Studies of Bacteriophage T4 “Antimutator” DNA Polymerases

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Regulation of DNA Polymerase Exonucleolytic Proofreading Activity: Studies of Bacteriophage T4 “Antimutator” DNA Polymerases

Linda J. Reha-Krantz Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9

NA polymerases replicate DNA with high fidelity lab, which demonstrated that single point mutations in D because of accurate nucleotide incorporation cou- the T4 DNA polymerase gene can increase or decrease pled with exonucleolytic proofreading to remove misin- mutation rates by 100-fold or more (Drake et al. 1969). corporated nucleotides. This statement is taken for Mutational analysis is a powerful method to probe granted today, in large part, because of groundbreaking enzyme function. Even today with the ready availability discoveries made 30 years ago that mutations in the of molecular techniques, genetic screens and selections DNA polymerase gene of bacteriophage T4 can have are useful because informative mutant enzymes can be dramatic effects on the fidelity of DNA replication. “Mu- identified without structural information or assump- tator” DNA polymerases (Speyer et al. 1966) are mutant tions about function. Most importantly, since classical ge- DNA polymerases that replicate DNA with less accuracy netic methods rely on detection and characterization of mutant than the wild-type enzyme, and “antimutator” DNA poly- phenotypes in vivo, alterations in function are observed within merases (Drake and Allen 1968; Drake et al. 1969) the context of the living organism. In the case of antimutator are mutant DNA polymerases that replicate DNA with DNA polymerases, these mutants provide a handle to more accuracy, at least for certain types of DNA replica- probe DNA polymerase function in vivo. tion errors. Biochemical characterization revealed that Genetic and biochemical techniques have continued the balance between the two DNA polymerase functions, to be used in studies of antimutator DNA polymerases. nucleotide incorporation and the opposing 3Ј 5Ј exonu- This research has led in a number of different directions → clease activity, was altered in the mutants compared to including studies of the evolution of spontaneous mu- the wild type enzyme. Decreased 3Ј 5Ј exonuclease activ- tation rates (Drake 1990, 1991a, 1991b), the effect of → ity was detected for many of the mutator DNA polymerases antimutator DNA polymerases on frameshift mutagene- (Muzyczka et al. 1972) while increased 3Ј 5Јexonuclease sis (Kaiser and Ripley 1995), the identification of anti- → activity, relative to polymerase activity, was observed for mutator DNA polymerases in other organisms (Fijal- the antimutator DNA polymerases (Muzyczka et al. 1972; kowska et al. 1993), and additional studies of DNA Gillin and Nossal 1976a,b). The correlation of de- polymerase function (Clayton et al. 1979; Spacciapoli creased 3Ј 5Ј exonuclease activity measured in in vitro and Nossal 1994a,b; Reha-Krantz and Nonay 1994; → assays with decreased accuracy of DNA replication in vivo, Reha-Krantz and Wong 1996). The focus of this short and increased 3Ј 5Ј exonuclease activity with increased review is the regulation of DNA polymerase proofread- → DNA replication fidelity indicates that the 3Ј 5Ј exo- ing activity (Stocki et al. 1995). Other aspects of T4 → nuclease activity of T4 DNA polymerase is an exonucleo- antimutator DNA polymerases are presented in this vol- lytic proofreading activity. ume (e.g., Nossal, Wang and Ripley). These observations were made at an exciting time in T4 antimutator DNA polymerases: Mutations within DNA polymerase research. I was fortunate then to be a genes result typically in mutant enzymes with reduced graduate student in Maurice Bessman’s lab at Johns activity and even loss of function; however, in the case Hopkins University and to see first hand the biochemi- of antimutator DNA polymerases, the mutant enzymes cal characterization of T4 mutator and antimutator appear to be working better, at least with respect to the DNA polymerases. But of equal importance was the op- accuracy of DNA replication. Since single point muta- portunity to meet Jan Drake and to learn about the tions can give rise to antimutator DNA polymerases, why use of genetics to study the fidelity of DNA replication. have more accurate DNA polymerases not evolved? The juxtaposition of biochemistry and genetic analyses Part of the answer must be that there are negative to elucidate the proofreading role for the 3Ј 5Јexonucle- consequences of the antimutator phenotype that reduce → ase activity