of the primer terminus to the exonuclease ( E ) active site. Step B represents the required removal of the mismatched bases at the 3”terminus of the primer by the 3’+ 5’-exonuclease activity of the T4 DNA polymerase. This results in the production of Species I. If at this point the T4 DNA polymerase dissociates from the P/T structure into solution (Step D), it will be prevented from rebinding to the P/T structure and Step E does not occur. Thus, Species I11 represents processively formed exonuclease products that represent a dead-end pathway as far as subsequent synthesis events are concerned (i.e. they are not switched). However, if after the processive removal of primer bases the terminus “relo- cates” to the synthesis active site, then Step C of the pathway results in the processive synthesis of products greater than the initial primer length of 17 bases (represented as Species 11). Formation of these various DNA species can be monitored and are shown in the inset for the interaction of T4 DNA polymerase with the 16C/25 P/T construct. The reaction in the left lane represents the profile obtained from processive exonuclease action only. This profile is obtained by simply initiating the reaction by the addition of Mg2‘ and heparin only. The right lane displays a “switch” experiment that differs in conditions from the left lane only by the inclusion of dNTPs in the preincubation protocol (see “Materials and Methods”).

substrate are displayed in the right lane of the inset to Fig. 3. Clearly a switch in catalytic modes has occurred, as evidenced by the “chasing” of the exonucleolytic products into synthesized products longer than the initial 17-mer primer.


We define the switching efficiency as the amount of synthesized products (defined as products greater than or equal to 18 nucleotide residues in length) formed directly from the processive degradation of the end-labeled primer strand (i.e. radiolabeled products that are 16, 15, and 14 nucleotide residues in length). That is, in terms of Fig. 3, the switch efficiency is expressed as the ratio of species I1 formed to the sum of species I1 plus species I11 present at the end of the reaction (see “Materials and Methods” for descriptions of the quantitation of these species). Therefore, the switching efficiency is 100% when only species I1 remains and is, of course, zero if no species I1 is produced. Thus, a switching efficiency of zero would be seen if (following the requisite exonuclease event) the T4 DNA polymerase molecule always dissociates into solution from the P/T substrate. This “dead-end” pathway is easily monitored by comparing the amount of Species I11 formed in reactions initiated with dNTPs (i.e. using the switch protocol) to the amount of Species I formed in reactions initiated without dNTPs (i.e. using the protocol designed to measure exonuclease processivity).

Various Switch Experiments on Singly Mismatched Primer- Templates-Since the value of the switching efficiency reflects directly the amount of processive synthesis taking place, it is apparent that there must be an effect of dNTP concentration on this parameter. This follows because processivity in the forward direction can be viewed as a competition between the rate of extension of the nascent DNA and the rate of dissociation of the polymerase from the primer-template construct (Newport et al., 1980; Mizrahi and Benkovic, 1986). Thus, if dNTP binding becomes a limiting step, the rate of dissociation from the primer-template will more effectively compete with incorporation rates, and the processivity of synthesis observed at low concentration of dNTPs will be reduced.

To test this effect we performed an experiment to correlate switching efficiency with dNTP concentration. The results, presented in Fig. 4, demonstrate that there is indeed a direct dependence of the switching efficiency on dNTP concentration, as evidenced by the observed decrease in amounts of exonucleolytic products and the concomitant increase in fully

2 3 4 5 1 c

25-

17-

14-

FIG. 4. Efficiency of switching displayed by T4 DNA polymerase correlates with dNTP concentration. Switching efficiency (see text for definition) as a function of increasing dNTP concentration was determined using the 16G/25 P/T substrate. In- dividual switch experiments (see “Materials and Methods”) were carried out at the following concentrations of dNTPs: 0 p~ (hne 1 ), 2.5 pM ( h e 2), 12.5 pM (hne 3), 25 pM (hne 4 ) , and 125 pM (hne 5).

extended template-length primer (25-mer). Switching efficiency attains a value of 85% (335%) a t 125 pM concentrations of dNTPs (shown in lune 5 of Fig. 4) and remains at this plateau level up to at least a 500 p~ dNTP concentration. These results again confirm that T4 DNA polymerase removes misincorporated bases primarily via a processive pathway.

