Download Regulation of DNA Polymerase Exonucleolytic Proofreading Activity

Copyright  1998 by the Genetics Society of America
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
D
NA polymerases replicate DNA with high fidelity
because of accurate nucleotide incorporation coupled with exonucleolytic proofreading to remove misincorporated nucleotides. This statement is taken for
granted today, in large part, because of groundbreaking
discoveries made 30 years ago that mutations in the
DNA polymerase gene of bacteriophage T4 can have
dramatic effects on the fidelity of DNA replication. “Mutator” DNA polymerases (Speyer et al. 1966) are mutant
DNA polymerases that replicate DNA with less accuracy
than the wild-type enzyme, and “antimutator” DNA polymerases (Drake and Allen 1968; Drake et al. 1969)
are mutant DNA polymerases that replicate DNA with
more accuracy, at least for certain types of DNA replication errors. Biochemical characterization revealed that
the balance between the two DNA polymerase functions,
nucleotide incorporation and the opposing 39→59 exonuclease activity, was altered in the mutants compared to
the wild type enzyme. Decreased 39→59 exonuclease activity was detected for many of the mutator DNA polymerases
(Muzyczka et al. 1972) while increased 39→59exonuclease
activity, relative to polymerase activity, was observed for
the antimutator DNA polymerases (Muzyczka et al. 1972;
Gillin and Nossal 1976a,b). The correlation of decreased 39→59 exonuclease activity measured in in vitro
assays with decreased accuracy of DNA replication in vivo,
and increased 39→59 exonuclease activity with increased
DNA replication fidelity indicates that the 39→59 exonuclease activity of T4 DNA polymerase is an exonucleolytic proofreading activity.
These observations were made at an exciting time in
DNA polymerase research. I was fortunate then to be a
graduate student in Maurice Bessman’s lab at Johns
Hopkins University and to see first hand the biochemical characterization of T4 mutator and antimutator
DNA polymerases. But of equal importance was the opportunity to meet Jan Drake and to learn about the
use of genetics to study the fidelity of DNA replication.
The juxtaposition of biochemistry and genetic analyses
to elucidate the proofreading role for the 39→59 exonuclease activity of T4 DNA polymerase made a strong impact
on my graduate studies and continues to do so today. I
am still intrigued by the genetic analyses from the Drake
Address for correspondence: Department of Biological Sciences, CW405
BioSciences Bldg., University of Alberta, Edmonton, Alberta T6G 2E9,
Canada. E-mail: [email protected]
Genetics 148: 1551–1557 (April, 1998)
lab, which demonstrated that single point mutations in
the T4 DNA polymerase gene can increase or decrease
mutation rates by 100-fold or more (Drake et al. 1969).
Mutational analysis is a powerful method to probe
enzyme function. Even today with the ready availability
of molecular techniques, genetic screens and selections
are useful because informative mutant enzymes can be
identified without structural information or assumptions about function. Most importantly, since classical genetic methods rely on detection and characterization of mutant
phenotypes in vivo, alterations in function are observed within
the context of the living organism. In the case of antimutator
DNA polymerases, these mutants provide a handle to
probe DNA polymerase function in vivo.
Genetic and biochemical techniques have continued
to be used in studies of antimutator DNA polymerases.
This research has led in a number of different directions
including studies of the evolution of spontaneous mutation rates (Drake 1990, 1991a, 1991b), the effect of
antimutator DNA polymerases on frameshift mutagenesis (Kaiser and Ripley 1995), the identification of antimutator DNA polymerases in other organisms (Fijalkowska et al. 1993), and additional studies of DNA
polymerase function (Clayton et al. 1979; Spacciapoli
and Nossal 1994a,b; Reha-Krantz and Nonay 1994;
Reha-Krantz and Wong 1996). The focus of this short
review is the regulation of DNA polymerase proofreading activity (Stocki et al. 1995). Other aspects of T4
antimutator DNA polymerases are presented in this volume (e.g., Nossal, Wang and Ripley).