of T4 DNA polymerase made a strong impact the efficiency of the DNA polymerase and the overall on my graduate studies and continues to do so today. I fitness of the organism. There is a cost associated with Fersht am still intrigued by the genetic analyses from the Drake DNA polymerase proofreading ( et al. 1982). For example, if T4 antimutator DNA polymerases replicate DNA more slowly than the wild-type enzyme, then fewer phage progeny may be produced. Higher concentra- Address for correspondence: Department of Biological Sciences, CW405 BioSciences Bldg., University of Alberta, Edmonton, Alberta T6G 2E9, tions of deoxynucleoside triphosphates (dNTPs) are re- Canada. E-mail: [email protected] quired to support DNA replication by T4 antimutator

Genetics 148: 1551–1557 (April, 1998) 1552 L. J. Reha-Krantz

DNA polymerases (Gauss et al. 1983; Beauchamp and Richardson 1988) due to increased exonucleolytic proofreading, which removes correct nucleotides in addition to incorrect nucleotides (Muzyczka et al. 1972; Gillin and Nossal, 1976a; reviewed in Goodman et al. 1993). Another potential disadvantage of increased DNA replication accuracy is the possible necessity of a certain minimal mutation rate that is sufficient to allow an organism to adapt to fluctuations in the environment (Drake 1990, 1991a, 1991b). Another possible problem with exonucleolytic proofreading stems from the observation by Drake et al. (1969) that the antimutator activity of the mutant T4 DNA polymerases appears not to be a general antimutator activity, but is directed to decreasing AT GC transitions and base analogue in- → duced mutations. Other mutational pathways such as GC AT transitions, transversions, and frameshifts are → not reduced at some sites by T4 antimutator DNA poly- Figure 1.—Locations of mutations in the bacteriophage T4 merases, and may even be increased (Drake and Green- DNA polymerase gene that confer mutator and antimutator ing 1970; Ripley 1975; Kaiser and Ripley 1995). Thus, phenotypes. More comprehensive listings of N-terminal muta- any gain in fitness obtained by a decrease in AT GC tor mutations and C-terminal antimutator mutations are given → Reha-Krantz transition mutations may be offset by increases in other in (1988, 1994, and 1995a). The codon number Drake and identities of the amino acid substitutions encoded by the types of DNA replication errors ( 1993). mutations are shown and more details are provided in the T4 DNA polymerase has a potent 3Ј 5Ј exonuclease → text. Increases and decreases in spontaneous mutation rates activity, about 1000-fold more active than the 3Ј 5Јexonu- are illustrated by bars extending upward (mutators) or down- → clease activity of Escherichia coli DNA polymerase I(Capson ward (antimutators). The spontaneous mutation rates for the et al. 1992); hence, a mechanism must be in place to antimutator A737V- and A777V-DNA polymerases are from Drake et al. (1969). Spontaneous mutation rates for the other prevent indiscriminate degradation of the newly synthe- mutant DNA polymerases are from Reha-Krantz and Nonay sized DNA and the other disadvantages of excessive proof- (1993), Stocki et al. (1995), and from Reha-Krantz and reading discussed above. This mechanism fails or regula- Wong (1996). tion is reduced in antimutator DNA polymerases. If the molecular basis of the antimutator phenotype can be learned, then this information can be applied to de- stitutions Thr for Ser411, Ile for Leu412, and Val for termining how the normal balance between polymerase Ile417 in the highly conserved DNA polymerase motif, and exonuclease activities is maintained. A first step in DXXS411L412YPSII417, that forms part of the polymerase understanding the regulation of exonucleolytic proof- active center. Another mutation that encodes a Cys sub- reading is to determine the location of mutations that stitution for Arg335 was also identified by this selection confer the antimutator phenotype. strategy (Reha-Krantz and Wong 1996). Thus two re- Locations of mutations within the T4 DNA polymerase gions of the DNA polymerase have been identified in gene that confer antimutator and mutator phenotypes: which single amino acid substitutions can produce a Drake (Drake and Allen 1968; Drake and Greening strong antimutator phenotype. 