There are clearly differences in the thermodynamics of mispaired constructs that differ in DNA sequence (i.e. see Topal and Fresco, 1975; Aboul-ela et al., 1985; Petruska et al., 1988). Therefore we have asked whether the switching efficiency of the T4 polymerase varies with the nature of the mismatch. This was tested by performing the switch experiment (i.e. at a dNTP concentration of 125 pM) on all three possible single mismatches opposite the A residue located at position 17 (counting from the 3’-end) of the template. These P/T mismatch constructs are shown in Table I. The switching efficiency under our standard conditions (60 mM monovalent cations; see “Materials and Methods”) was 88% with the 16A/ 25 construct (data not shown); 85% with 16G/25 (lane 5 of Fig. 4), and 80% with 16C/25 (see inset, Fig. 3). Therefore, we conclude, at least within our experimental error and at the template position and salt concentration tested, that the switching efficiency of the T4 DNA polymerase is independent of mismatch sequence.

Relevant to this same point, although by no means provid- ing a complete description of the thermodynamic properties of these constructs, we did determine the melting temperature (T,,,) of this set of P/T mismatch structures (see Table I). Since we have only measured the T,,, at one strand concentration, we cannot perform a van’t Hoff analysis to obtain enthalpy and entropy changes. However, consistent with the results of Petruska et al. (1988), the small differences in T,,, that we have observed for the single-base mismatched structures compared to the T,,, of the fully base-paired P/T structure suggest that the difference in free energy between the “matched” and “mismatched” structures is very small in aqueous solution.

Other solution parameters that may influence switching between catalytic modes were also investigated. In many DNA-protein interactions (e.g. see Kowalczykowski et al., 1981; Leirmo et al., 1987; Overman et al., 1988; Der Garabe- dian et al., 1991) and other DNA replication systems (Griep and McHenry, 1989), the nature of the anion components of the solvent environment can have a significant effect. There- fore we repeated the switching experiments presented in Fig. 3 in buffers containing chloride or glutamate instead of ace- tate. The results showed that, within experimental error, the nature of the anion does not alter the switching efficiency of the T4 polymerase for the constructs tested (data not shown). We interpret this observation by suggesting that, although anions may affect the association reaction between a polymerase and its substrate, the processivity of the reaction (which is independent of the association process) is unaffected by the nature of the anion. A comparable experiment was also run to determine whether the replacement of Mg2‘ with Mn2+ changes the switch pattern. This experiment is of interest since it has been proposed that the substitution of Mg2‘ by Mn2+ may have mutagenic effects (Goodman et al., 1983). Again we found that this change in the reaction protocol did not alter the observed switching efficiency (data not shown).

Switching Efficiency on Multiply Mismatched Substrates- We now consider the interaction of the T4 DNA polymerase with P/T constructs carrying two or three mismatches at the 3‘ terminus of the primer. We recognize that this multiple mismatch situation is one that a T4 holoenyzme complex is


unlikely to encounter in vivo. However, the use of such substrates could provide structural insight into the optimal length of the single-stranded DNA required to occupy the exonuclease active site of the polymerase.

Reactions of the polymerase with multiply mismatched P/ T constructs of this type were carried out with the same switch protocol used for the single-mismatch experiments described in Fig. 3. A trap experiment to determine the exonucleolytic processivity of T4 DNA polymerase on the 15CC/25 P/T substrate (Table I) established that both mismatched dCMP residues (lane 2, Fig. 5) are removed in a totally processive manner. Similarly, as seen with the single base mismatch constructs described previously, a very processive switch interaction is observed when the polymerase acts upon this structure, as evidenced by the polymerization of the degraded primer to a full length product (compare lanes 2 and 3 of Fig. 5) .