T4 antimutator DNA polymerases: Mutations within
genes result typically in mutant enzymes with reduced
activity and even loss of function; however, in the case
of antimutator DNA polymerases, the mutant enzymes
appear to be working better, at least with respect to the
accuracy of DNA replication. Since single point mutations can give rise to antimutator DNA polymerases, why
have more accurate DNA polymerases not evolved?
Part of the answer must be that there are negative
consequences of the antimutator phenotype that reduce
the efficiency of the DNA polymerase and the overall
fitness of the organism. There is a cost associated with
DNA polymerase proofreading (Fersht 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 concentrations of deoxynucleoside triphosphates (dNTPs) are required to support DNA replication by T4 antimutator
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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 induced mutations. Other mutational pathways such as
GC→AT transitions, transversions, and frameshifts are
not reduced at some sites by T4 antimutator DNA polymerases, and may even be increased (Drake and Greening 1970; Ripley 1975; Kaiser and Ripley 1995). Thus,
any gain in fitness obtained by a decrease in AT→GC
transition mutations may be offset by increases in other
types of DNA replication errors (Drake 1993).
T4 DNA polymerase has a potent 39→59 exonuclease
activity, about 1000-fold more active than the 39→59exonuclease activity of Escherichia coli DNA polymerase I (Capson
et al. 1992); hence, a mechanism must be in place to
prevent indiscriminate degradation of the newly synthesized DNA and the other disadvantages of excessive proofreading discussed above. This mechanism fails or regulation is reduced in antimutator DNA polymerases. If the
molecular basis of the antimutator phenotype can be
learned, then this information can be applied to determining how the normal balance between polymerase
and exonuclease activities is maintained. A first step in
understanding the regulation of exonucleolytic proofreading is to determine the location of mutations that
confer the antimutator phenotype.
Locations of mutations within the T4 DNA polymerase
gene that confer antimutator and mutator phenotypes:
Drake (Drake and Allen 1968; Drake and Greening
1970) mapped mutations that confer the antimutator
phenotype by recombination. Two of the mutations that
produce a strong antimutator phenotype encode Val to
Ala substitutions at codons 737 and 777 (Reha-Krantz
1989). The locations of these mutations in the primary
structure of T4 DNA polymerase are illustrated in Figure
1. Additional mutations that confer the antimutator
phenotype have also been located in this region at codons 730, 731, 827, and 844 (Reha-Krantz 1988, 1994,
1995a). Mutations in the T4 DNA polymerase that confer the antimutator phenotype have also been identified
by genetic selection for second-site mutations that suppress sensitivity to the pyrophosphate analogue, phosphonoacetic acid (Reha-Krantz and Nonay 1994).
These mutations are located near the center of the gene
(Figure 1) and encode the conservative amino acid sub-
Figure 1.—Locations of mutations in the bacteriophage T4
DNA polymerase gene that confer mutator and antimutator
phenotypes. More comprehensive listings of N-terminal mutator mutations and C-terminal antimutator mutations are given
in Reha-Krantz (1988, 1994, and 1995a). The codon number
and identities of the amino acid substitutions encoded by the
mutations are shown and more details are provided in the
text. Increases and decreases in spontaneous mutation rates
are illustrated by bars extending upward (mutators) or downward (antimutators). The spontaneous mutation rates for the
antimutator A737V- and A777V-DNA polymerases are from
Drake et al. (1969). Spontaneous mutation rates for the other
mutant DNA polymerases are from Reha-Krantz and Nonay
(1993), Stocki et al. (1995), and from Reha-Krantz and
Wong (1996).
stitutions Thr for Ser411, Ile for Leu412, and Val for
Ile417 in the highly conserved DNA polymerase motif,
DXXS411L 412YPSII417, that forms part of the polymerase
active center. Another mutation that encodes a Cys substitution for Arg335 was also identified by this selection
strategy (Reha-Krantz and Wong 1996). Thus two regions of the DNA polymerase have been identified in
which single amino acid substitutions can produce a
strong antimutator phenotype.
