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Recognition of Aberrant DNA Termini by the Klenow Fragment Of Proc. Nati. Acad. Sci. USA Vol. 91, pp. 10670-10674, October 1994 Biophysics Proofreading DNA: Recognition of aberrant DNA termini by the Klenow fragment of DNA polymerase I (polymerase and 3'-5' exonuclease sites/DNA partitioning/nismatched base pairs/time-resolved fluorescence spectroscopy) THEODORE E. CARVER, JR., REMO A. HOCHSTRASSERt, AND DAVID P. MILLAR* Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037 Communicated by Bruno H. Zimm, June 14, 1994 (receivedfor review September 22, 1993) ABSTRACT Fluorescence depolarization decays were volves an 8-bp translocation of the protein upstream on the measured for 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) DNA (8). However, a recent structure ofduplex DNA bound probes attached internally to 17-mer27-mer oligonucleotides to Klenow fragment shows the primer terminus bound in the bound to Klenow framet of DNA polymerase I. The time- cleft adjacent to the exonuclease site, much closer to both resolved motions of the dansyl probes were sensitive indicators active sites than thought (9). Currently, the mechanism of of DNA-protein contacts, showing that the protein binds to DNA movement within the protein remains unclear. In DNA with two footprints, corresponding to primer termini at addition, the effects of protein and DNA structure upon the either the polymerase or 3'-5' exonuclease sites. We examined binding of DNA to each site have not been resolved. The kinetics of misincorporation and proofreading in the complexes of Klenow fragment with DNAs containing various Klenow fragment have been examined in detail using base mismatches. Single mismatches at the primer terminus quenched-flow techniques, and the scheme required to de- caused a 3- to 4-fold increase in the equilibrium partitioning of scribe interconversion of the intermediate states is complex DNA into the exonuclease site; the largest effects were observed (10, 11). For a simple model in which only DNA binding is for purine-purine mismatches. Two or more consecutive G-G considered, preferential excision of mismatched bases is mismatches caused the DNA to bind exclusively at the exonu- achieved by increasing the rate of exonucleolysis of the clease site, with a partitioning constant at least 250-fold greater terminal nucleotide relative to the rate ofaddition of the next than that of the corresponding matched DNA sequence. Inter- nucleotide. An increase in the ratio of exonuclease to poly- nal single mismatches produced larger effects than the same merase rates could be due to a decrease in the intrinsic rate mismatch at the primer terminus, with a AAG relative to the ofpolymerization, kpol, or to an increase in the partitioning of matched sequence of -1.1 to -1.3 kcal/mol for mismatches DNA from the polymerase site into the exonuclease site, or located 2, 3, or 4 bases from the primer terminus. Although to a combination ofthese effects (Fig. 1). The greater melting part of the observed effects may be attributed to the increased capacity of mispaired termini could contribute to increased melting capacity ofthe DNA, it appears that the polymerase site partitioning of DNA into the exonuclease site (12). In addi- also promotes movement of DNA into the exonuclease site by tion, it is important to note that any structural feature of the rejecting aberrant primer termini. These effects suggest that DNA favoring DNA binding at the exonuclease site over the polymerase and exonuclease sites act together to recognize binding at the polymerase site will increase the rate of specific errors that distort the primer terminus, such as exonucleolysis relative to polymerization. Because move- frameshifts, in addition to proofreading misincorporated ment of DNA between the polymerase and exonuclease sites bases. appears markedly faster than the intrinsic rates of the reac- tions occurring at either site, the partitioning step is at A necessary feature of all DNA-dependent DNA polymer- equilibrium relative to the other processes and can be rep- ases is the ability to copy DNA with high fidelity; for DNA resented by the equilibrium constant for partitioning ofDNA polymerase I, the probability of an error during replication is termini between the polymerase and exonuclease sites, Kpe <10-6 per nt addition (1). Exonucleolytic proofreading con- (Fig. 1). tributes to replication fidelity through the preferential exci- In this study, we examine the effects of base mismatches sion of mismatched bases after mispair synthesis occurs (for and sequence variation upon binding of DNA to the poly- reviews, see refs. 2-4). In DNA polymerase I, proofreading merase and exonuclease sites of Klenow fragment. The is accomplished by a 3'-5' exonuclease in a separate domain time-resolved fluorescence depolarization of a 5-dimeth- of the same polypeptide with 5'-3' polymerase activity. ylaminonaphthalene-l-sulfonyl (dansyl) probe covalently at- The