We then extended this series by examining the interaction of the polymerase with a P/T construct carrying three mispaired dCMP residues at the 3‘ terminus of the primer (14CCC/25; see Table I). The switch efficiency on this substrate was monitored by carrying out three sets of identical switch experiments at different reaction temperatures, as shown at the top of Fig. 6. The extent of processive exonuclease digestion at each temperature is shown in lanes 2, 4, and 6. The corresponding switch reactions are adjacent in lanes 3, 5, and 7. Note that the extent of processive exonuclease digestion varies with temperature, the overall result being an increase from a processive removal of only 2 of the mismatched dC residues a t 10 “C to the processive removal of all 3 mismatched residues at the highest temperature tested (37 “C). However, even a t 37 “C, there is a significant amount of product remaining from which only two of the three mismatches have been removed. This result contrasts sharply with the temperature independence over this same range observed for the processive digestion by T4 polymerase of single-stranded DNA oligonucleotides, as well as of the doubly mismatched 15CC/25 P/T (data not shown).

These results with triply mismatched P/T constructs a t each temperature (lanes 3,5, and 7) illustrate the point made earlier which is central to establishing the authenticity of the switch protocol; i.e. T4 DNA polymerase does not polymerize from a mispaired terminus. This point is illustrated in the experiments of Fig. 6 by comparing bands in adjacent lanes that correspond to primers that have had 1, 2, or all 3 dC residues removed. Primers that are shortened by only 1 or 2

1 2 3

25 *-

15 *llllllllllllll

FIG. 5. Enzymatic interactions of T4 DNA polymerase with a doubly mismatched primer-template structure are extremely processive. Lane 1 shows the initial end-labeled 17-nucleotide primer of the 15CC/25 P/T construct. A diagram representing this substrate is depicted at the left of the gel. Lane 2 displays the product resulting from a single-turnover exonuclease reaction with this P/T substrate. This product results from the processive removal of the two mismatched dCMP residues and is depicted at the right of the gel. Lane 3 displays the results of a single-turnover switch reaction showing the production of a full length 25-nucleotide product that is also depicted at the right of the gel.

14 *,,,,,,,,,,,,,,

T E M P P C ) I IO I 25 I 37 1 1 2 3 4 5 6 7 8

FIG. 6. Removal of three mismatched primer residues by T4 DNA polymerase is not totally processive. This figure displays products formed under single-turnover conditions between T4 DNA polymerase and the 14CCC/25 P/T construct as a function of reaction temperature (indicated at the top of the figure). Products from single- turnover exonuclease reactions are shown in lnnes 2, 4 , and 6. Prod- ucts from single-turnover switch reactions are shown in lanes 3, 5, and 7. Lune 1 shows the initial end-labeled 17-nucleotide primer of the 14CCC/25 P/T construct. Lune 8 shows the full-length product (25-mer) formed from multiple turnovers under synthesis conditions. The diagram at the left of the gel depicts P/T products resulting from the processive exonuclease activity of the polymerase.

residues still carry a terminal mismatch (see diagram on the left of Fig. 6). This prevents them from being substrates for polymerization, and therefore switching on such P/T substrates is not observed. Only the P/T construct carrying a primer from which all 3 mismatched dC residues have been removed permits subsequent polymerization. By following the fate of this fully base-paired 14-mer, in contrast to that of the singly or doubly mismatched 15- and 16-mer structures (compare lanes 4 and 5 as well as 6 and 7), we can see that only the fully base-paired 14-mer primer is elongated in the switch reaction.

Binding-Finally, we have measured directly the binding constant of T4 DNA polymerase to the various DNA substrates employed in this study. We were particularly inter- ested in any differences in binding behavior to the fully base- paired 17/25 P/T substrate and those substrates carrying an increased number of 3’-terminal mismatches. Such differences could provide mechanistic insight into why extension from a mismatched terminus is not observed. Binding constants were determined in the identical buffer used for all the above switch reactions, except for the necessary omission of M e to prevent degradation of the starting substrate, em- ploying “traditional” (Fried and Crothers, 1981; Garner and Revzin, 1981) band shift assays. These experiments revealed binding constants with polymerase that are, within experimental error, identical for the various P/T structures exam- ined. These binding results and others will be described in detail el~ewhere.~