1970) mapped mutations that confer the antimutator Most mutations that produce a strong mutator pheno- phenotype by recombination. Two of the mutations that type encode amino acid substitutions in the N-terminal produce a strong antimutator phenotype encode Val to region of T4 DNA polymerase (Reha-Krantz 1988, Ala substitutions at codons 737 and 777 (Reha-Krantz 1989). Alanine substitutions for aspartate residues D112, 1989). The locations of these mutations in the primary D219, and D324 (Figure 1) that are required for the structure of T4 DNA polymerase are illustrated in Figure hydrolysis reaction increase the mutation rate up to several 1. Additional mutations that confer the antimutator hundredfold, as expected if exonucleolytic proofreading phenotype have also been located in this region at co- activity is prevented (Reha-Krantz et al. 1991; Frey et al. dons 730, 731, 827, and 844 (Reha-Krantz 1988, 1994, 1993; Reha-Krantz and Nonay 1993). 1995a). Mutations in the T4 DNA polymerase that con- The recent determination of the structure of the RB69 fer the antimutator phenotype have also been identified DNA polymerase (Wang et al. 1997), a T4-like DNA by genetic selection for second-site mutations that sup- polymerase, allows placement of residues that are im- press sensitivity to the pyrophosphate analogue, phos- portant for DNA replication fidelity in a three-dimen- phonoacetic acid (Reha-Krantz and Nonay 1994). sional, structural context. Residues in the C-terminal These mutations are located near the center of the gene domain, as well as residues in the exonuclease domain, (Figure 1) and encode the conservative amino acid sub- make contacts with single-stranded DNA bound in the DNA Polymerase Proofreading 1553 exonuclease catalytic center (Wang et al. 1997). Thus, merase at the rapid rate of about 400 secϪ1 (Capson some residues in the C-terminal domain, the location et al. 1992), but elongation of a mismatched primer- of residues A737 and A777 (Figure 1), are in close con- terminus is extremely slow (reviewed by Goodman et al. tact with the exonuclease domain (see Nossal 1998). 1993). The greatly reduced ability of a DNA polymerase Amino acid substitutions that produce the antimutator to extend a mismatched primer-terminus provides a phenotype were also identified in the polymerase active window of opportunity to transfer the primer-terminus center, but the polymerase and exonuclease active cen- from the polymerase to the exonuclease active center, a ters are separated by a relatively great distance, about slower reaction measured at the apparent rate of 4 secϪ1 30 A˚ (Wang et al. 1997). Proofreading requires that the (Capson et al. 1992; Marquez and Reha-Krantz 1996). primer strand be moved from the polymerase to the The relatively slow transfer of DNA from the polymerase exonuclease active center. The locations of mutations to the exonuclease active center serves as a kinetic bar- that confer the antimutator phenotype suggest the possi- rier to the exonucleolytic proofreading pathway that bility that residues in the polymerase active center and protects correct primer-termini from indiscriminate in the C-terminal domain affect how the DNA is trans- degradation. ferred between the two active centers. The antimutator phenotype can be explained in the Antimutator DNA polymerases have more opportunity context of the kinetic scheme by proposing that the to proofread: Biochemical characterizations of the anti- antimutator DNA polymerases have even less ability than mutator DNA polymerases revealed that the balance the wild-type DNA polymerase to extend a mismatched between nucleotide incorporation and exonuclease ac- primer-terminus. One prediction of this proposal is that tivities was shifted toward increased exonuclease activity antimutator DNA polymerases would also have less abil- in the mutant enzymes by providing more opportunity ity to extend a correctly matched primer-terminus. Both Gillin Nossal to proofread ( and 1976a). The relative the I417V-DNA polymerase, which has decreased ability opportunities to proofread or to extend a primer-termi- to form the Enzpol·DNA·dNTP complex (Reha-Krantz nus can be demonstrated by preforming Enz·DNA com- and Nonay 1994), and the A737V-DNA polymerase, plexes in the presence of dNTPs but in the absence of which is defective in translocation (Gillin and Nossal Mg2ϩ, which is required for both nucleotide incorpora- 1976a; Spacciapoli and Nossal 1994a), have high rates tion and exonucleolytic proofreading activities. The ad- of turnover of correct nucleotides (Muzyczka et al. dition of Mg2ϩ then allows preformed Enz·DNA com- 1972; Gillin and Nossal 1976a; reviewed in Goodman plexes in which the primer-terminus resides in the et al. 1993). The high turnover of correct nucleotides polymerase active center (Enzpol·DNA·dNTP complex) by T4 antimutator DNA polymerases was used as the to incorporate nucleotides and Enz·DNA