Most mutations that produce a strong mutator phenotype encode amino acid substitutions in the N-terminal
region of T4 DNA polymerase (Reha-Krantz 1988,
1989). Alanine substitutions for aspartate residues D112,
D219, and D324 (Figure 1) that are required for the
hydrolysis reaction increase the mutation rate up to several
hundredfold, as expected if exonucleolytic proofreading
activity is prevented (Reha-Krantz et al. 1991; Frey et al.
1993; Reha-Krantz and Nonay 1993).
The recent determination of the structure of the RB69
DNA polymerase (Wang et al. 1997), a T4-like DNA
polymerase, allows placement of residues that are important for DNA replication fidelity in a three-dimensional, structural context. Residues in the C-terminal
domain, as well as residues in the exonuclease domain,
make contacts with single-stranded DNA bound in the
DNA Polymerase Proofreading
exonuclease catalytic center (Wang et al. 1997). Thus,
some residues in the C-terminal domain, the location
of residues A737 and A777 (Figure 1), are in close contact with the exonuclease domain (see Nossal 1998).
Amino acid substitutions that produce the antimutator
phenotype were also identified in the polymerase active
center, but the polymerase and exonuclease active centers are separated by a relatively great distance, about
30 Å (Wang et al. 1997). Proofreading requires that the
primer strand be moved from the polymerase to the
exonuclease active center. The locations of mutations
that confer the antimutator phenotype suggest the possibility that residues in the polymerase active center and
in the C-terminal domain affect how the DNA is transferred between the two active centers.
Antimutator DNA polymerases have more opportunity
to proofread: Biochemical characterizations of the antimutator DNA polymerases revealed that the balance
between nucleotide incorporation and exonuclease activities was shifted toward increased exonuclease activity
in the mutant enzymes by providing more opportunity
to proofread (Gillin and Nossal 1976a). The relative
opportunities to proofread or to extend a primer-terminus can be demonstrated by preforming Enz·DNA complexes in the presence of dNTPs but in the absence of
Mg21, which is required for both nucleotide incorporation and exonucleolytic proofreading activities. The addition of Mg21 then allows preformed Enz·DNA complexes in which the primer-terminus resides in the
polymerase active center (Enzpol·DNA·dNTP complex)
to incorporate nucleotides and Enz·DNA complexes in
which the primer-terminus resides in the exonuclease
active center (Enzexo·DNA complex) to remove the terminal nucleotide. The antimutator I417V-DNA polymerase, Val substitution for Ile417 within the polymerase active center, forms active exonuclease complexes
almost twofold more frequently than the wild-type enzyme (Reha-Krantz and Nonay 1994).
These results were interpreted to indicate that the
I417V substitution destabilizes interactions with the
primer-terminus within the polymerase active center
and thus increases the opportunity of the enzyme to
form complexes in which the primer-terminus resides
in the exonuclease active center. Reduced dNTP concentrations can also enhance exonuclease activity by
decreasing the formation of Enzpol·DNA·dNTP complexes which then favors increased formation of active
Enzexo·DNA complexes (Clayton et al. 1979).
Kinetic partitioning between polymerase and exonuclease activities: The studies above and numerous
additional studies of T4 DNA polymerase and other
DNA polymerases (Hopfield 1974; Fersht et al. 1982;
Capson et al. 1992; Johnson 1993; Goodman et al. 1993)
have resulted in formulation of a kinetic scheme that
describes the regulation between nucleotide incorporation and exonucleolytic proofreading activities. A
primer-terminus is normally extended by T4 DNA poly-
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merase at the rapid rate of about 400 sec 21 (Capson
et al. 1992), but elongation of a mismatched primerterminus is extremely slow (reviewed by Goodman et al.
1993). The greatly reduced ability of a DNA polymerase
to extend a mismatched primer-terminus provides a
window of opportunity to transfer the primer-terminus
from the polymerase to the exonuclease active center, a
slower reaction measured at the apparent rate of 4 sec2 1
(Capson et al. 1992; Marquez and Reha-Krantz 1996).