DISCUSSION

The polymerase accessory proteins of the T4 DNA replication complex (the proteins coded by T4 genes 44, 62, and 45) interact with the T4 DNA polymerase (gene 43 protein) in the presence of ATP, and, together with gene 32 protein (the T4-coded single-stranded DNA binding protein), form a T4 holoenzyme assembly that is capable of synthesizing DNA under physiological conditions with rates, fidelities, and pro-

‘ M. K. Reddy, s. E. Weitzel, M. K. Dolejsi, and P. H. von Hippel, manuscript in preparation.


cessivities characteristic of the in vivo T4 DNA replication system (Sinha et al., 1980; Newport et al., 1980; for a recent mechanistic review see Footnote 2). The DNA replication systems of other organisms contain subunits that seem to increase the processivity of their associated polymerase in very similar ways (Kornberg and Baker, 1991). The mechanisms by which the accessory proteins induce these functional changes in the properties of the polymerase are presently not understood.

A central problem in studying the development of processivity within a holoenzyme complex is to determine whether the elements of processive function are intrinsic to the interactions of the polymerase itself with the primer-template junction, or whether the polymerase is inherently dispersive and development of processivity requires the action of a distinct set of subunits of the replication complex that bind to the polymerase and physically “move-it-along” the DNA template. If conditions can be found to demonstrate unequiv- ocally that all the elements of processivity can, in principle, be manifested by the polymerase alone, the mechanistic requirements imposed on the accessory proteins subassembly become much simpler. Then we need only require that the accessory proteins interact with the polymerase within the holoenzyme complex in a manner that brings out the latent processivity of the polymerase itself.

Newport et al. (1980) demonstrated that T4 polymerase alone can synthesize DNA with a substantial degree of processivity at low salt concentrations (<loo mM monovalent cations). In the present study, we have established that the processive elements required for efficient proofreading are also intrinsic to the T4 DNA polymerase at these same salt concentrations and that, in fact, this enzyme can move between editing and synthesis modes with high efficiency and without dissociating from the DNA template. These key observations are in direct accord with other lines of evidence demonstrating that in multicomponent prokaryotic systems the DNA polymerase itself is the major contributor to the fidelity of DNA replication (see Loeb and Kunkel, 1982, for a review).

Processivity on a Base-paired Substrate-An important as- pect of our results (see Fig. 1) has been to establish that T4 DNA polymerase, when pre-equilibrated with a totally base- paired P/T construct, is poised to catalyze single nucleotide addition or removal reactions in both directions along the DNA template. That is, we can observe either processive synthesis or processive exonuclease activity, depending upon the presence or absence of dNTPs in the reaction protocol. We suggest two distinct molecular mechanisms that might account for this observed bilateral processivity of the T4 enzyme.

The first scenario is equivalent to the “melt-and-slide” model that has been put forward for Pol I by Joyce et al. (1988). In this model, it was proposed that the primer moves rapidly between the two active sites of the enzyme and is distributed between them in a “binding equilibrium” pattern. Catalysis then occurs “instantaneously” with the addition of Mg2+ and the resulting reaction products merely reflect the pre-equilibrium distribution of the primer terminus between the “synthesis” and the “editing” active sites of the enzyme. Alternatively, we can assume that the nascent primer terminus is prebound exclusively at the polymerase active site of T4 DNA polymerase, as shown at the top of Fig. 3. Here, since we are operating under single-turnover conditions, the observed removal of correctly paired bases must reflect the fact that the rate of transfer from the polymerase site to the

exonuclease site is faster than dissociation from the DNA template.

The latter mechanism draws on insights obtained from recent comprehensive pre-steady state kinetic analyses of Pol I (Kuchta et al., 1988; Catalan0 et al., 1990) and T7 DNA polymerase (Donlin et al. 1991). Based on their findings, these latter investigators proposed an elegant proofreading mechanism to account for the high fidelity displayed by the isolated T7 DNA polymerase. A major premise of this mechanism, which they have called “kinetic partitioning,” is that, at equilibrium, binding of the 3”terminus of the DNA primer at the synthesis site of the enzyme is strongly favored over binding at the editing site.