complexes in basis of a genetic selection strategy to isolate second- which the primer-terminus resides in the exonuclease site mutations that suppress the excessive proofreading active center (Enz ·DNA complex) to remove the ter- exo of antimutator DNA polymerases (Stocki et al. 1995). minal nucleotide. The antimutator I417V-DNA poly- Suppressors of excessive DNA polymerase proofread- merase, Val substitution for Ile417 within the polymer- ing decrease transfer of the primer-terminus from the ase active center, forms active exonuclease complexes polymerase to the exonuclease active center: Excessive almost twofold more frequently than the wild-type en- proofreading by T4 antimutator DNA polymerases is zyme (Reha-Krantz and Nonay 1994). These results were interpreted to indicate that the detected in vivo in infections of a host bacterial strain, optA1, which has increased ability to degrade dGTP I417V substitution destabilizes interactions with the Gauss Beauchamp Richardson primer-terminus within the polymerase active center ( et al. 1983; and 1988). and thus increases the opportunity of the enzyme to The reduced dGTP concentration in the restrictive host form complexes in which the primer-terminus resides is not sufficient to support DNA replication by antimuta- in the exonuclease active center. Reduced dNTP con- tor DNA polymerases and prevents replication of the centrations can also enhance exonuclease activity by phage genome. We have used this conditional lethal decreasing the formation of Enzpol·DNA·dNTP com- phenotype to identify second-site mutations that sup- plexes which then favors increased formation of active press the excessive exonucleolytic proofreading activity Clayton Stocki Enzexo·DNA complexes ( et al. 1979). of four mutant T4 DNA polymerases ( et al. Kinetic partitioning between polymerase and exo- 1995). Two antimutator DNA polymerases identified by nuclease activities: The studies above and numerous Drake, the A737V- and A777V-DNA polymerases, were additional studies of T4 DNA polymerase and other part of the study. Nineteen second-site, suppressor mu- DNA polymerases (Hopfield 1974; Fersht et al. 1982; tations were found. Although the suppressor mutations Capson et al. 1992; Johnson 1993; Goodman et al. 1993) are located in four different regions of the DNA poly- have resulted in formulation of a kinetic scheme that merase, the mechanism of suppression appears to be the describes the regulation between nucleotide incorpo- same—to raise the kinetic barrier to the proofreading ration and exonucleolytic proofreading activities. A pathway. One of the suppressor mutations will be dis- primer-terminus is normally extended by T4 DNA poly- cussed here to illustrate this point. 1554 L. J. Reha-Krantz

Figure 2.—Dynamics of DNA polymerase proofreading. For nucleotide incorporation, DNA is bound in the polymerase active center in a base-paired configuration. For exonucleolytic proofreading, the primer-terminus is partially separated from the template strand and transferred to the exonuclease active center. The exonuclease active center is illustrated as an inverted “V” in order to mimic the single-stranded binding groove observed in crystallographic structures of the T4 DNA polymerase proofreading complex (Wang et al. 1996, 1997). Amino acid residue Gly255 resides in a novel protein loop Figure 3.—Location of the G255 protein loop. A similar involved in transfer of the primer-terminus from the polymer- figure was published by Marquez and Reha-Krantz (1996) ase to the exonuclease active center (Marquez and Reha- and is republished here with permission from The Journal Krantz 1996). of Biological Chemistry. This structure is of the N-terminal and exonuclease domains of T4 DNA polymerase with an oligonucleotide, [p(dT4)], bound in the exonuclease active center (Wang et al. 1996). The position of Gly at codon 255 The most frequently isolated second-site suppressor is highlighted. The G255 loop appears to be located at a mutation of excessive proofreading activity encodes a branch point between the observed proofreading complex Ser substitution for residue Gly255, which resides in and duplex DNA in the polymerase active center based on the exonuclease domain (Figure 1). The G255S-DNA modeling the structures of duplex DNA bound by pol ␤ and polymerase in vivo displays a strong mutator phenotype HIV reverse transcriptase with the structure of the RB69 DNA polymerase, a T4-like DNA polymerase (Wang et al. 1997; en par with some of the exonuclease-deficient DNA poly- J. Wang and T. A. Steitz, personal communication). merases. The purified