The relatively slow transfer of DNA from the polymerase
to the exonuclease active center serves as a kinetic barrier to the exonucleolytic proofreading pathway that
protects correct primer-termini from indiscriminate
degradation.
The antimutator phenotype can be explained in the
context of the kinetic scheme by proposing that the
antimutator DNA polymerases have even less ability than
the wild-type DNA polymerase to extend a mismatched
primer-terminus. One prediction of this proposal is that
antimutator DNA polymerases would also have less ability to extend a correctly matched primer-terminus. Both
the I417V-DNA polymerase, which has decreased ability
to form the Enzpol·DNA·dNTP complex (Reha-Krantz
and Nonay 1994), and the A737V-DNA polymerase,
which is defective in translocation (Gillin and Nossal
1976a; Spacciapoli and Nossal 1994a), have high rates
of turnover of correct nucleotides (Muzyczka et al.
1972; Gillin and Nossal 1976a; reviewed in Goodman
et al. 1993). The high turnover of correct nucleotides
by T4 antimutator DNA polymerases was used as the
basis of a genetic selection strategy to isolate secondsite mutations that suppress the excessive proofreading
of antimutator DNA polymerases (Stocki et al. 1995).
Suppressors of excessive DNA polymerase proofreading decrease transfer of the primer-terminus from the
polymerase to the exonuclease active center: Excessive
proofreading by T4 antimutator DNA polymerases is
detected in vivo in infections of a host bacterial strain,
optA1, which has increased ability to degrade dGTP
(Gauss et al. 1983; Beauchamp and Richardson 1988).
The reduced dGTP concentration in the restrictive host
is not sufficient to support DNA replication by antimutator DNA polymerases and prevents replication of the
phage genome. We have used this conditional lethal
phenotype to identify second-site mutations that suppress the excessive exonucleolytic proofreading activity
of four mutant T4 DNA polymerases (Stocki et al.
1995). Two antimutator DNA polymerases identified by
Drake, the A737V- and A777V-DNA polymerases, were
part of the study. Nineteen second-site, suppressor mutations were found. Although the suppressor mutations
are located in four different regions of the DNA polymerase, the mechanism of suppression appears to be the
same—to raise the kinetic barrier to the proofreading
pathway. One of the suppressor mutations will be discussed here to illustrate this point.
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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
involved in transfer of the primer-terminus from the polymerase to the exonuclease active center (Marquez and RehaKrantz 1996).
The most frequently isolated second-site suppressor
mutation of excessive proofreading activity encodes a
Ser substitution for residue Gly255, which resides in
the exonuclease domain (Figure 1). The G255S-DNA
polymerase in vivo displays a strong mutator phenotype
en par with some of the exonuclease-deficient DNA polymerases. The purified G255S-DNA polymerase, however, has near wild-type levels of 39→59exonuclease activity on single-stranded DNA substrates, but reduced
activity on duplex DNA substrates. This observation indicates that the G255S-DNA polymerase retains the ability
to catalyze hydrolysis of the phosphodiester bond, but
the mutant is deficient in converting duplex DNA to the
partially strand-separated DNA substrate that is required
for exonuclease activity (Figure 2).
Kinetic studies using the fluorescence of the base analogue 2-aminopurine were used to demonstrate that
the rate of movement of the primer-terminus from the
polymerase to the exonuclease active center was re-
Figure 3.—Location of the G255 protein loop. A similar
figure was published by Marquez and Reha-Krantz (1996)
and is republished here with permission from The Journal
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
is highlighted. The G255 loop appears to be located at a
branch point between the observed proofreading complex
and duplex DNA in the polymerase active center based on
modeling the structures of duplex DNA bound by pol b and
HIV reverse transcriptase with the structure of the RB69 DNA
polymerase, a T4-like DNA polymerase (Wang et al. 1997;
J. Wang and T. A. Steitz, personal communication).
duced by about 10-fold for the G255S-DNA polymerase
compared to the wild-type enzyme (Marquez and
Reha-Krantz 1996). The slow transfer rate was linked
to reduced ability of the mutant DNA polymerase to
separate the primer strand from the template strand.