The central observation of the kinetic partitioning mechanism is that the rate of transfer of the 3’4erminus of the primer from the polymerase active site to the exonuclease active site (kFx in the terminology of Dolin et al., 1991) is faster than dissociation of the polymerase from the DNA template (kOff) only when the polymerase encounters a mismatched terminus (Donlin et al., 1991). That is, when T7 DNA polymerase is equilibrated with a base-paired terminus, kFx is smaller than kOff of the DNA from the polymerase site (Wong et al., 1991). This means that on properly base-paired DNA molecules exonucleolytic cleavage occurs essentially intermolecularly; this “restraint” ensures that maximum fidelity is coupled with a minimum use of chemical energy.

If the kinetic partitioning scheme outlined above proves to be correct, and T4 DNA polymerase is preferentially bound at the synthesis site of the enzyme, then such partitioning would not occur if the rate of transfer between active sites ( kpx) is faster than the dissociation rate (kg), even when T4 DNA polymerase is bound to a base-paired P/T structure. The absence of a kinetic partitioning mechanism could account for the significant energetic cost of the exonucleolytic cleavage of correctly base-paired P/T junctions by T4 DNA polymerase (Clayton et al., 1979; Fersht et al., 1982). In this connection we note also the possibility that the altered exonuclease to polymerase ratios of mutator and antimutator T4 DNA polymerase mutants (Muzyczka et al., 1972), long be- lieved to reflect differences in the actual intrinsic rates of exonuclease and polymerase activities, may instead be mani- festations of different rates of transfer of the primer terminus between the two active sites in these enzymes. Precise determinations of the kinetic parameters kpr and koff for wild type and mutant T4 DNA polymerases will require a careful pre- steady state kinetic analysis of the T4 DNA system.

The Size of the Exonuclease Active Site-Results of a study by Cowart et al. (1989), using chemically cross-linked P/T substrates, can be interpreted to suggest that the exonuclease active site of T4 DNA polymerase can bind either one or two unpaired nucleotide residues. Our findings presented here that are based on monitoring the processivity of the exonuclease reaction are quite consistent with this proposal, since our results suggest that the exonuclease active site of T4 DNA polymerase bound to a P/T substrate can accommodate no more than 2 residues of single-stranded DNA at the 3’-end of the primer. The following evidence from our study supports this suggestion.

First, the observed processivity for both the matched and the mismatched P/T substrates results in the removal of 1 and 2 nucleotide residues (lanes 2 and 4, Fig. 1). In fact, of all the single mismatches we have employed, the predominant product is the species that is formed by the removal of 2 nucleotide residues (i.e. the 15-mer). In addition, our results based on the interaction of T4 DNA polymerase with the multiply mismatched P/T structures are also consistent with


an exonuclease site size of 2 residues. Using the doubly mismatched P/T (15CC/25) construct, we consistently observe, even at lower temperatures and higher salt concentrations, the potent and totally processive removal of 2 (and only 2) mismatched dC residues (Fig. 5 and data not shown). Finally, our results on the triple mismatch (14cCc/25; see Fig. 6) reveal that with this construct all 3 dC residues are not removed together. Rather, there appears to be a preference for the processive removal of only 2 nucleotide residues a t a time, especially at temperatures below 37 “C.

These results suggest that T4 DNA polymerase bound to a P/T construct can “accept” no more than 2 nucleotide residues at a time into its exonuclease active site. When, as shown with the triple mismatch, the exonuclease active site is presented with more than two bases, it must “reload this site after nucleolytic cleavage of the first or first two bases. At this point, dissociation becomes a faster event than translo- cation and therefore the intrinsic processivity of the exonuclease activity of T4 DNA polymerase will be limited to the hydrolysis of one or two bases. This limitation, of course, makes physiological sense.