G255S-DNA polymerase, however, has near wild-type levels of 3Ј 5Јexonuclease activ- → ity on single-stranded DNA substrates, but reduced duced by about 10-fold for the G255S-DNA polymerase activity on duplex DNA substrates. This observation indi- compared to the wild-type enzyme (Marquez and cates that the G255S-DNA polymerase retains the ability Reha-Krantz 1996). The slow transfer rate was linked to catalyze hydrolysis of the phosphodiester bond, but to reduced ability of the mutant DNA polymerase to the mutant is deficient in converting duplex DNA to the separate the primer strand from the template strand. partially strand-separated DNA substrate that is required The defect in moving the primer-terminus from the for exonuclease activity (Figure 2). polymerase to the exonuclease active center, called “ac- Kinetic studies using the fluorescence of the base ana- tive-site-switching” (Stocki et al. 1995), is correlated logue 2-aminopurine were used to demonstrate that with structure. Residue G255 is located in a novel pro- the rate of movement of the primer-terminus from the tein loop structure (Figure 3) that resides away from polymerase to the exonuclease active center was re- the exonuclease active center (Wang et al. 1996). The DNA Polymerase Proofreading 1555 reduced ability of the G255S-DNA polymerase to trans- DNA polymerases: One way to answer this question is fer the primer-terminus to the exonuclease active center to propose that the in vivo AT GC mutational specific- → indicates that the protein loop is involved in some aspect ity of T4 antimutator DNA polymerases identifies a class of this process (Figure 2). or classes of mispairs that more frequently escape proof- If the excessive proofreading activity of antimutator reading by the wild-type level of exonucleolytic proof- DNA polymerases can be corrected by alterations in the reading activity (Reha-Krantz 1995b). Mutations at enzyme that raise the kinetic barrier to the proofreading sites that are not reduced by antimutator DNA polymer- pathway, then alterations that produce antimutator ases may then identify mutational pathways that are DNA polymerases may lower the kinetic barrier by in- not sensitive to increased exonucleolytic proofreading. creasing the rate of transfer of the primer-terminus from Drake et al. (1969) pointed out that while the GC AT → the polymerase to the exonuclease active center. The mutational pathway at certain sites appears unaffected antimutator I417V-DNA polymerase, which has a conser- by antimutator DNA polymerases, antimutator DNA vative amino acid substitution in the polymerase active polymerases do strongly reduce base-analogue-induced center, appears to directly affect transfer of DNA from GC AT mutations. These observations suggest that → the polymerase to the exonuclease active center, be- base-analogue-induced GC AT mutations arise by a → cause an increased rate of transfer was detected with proofreading-sensitive mechanism, but that spontane- the 2-aminopurine fluorescence assay that was used to ous GC AT mutations at certain sites arise by a proof- → study the G255S-DNA polymerase (L. A. Marquez and reading-insensitive mechanism. L. J. Reha-Krantz, unpublished observations). An example of a proofreading-insensitive type of path- An important future experiment is to determine if way is one in which mutations arise from slippage be- amino acid substitutions in the C-terminal region, for tween the primer and template strands to form a tran- example A737V or A777V, also increase the rate of trans- sient misaligned primer-terminus. Misalignment of the fer of DNA from the polymerase to the exonuclease primer and template DNA strands was originally pro- active center or if these substitutions affect another as- posed by Streisinger et al. (1966) as a mechanism to pect of proofreading. For example, translocation, the generate frameshift mutations in DNA sequences with ability of the DNA polymerase to move along the DNA monotonic runs of nucleotides or simple repeats. To- template, is reduced for the A737V-DNA polymerase day, misalignment mutagenesis is recognized as a sig- (Gillin and Nossal 1976a; Spacciapoli and Nossal nificant contributor not just to insertions and deletions 1994a). A delay in translocation is predicted to provide of nucleotides but also to transitions and transversions more opportunity to initiate the proofreading pathway, (note reviews by Ripley 1990; Drake 1991b; Kunkel since reduction in the rate of nucleotide incorporation 1990, 1993). Thus, base substitution DNA replication would make initiation of the proofreading pathway