The defect in moving the primer-terminus from the
polymerase to the exonuclease active center, called “active-site-switching” (Stocki et al. 1995), is correlated
with structure. Residue G255 is located in a novel protein loop structure (Figure 3) that resides away from
the exonuclease active center (Wang et al. 1996). The
DNA Polymerase Proofreading
reduced ability of the G255S-DNA polymerase to transfer the primer-terminus to the exonuclease active center
indicates that the protein loop is involved in some aspect
of this process (Figure 2).
If the excessive proofreading activity of antimutator
DNA polymerases can be corrected by alterations in the
enzyme that raise the kinetic barrier to the proofreading
pathway, then alterations that produce antimutator
DNA polymerases may lower the kinetic barrier by increasing the rate of transfer of the primer-terminus from
the polymerase to the exonuclease active center. The
antimutator I417V-DNA polymerase, which has a conservative amino acid substitution in the polymerase active
center, appears to directly affect transfer of DNA from
the polymerase to the exonuclease active center, because an increased rate of transfer was detected with
the 2-aminopurine fluorescence assay that was used to
study the G255S-DNA polymerase (L. A. Marquez and
L. J. Reha-Krantz, unpublished observations).
An important future experiment is to determine if
amino acid substitutions in the C-terminal region, for
example A737V or A777V, also increase the rate of transfer of DNA from the polymerase to the exonuclease
active center or if these substitutions affect another aspect of proofreading. For example, translocation, the
ability of the DNA polymerase to move along the DNA
template, is reduced for the A737V-DNA polymerase
(Gillin and Nossal 1976a; Spacciapoli and Nossal
1994a). A delay in translocation is predicted to provide
more opportunity to initiate the proofreading pathway,
since reduction in the rate of nucleotide incorporation
would make initiation of the proofreading pathway
more competitive with primer extension. Yet, the A737VDNA polymerase also has a more processive exonuclease
activity, which means that sometimes additional nucleotides besides the terminal 39-nucleotide are excised
(Spacciapoli and Nossal 1994b). The T4 DNA polymerase proofreading activity normally removes just the
terminal nucleotide, but sometimes the penultimate nucleotide is also excised (Reddy et al. 1992). If removal
of multiple nucleotides is increased by the A737V substitution, this may account for the antimutator phenotype
in cases where the mismatch is not at the primer-terminus.
These observations indicate that there is a delicate
balance between primer extension and movement of
the primer-terminus between the polymerase and exonuclease active centers, which means that there is also
a delicate balance between more or less accurate DNA
replication. This model, however, does not explain the
apparent AT→GC mutational specificity of T4 antimutator DNA polymerases (Drake et al. 1969). How can
antimutator DNA polymerases reduce AT→GC transition mutations 100-fold, but not spontaneous GC→AT
transitions, transversions or other types of DNA replication errors?
The AT→GC mutational specificity of T4 antimutator
1555
DNA polymerases: One way to answer this question is
to propose that the in vivo AT→GC mutational specificity of T4 antimutator DNA polymerases identifies a class
or classes of mispairs that more frequently escape proofreading by the wild-type level of exonucleolytic proofreading activity (Reha-Krantz 1995b). Mutations at
sites that are not reduced by antimutator DNA polymerases may then identify mutational pathways that are
not sensitive to increased exonucleolytic proofreading.
Drake et al. (1969) pointed out that while the GC→AT
mutational pathway at certain sites appears unaffected
by antimutator DNA polymerases, antimutator DNA
polymerases do strongly reduce base-analogue-induced
GC→AT mutations. These observations suggest that
base-analogue-induced GC→AT mutations arise by a
proofreading-sensitive mechanism, but that spontaneous GC→AT mutations at certain sites arise by a proofreading-insensitive mechanism.