Possible Mechanisms of Fidelity-The observation that T4 DNA polymerase will not extend from a mismatched terminus until the mismatched base has been removed by the 3‘+5‘- exonuclease activity of the polymerase was initially based on experiments with synthetic homopolymers (Brutlag and Kornberg, 1972). A subsequent study on the “next nucleotide effect” (Kunkel et al., 1981) indirectly confirmed this fact. However, neither of these previous studies was performed under single-turnover conditions and so nothing could be discerned about the intrinsic processivity of the editing process.

In this study we have carried out single-turnover experiments that are made possible by the strategic use of heparin to trap T4 DNA polymerase molecules that have dissociated from the DNA template. An outcome of this experimental strategy has been to demonstrate that T4 DNA polymerase can switch intramolecularly between exonuclease and synthesis modes. This observation supports an earlier suggestion, derived from a study on T5 DNA polymerase, that the pathway of editing for replicative polymerases is processive (Das and Fujimura, 1980). This pathway, also established for T7 DNA polymerase (Donlin et al., 1991), is distinct from the primary editing pathway of the Klenow fragment of Pol I (Joyce et al., 1988), which, as a repair enzyme displays lower processivity and a slower polymerization rate than replicative polymerases.

We are aware that we have not ruled out the possibility that T4 DNA polymerase could misincorporate a base, remain bound to the DNA during a subsequent excision step, and then dissociate prior to addition of a correct nucleotide at the original site of the mispair. A difficulty in performing this experiment results from the fact that one needs to add high amounts (>0.5 mM) of a dNTP to observe a misincorporation event at all. At these nonphysiological levels of dNTPs, un- known secondary effects on the exonuclease activity and/or dissociation rates cloud the interpretation.

Finally, our observation that T4 DNA polymerase binds with equal affinity to base-paired and mismatched P/T structures is consistent with the notion that the process of “dis- crimination against extension” (Kunkel, 1988) is a result of kinetic barriers and not of an intrinsic inability of T4 DNA polymerase to bind to mismatched termini. Although such equal binding to correct and incorrect primer termini has also been observed for avian myeloblastosis virus reverse tran- scriptase (Creighton et al., 1992) and for T7 DNA polymerase

(Donlin et al., 1991), data from this and many previous studies indicate clearly that DNA polymerases “handle” 3”terminal mismatches quite differently from base-paired termini. Thus, DNA polymerases can somehow “magnify” free energy differences between base-paired and mismatched DNA termini.

An explanation put forward by Petruska et al. (1986) is that these small free energy differences are magnified by the exclusion of water from the polymerase active site (in essence, by a change in local dielectric constant). However, there is no direct evidence for this suggestion, and we conclude that the molecular mechanisms by which T4 DNA polymerase distin- guish between base-paired and mismatched primer termini remain to be elucidated.

Acknowledgments-We thank members of our laboratory, especially Shirley Daube, Yan Wang, and Mark Young, for invaluable discussions of the issues under consideration here. We are most grateful to Shirley Daube for preparing all of the figures contained in this paper.

REFERENCES Aboul-ela, F., Koh, D., and Tinoco, I. (1985) Nucleic Acids Res. 13 , 4811-4824 Brutlag, D., and Kornberg, A. (1972) J. Biol. Chem. 247,241-248 Catalano, C. E., Allen, D. J., and Benkovic, S. J. (1990) Biochemutry 29,3612-

R R 9 l Clayton, L. K., Goodman, M. F., Branscomb, E. W., and Galas, D. J. (1979) J.