errors can arise not only from direct misinsertion of more competitive with primer extension. Yet, the A737V- nucleotides, for example to produce G-T or A-C mis- DNA polymerase alsohas a more processive exonuclease pairs, but also from insertion of correct nucleotides activity, which means that sometimes additional nucleo- within incorrect alignments of the primer-template. tides besides the terminal 3Ј-nucleotide are excised Since the base pairing is correct in the misaligned DNA (Spacciapoli and Nossal 1994b). The T4 DNA poly- strands, these matched primer-termini are not expected merase proofreading activity normally removes just the to be substrates for DNA polymerase exonucleolytic terminal nucleotide, but sometimes the penultimate nu- proofreading. cleotide is also excised (Reddy et al. 1992). If removal If reduction in AT GC mutations by antimutator → of multiple nucleotides is increased by the A737V substi- DNA polymerases is due to increased exonucleolytic tution, this may account for the antimutator phenotype proofreading without a change in nuclease specificity, in cases where the mismatch is not at the primer-ter- then any mechanism that increases exonucleolytic minus. proofreading should parallel the mutational specificity These observations indicate that there is a delicate observed for the antimutator DNA polymerases. An in- balance between primer extension and movement of crease in temperature from 20Њ to 42Њ enhances exo- the primer-terminus between the polymerase and exonucleolytic proofreading to a greater extent than nucle- nuclease active centers, which means that there is also otide incorporation (Brutlag and Kornberg 1972; a delicate balance between more or less accurate DNA Bessman and Reha-Krantz 1977). Increased tempera- replication. This model, however, does not explain the ture is proposed to favor proofreading by destabilizing apparent AT GC mutational specificity of T4 antimuta- the primer-terminus and in assisting in strand separa- → tor DNA polymerases (Drake et al. 1969). How can tion. The temperature effect on exonucleolytic proof- antimutator DNA polymerases reduce AT GC transi- reading was observed for the wild-type T4 DNA polymer- tion mutations 100-fold, but not spontaneous→ GC AT ase in vitro and in vivo. In vivo, the largest reductions → transitions, transversions or other types of DNA replica- in mutation rates produced by high temperature were tion errors? detected for AT GC mutations at sites that also showed → The AT GC mutational specificity of T4 antimutator the largest reductions in reversion rates by T4 antimuta- → 1556 L. J. Reha-Krantz tor DNA polymerases (Bessman and Reha-Krantz reading activity and the mechanism of “active-site- 1977). Sites that were not sensitive to antimutator DNA switching.” Perhaps an even more important benefit of polymerases were also not affected by temperature. studies of antimutator DNA polymerases is an awareness Since the same mutational specificity was detected for that errors in DNA replication can arise by a variety antimutator DNA polymerases and for the wild-type of mechanisms in addition to nucleotide misinsertion. DNA polymerase at high temperature, the AT GC mu- Although the level of exonucleolytic proofreading de- tational specificity can be attributed to a general→ effect tected for wild-type T4 DNA polymerase reduces DNA on the level of proofreading rather than to a specific replication errors by 100-fold or more, increased proof- effect by a mutant DNA polymerase on a specific muta- reading as detected for antimutator DNA polymerases tional pathway. cannot further reduce the frequency of all DNA replica- Errors in DNA replication at one antimutator- and tion errors. Some types of replication errors are insensi- temperature-sensitive site have been sequenced (Reha- tive to exonucleolytic proofreading. The cost and limita- Krantz 1995b). This site is an ochre codon and any of tions of DNA polymerase proofreading may be linked the three base pairs in the codon can be mutated to to the development of mismatch-repair systems that cor- provide protein function. Yet, about 90% of the re- rect replication errors that are missed by proofreading. vertants are AT GC mutations at the first base pair → I thank current and former lab members for research and for position of the codon, which means that this position helpful comments on the manuscript. I also extend special thanks to is a “hotspot” for AT GC mutations. For the wild-type Jimin Wang and Tom Steitz for providing structural information and DNA polymerase, hotspot→ AT GC transitions were de- to T.