crystal structure of the 70-kDa Klenow fragment of tached to DNA contains information about partitioning ofthe DNA polymerase I has been determined. Klenow fragment DNA between the polymerase and 3'-5' exonuclease sites of consists of two putative DNA-binding clefts divided by a the enzyme (13). This technique is used here to estimate the "thumb-like" domain (5). The 5'-3' polymerase and 3'-5' internal partitioning constant Kpc for a series of defined exonuclease activities are separated by 30-40 A, indicating oligonucleotide primer/templates containing either matched that shuttling of the DNA between the two sites is a require- or mismatched sequences. ment for proofreading. Joyce and Steitz (6) have proposed a structural model for movement ofDNA from the polymerase EXPERIMENTAL METHODS site to the exonuclease site. In this model, 3- to 4-bp melt at the primer terminus, followed by movement of the resulting Materials. The exonuclease-deficient D424A mutant of single-stranded region of DNA into the exonuclease site (7). Klenow fragment was purified from Escherichia coli CJ376, From assays using biotinylated DNA substrates, there is evidence that the polymerase-exonuclease transition in- Abbreviations: dansyl, 5-dimethylaminonaphthalene-1-sulfonyl; Kpe, equilibrium constant for partitioning of DNA termini between the polymerase and exonuclease sites. The publication costs of this article were defrayed in part by page charge tPresent address: Biophysikalische Chemie, Biozentrum, CH-4056 payment. This article must therefore be hereby marked "advertisement" Basel, Switzerland. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 10670 Downloaded by guest on September 24, 2021 Biophysics: Carver et al. Proc. Nati. Acad. Sci. USA 91 (1994) 10671 where III(t) and IL(t) are the observed intensity decays when A<~ D the observing polarizer is oriented parallel (11) or perpendic- ~~~~~~~ A,, ular (I) to the direction of the vertically polarized exciting light. For a homogeneous population of dansyl probes at- POL Site tached to DNA, the contributions made by various types of motion to the time-dependent fluorescence anisotropy r(t) can be represented as KF X r(t) = P, exp(-t/4,) + P2 exp(-t/02), [2] DNA i\ where Xh is the correlation time for local rotation ofthe probe (1 - 2 ns), and 02 is the correlation time for overall tumbling of the DNA-protein complex (=60 ns). Pi and P2 are the 11,7' kexo corresponding anisotropy amplitudes for the two compo- IED >DNAn-1 nents. If the probe population is heterogeneous, each sub- population ofprobes will have its own associated amplitudes EXO Site and decay times. For a two-state model, in which one state FIG. 1. Simplified scheme for a single round of DNA processing corresponds to probe molecules that are largely exposed to by Klenow fragment of DNA polymerase I. kpol is the intrinsic rate solvent, whereas the other state corresponds to probes that constant for addition of the next nucleotide to DNA bound at the are buried within the protein, the time-dependent anisotropy polymerase site, and kxo is the intrinsic rate constant for exonucle- is olysis ofDNA bound at the exonuclease site. The active sites for the polymerization reaction and the 3'-5' exonuclease reaction are r(t) = fe(t)re(t) + fb(t)rb(t), [31 denoted P and E, respectively. The DNA primer and template strands are shown as stippled and solid lines, respectively. The circle wheref (t) is the fraction offluorescence at time t due to the located within the DNA helix represents an internal dansyl probe exposed probes, r.(t) describes the anisotropy decay of the attached via a 3-carbon linker to a uridine base 7 bp from the primer exposed probes, andfb(t), rb(t) are the corresponding quan- terminus. When DNA termini are bound to the polymerase site, the dansyl probe is exposed to solvent. Movement ofthe DNA terminus tities for the buried probes (17). The fractionsf.(t) andfb(t) into the exonuclease site shifts the footprint of the protein, drawing are complex functions of the fluorescence lifetimes of the the probe into the protein interior. The different footprints for exposed and buried probes (13): polymerase- and exonuclease-bound DNA could result from either a translocation ofthe DNA or a conformational shift in the enzyme that N exposes different faces ofthe DNA helix to solvent. The 3' terminal xeSa ajeexp(-t/Tje) strand be within region of the primer may single-stranded the fe(t) 9 [4] exonuclease site. Note that the polymerase and exonuclease reac- N M tions are inhibited during the present equilibrium partitioning mea- Xel>ajyexp(-t/Tre) + XbEaJbexp(-t/Tjb) surements. j=1 j=1 where Tib, ajb, and ij,, aj are the corresponding lifetimes and as described (14). The E. coli CJ376 cells were provided by amplitudes for the two populations. An analogous expression C. Joyce (Yale University). Protein samples were found to be was used forfb(t). Immediately after excitation (t = 0),f.(t) >95% pure by PAGE.
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