Cowart, M., Gibson, K. J., Allen, D. J., and Benkovic, S. J. (1989) Biochemistry

Creighton, S., Huang, M.-M., Cai, H., Arnheim, N., andGoodman, M. F. (1992)

Das, S. K., and Fujimura, R. K. (1980) J. Biol. Chem. 255 , 7149- 7154 Der Garabedian, P. A., Mirambeau, G., and Vermeersch, J. J . (1991) Biochem-

Derbyshire, V., Freemont, P. S., Sanderson, M. R., Beese, L., Friedman, J. M.,

Dolejsi, M. K. (1988) Molecular Interactwns between the T4 DNA Polymerase

Donlin, M. J., Patel, S. S., and Johnson, K. A. (1991) Biochemistry 30 , 538-

””

Biol. Chem. 264,1902-1912

2 8 , 1975-1983

J. Biol. Chem. 267,2633-2639

istry 30,9940-9947

Joyce, C. M., and Steitz, T. A. (1988) Scrence 2 4 0 , 199-201

and D N A Substrates. Ph. D. thesis, University of Oregon

RAfi Fay, P. J., Johanson, K. O., McHenry, C. S., and Bambara, R. A. (1981) J. Biol.

Fay, P. J., Johanson, K. O., McHenry, C. S., and Bambara, R. A. (1982) J. Bid.

Fersht, A. R., and Knlll-Jones, J. W. (1981) Proc. Natl. Acad. Sei. U. S. A. . 78 ,

-”

Chem. 256,976-983

Chem. 257,5692-5699

Fersht, A. R., Knill-Jones, J. W., and Tsui, W.-C. (1982) J. Mol. Biol. 156,37- 4251-4255

51 Fried, M., and Crothers, D. M. (1981) Nucleic Acids Res. 9,6505- 6525

Gill, S. C., and von Hippel, P. H. (1989) Anal. Biochem. 182,319-326 Garner, M. M., and Revzin, A. (1981) Nucleic Acids Res. 9,3047-3059

Goodman, M. F., Keener, S., Guidotti, S., and Branscomb, E. W. (1983) J. Biol.

Goulian, M., Lucas, Z. J., and Kornberg, A. (1968) J. Biol. Chem. 243 , 627-

Greip, M. A,, and McHenry, C. S. (1989) J. Biol. Chem. 2 6 4 , 11294-11301 Herendeen, D. R., Williams, K. P., Kassavetis, G. A,, and Geiduschek, E. P.

Huang, W. M., and Lehman, I. R. (1972) J. Biol. Chem. 247,3139- 3146 Huber, H. E., Tabor, S., and Richardson, C. C. (1987) J. Biol. Chem. 2 6 2 ,

Jarvis, T. C., Paul, L. S., and von Hippel, P. H. (1989) J. Biol. Chem. 264 , 16224-16232

Jarvis, T. C., Dolejsi, M. K., Kubasek, W. L., Paul, L. S., Reddy, M. K., and 12709-12716

von Hippel, P. H. (1990a) in Molecular Mechanisms in DNA Replication and Recombination, UCLA Symposium on Molecular and Cellular Biology (Rich- ardson, C. C., and Lehman, I. R., eds) New series, Vol. 127, pp. 261-275, Alan R. Liss, Inc., New York

Jarvis, T. C., Ring, D. M, Daube, S. S., and von Hippel, P. H. (1990b) J. Biol. Chem. 265,15160-15167

Jarvis, T. C., Newport, J. W., and von Hippel, P. H. (1991) J. Biol. Chem. 266 , 1830-1840

Chem. 258,3469-3475

638

(1990) Science 248,573-578

Joyce, C. M., Freidman, J. M., Beese, L., Freemont, P. S., and Steitz, T. A. (1988) in DNA Replication and Mutagenesis (Moses, R. E., and Summers, W. C., eds) pp. 220-226, American Society for Microbiology, Washington, D. C.

Kornberg, A., and Baker, T. A. (1992) in DNA Replication, 2nd Ed, W. H. Freeman and Co., New York

Kowalczykowski, S. C., Lonberg, N., Newport, J. W., and von Hippel, P. H. (1981) J. Mol. Biol. 1 4 5 , 75-104

Kuchta, R. D., Benkovic, P. A,, and Benkovic, S. J. (1988) Biochemistry 2 7 , 6716-6725

Kunkel, T. A. (1988) Cell 53,837-840 Kunkel, T. A., Schaaper, R. M., Beckman, R. A,, and Loeb, L. A. (1981) J. Biol.