-C. Lin and W. Konigsberg for T4 DNA polymerase expression → vectors. Research in my lab has been supported by grants from the tected at a frequency of about 1 per 106 while transver- Natural Sciences and Engineering Research Council of Canada and sion mutations at this position and transition and trans- the National Cancer Institute of Canada with funds from the Canadian version mutations at the other two base pairs of the Cancer Society. L.R.-K. is a Scientist of the Alberta Heritage Founda- codon were detected 10- to Ͼ100-fold less frequently. tion for Medical Research. The antimutator A737V-DNA polymerase reduced the hotspot AT GC transitions about 100-fold, but this site → was still a relative hotspot since other mutations within LITERATURE CITED the three base pair codon were also reduced about 100- Beauchamp, B. B., and C. C. Richardson, 1988 A unique deoxygua- fold. Thus, the antimutator DNA polymerase reduces nosine triphosphatase is responsible for the optA1 phenotype of the hotspot AT GC mutation as well as other less fre- → Escherichia coli. Proc. Natl. Acad. Sci. USA 85: 2563–2567. quent replication errors equivalently. Bessman, M. J., and L. J. Reha-Krantz, 1977 Studies on the biochemical basis of spontaneous mutation V. Effect of temperature Together, these observations suggest that the in- on mutation frequency. J. Mol. Biol. 116: 115–123. creased exonucleolytic proofreading of antimutator Brutlag, D., and A. Kornberg, 1972 Enzymatic synthesis of deoxy- DNA polymerases reduces both transition and transver- ribonucleic acid. XXXVI. A proofreading function for the 3Ј 5Ј exonuclease activity of deoxyribonucleic acid polymerases. J. Biol.→ sion DNA replication errors, but there are sites in which Chem. 247: 241–248. certain types of DNA replication errors are refractory Capson, T. L., J. A. Peliska, B. F. Kaboord, M. W. Frey, C. Lively to proofreading. Mutations at proofreading-refractory et al., 1992 Kinetic characterization of the polymerase and exonuclease activities of the gene 43 protein of bacteriophage T4. sites may be produced by mechanisms in which “right Biochemistry 31: 10984–10994. bases are in the wrong places” due to misalignment of Clayton, L. K., M. F. Goodman, E. W. Branscomb and D. J. Galas, the primer and template DNA strands (Drake 1991b). 1979 Error induction and correction by mutant and wild-type T4 DNA polymerases. J. Biol. Chem. 254: 1902–1912. A goal of future experimentation will be to determine Drake, J. W., 1990 Evolving mutation rates and prospects of antimu- why certain sites are hotspots for AT GC mutations → tagenesis, pp. 139–149 in Mechanisms of Environmental Mutagenesis- and why these sites escape proofreading by the wild- Carcinogenesis, edited by A. Kappas. Plenum Press, New York. Drake, J. W., 1991a A constant rate of spontaneous mutation in type DNA polymerase. Another goal is to understand DNA-based microbes. Proc. Natl. Acad. Sci. USA 88: 7160–7164. how the antimutator A737V-DNA polymerase increases Drake, J. W., 1991b Spontaneous mutation. Annu. Rev. Genet. 25: the frequency of other types of mutations (see Wang 125–146. Ripley Drake, J. W., 1993 General antimutators are improbable. J. Mol. and 1998). Biol. 229: 8–13. Summary: Only a relatively few discoveries or observa- Drake, J. W., and E. F. Allen, 1968 Antimutator DNA polymerases tions in science have great impact in advancing knowl- of bacteriophage T4. Cold Spring Harbor Symp. Quant. Biol. 33: edge and in stimulating new lines of thought. Such 339–344. Drake, J. W., and E. O. Greening, 1970 Suppression of chemical was the discovery of T4 antimutator DNA polymerases mutagenesis in bacteriophage T4 by genetically modified DNA (Drake and Allen 1968; Drake et al. 1969). The avail- polymerases. Proc. Natl. Acad. Sci. USA 66: 823–829. Drake, J. W., E. F. Allen, S. A. Forsberg, R.-M. Preparata ability of antimutator DNA polymerases was critical in and E. O. Greening, 1969 Spontaneous mutation. Nature 221: the elucidation of the role played by the DNA polymer- 1128–1132. ase 3Ј 5Ј exonuclease activity in proofreading newly Fijalkowska, I. J., R. L. Dunn and R. M. Schaaper, 1993 Mutants of → synthesized DNA. Continued biochemical characteriza- Escherichia coli with increased fidelity of DNA replication. Genetics 134: 1023–1030. tion of antimutator DNA polymerases is providing fur- Fersht, A. R., J. W. Knill-Jones and W.-C. Tsui, 1982 Kinetic basis ther insights into the regulation of exonucleolytic proof- ofspontaneous mutation. Misinsertion frequencies, proofreading DNA Polymerase Proofreading 1557

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