Kunkel, T. A,, Loeb, L. A,, and Goodman, M. F. (1984) J. Biol. Chem. 259 ,

Leirmo, S., Harrison, C., Cayley, D. S., Burgess, R. R., and Record, M. T., Jr.

Loeb, L. A,, and Kunkel, T. A. (1982) Annu. Reu. Biochem. 62,429- 457 Maxam, A. M., and Gilbert, W. (1980) Methods Enzymol. 65,499-559

Joyce, C. M. (1989) J. Biol. Chem. 264,10858-10866

Chem. 256,9883-9889

1539-1545

(1987) Biochemistry 26,2095-2101

14166 Processive Proofreading with T4 DNA Polymerase McCarthy, D., Minner, C., Bernstein, H., and Bernstein, C. (1976) J. Mol. Biol.

Mizrahi, V., and Benkovic, S. J. (1986) Ado. Enzymol. Re&. Areas Mol. Biol.

Muzyczka, N., Poland, R. L., and Bessman, M. J. (1972) J. Biol. Chem. 247,

106,963-981

61,437-457

7116-7122 Newport, J. W., Kowalczykowski, S. C., Lonberg, N., Paul, L. S., and von

Hippel, P. H. (1980) in Mechanistic Studies of DNA Replication and Genetic Recombinution ICN-UCLA Symp. Mol. Cell. Biol. (Alberts, B. M., ed) Vol. 19, pp. 485-505, Academic Press, New York

Nossal, N., and Hershfield, M. S. (1971) J. Biol. Chem. 246,5414-5426 Ollis, D. L., Brick, P., Hamlin, R., Xuong, N. G., and Steitz, T. A. (1985) Nature

Overman, L. B., Bujalowski, W., and Lohman, T. M. (1988) Biochemistry 34 ,

Petruska, J., Sowers, L. C., and Goodman, M. F. (1986) Proc. Natl. Acud. Sci.

Petruska, J., Goodman, M. F., Boosalis, M. S., Sowers, L. C., Cheong, C., and

313,762-766

440-447

U. S. A. 83, 1559-1562

Tinoco, I. (1988) Proc. Natl. Acud. Sci. U. S. A. 86,6252-6256

Reha-Krantz, L. J. (1988) J. Mol. Biol. 202, 711-724 Reha-Krantz L. J., Stocki S. Nonay, R. L., Dimayuga, E., Goodrich, L. D.,

Konigsberi, W. H., and 'spiher, E. K. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,2417-2421

Rush, J., and Konigsber , W H (1989) Pre Biochem. 19,329-340 Sinba, N. K., Morris, 8 F.; and Alberts, f;: M. (1980) J. Biol. Chem. 266,

A 7 9 ~ - A W I . 1 Spicer E. K. Rush J Fung C., Reha-Krantz L. J. Karam, J. D., and

Tabor, 8, and Richardson, C. C.,(1989) J. Biol. Chem. 264,6447-6458 Tabor, S., Huber, H. E., and Rlchardson, C. C. (1987) J. Bcol. Chem. 262,

1"- -I_"

Kon'i berg' W. H.'(1988) J. Biol. Chem. 263,7i78-7&6

16212-16223 Topal, M. D., and Fresco, J. R. (1976) Nature 262,285-289 van de Sande, J. H., Kleppe, K., and Khorana, H. G. (1973) Biochemistry 12, mm-mm

von Hippel, P. H., Revzin, A., Gross, C. A., and Wang, A. C. (1975) in Protein- Ligand Interactions (Sund, H., and Blauer, G., eds) pp. 270-288, Walter de

"" ""

Gfuyter Berlin Warshaw,". M. and Cantor, C. R. (1970) Biop l mers 9,. 1079-1103 Wong, I., Patel, 8. S., and Johnson, K. A. (1991) &chemlstry 30,526-537

the journal of biological vol. 267, no. 20. of pp. 1992 ... · processive proofreading is intrinsic...

Documents