An example of a proofreading-insensitive type of pathway is one in which mutations arise from slippage between the primer and template strands to form a transient misaligned primer-terminus. Misalignment of the
primer and template DNA strands was originally proposed by Streisinger et al. (1966) as a mechanism to
generate frameshift mutations in DNA sequences with
monotonic runs of nucleotides or simple repeats. Today, misalignment mutagenesis is recognized as a significant contributor not just to insertions and deletions
of nucleotides but also to transitions and transversions
(note reviews by Ripley 1990; Drake 1991b; Kunkel
1990, 1993). Thus, base substitution DNA replication
errors can arise not only from direct misinsertion of
nucleotides, for example to produce G-T or A-C mispairs, but also from insertion of correct nucleotides
within incorrect alignments of the primer-template.
Since the base pairing is correct in the misaligned DNA
strands, these matched primer-termini are not expected
to be substrates for DNA polymerase exonucleolytic
proofreading.
If reduction in AT→GC mutations by antimutator
DNA polymerases is due to increased exonucleolytic
proofreading without a change in nuclease specificity,
then any mechanism that increases exonucleolytic
proofreading should parallel the mutational specificity
observed for the antimutator DNA polymerases. An increase in temperature from 208 to 428 enhances exonucleolytic proofreading to a greater extent than nucleotide incorporation (Brutlag and Kornberg 1972;
Bessman and Reha-Krantz 1977). Increased temperature is proposed to favor proofreading by destabilizing
the primer-terminus and in assisting in strand separation. The temperature effect on exonucleolytic proofreading was observed for the wild-type T4 DNA polymerase in vitro and in vivo. In vivo, the largest reductions
in mutation rates produced by high temperature were
detected for AT→GC mutations at sites that also showed
the largest reductions in reversion rates by T4 antimuta-
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L. J. Reha-Krantz
tor DNA polymerases (Bessman and Reha-Krantz
1977). Sites that were not sensitive to antimutator DNA
polymerases were also not affected by temperature.
Since the same mutational specificity was detected for
antimutator DNA polymerases and for the wild-type
DNA polymerase at high temperature, the AT→GC mutational specificity can be attributed to a general effect
on the level of proofreading rather than to a specific
effect by a mutant DNA polymerase on a specific mutational pathway.
Errors in DNA replication at one antimutator- and
temperature-sensitive site have been sequenced (RehaKrantz 1995b). This site is an ochre codon and any of
the three base pairs in the codon can be mutated to
provide protein function. Yet, about 90% of the revertants are AT→GC mutations at the first base pair
position of the codon, which means that this position
is a “hotspot” for AT→GC mutations. For the wild-type
DNA polymerase, hotspot AT→GC transitions were detected at a frequency of about 1 per 106 while transversion mutations at this position and transition and transversion mutations at the other two base pairs of the
codon were detected 10- to .100-fold less frequently.
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
the three base pair codon were also reduced about 100fold. Thus, the antimutator DNA polymerase reduces
the hotspot AT→GC mutation as well as other less frequent replication errors equivalently.
Together, these observations suggest that the increased exonucleolytic proofreading of antimutator
DNA polymerases reduces both transition and transversion DNA replication errors, but there are sites in which
certain types of DNA replication errors are refractory
to proofreading. Mutations at proofreading-refractory
sites may be produced by mechanisms in which “right
bases are in the wrong places” due to misalignment of
the primer and template DNA strands (Drake 1991b).
A goal of future experimentation will be to determine
why certain sites are hotspots for AT→GC mutations
and why these sites escape proofreading by the wildtype DNA polymerase. Another goal is to understand
how the antimutator A737V-DNA polymerase increases
the frequency of other types of mutations (see Wang
and Ripley 1998).
Summary: Only a relatively few discoveries or observations in science have great impact in advancing knowledge and in stimulating new lines of thought. Such
was the discovery of T4 antimutator DNA polymerases
(Drake and Allen 1968; Drake et al. 1969). The availability of antimutator DNA polymerases was critical in
the elucidation of the role played by the DNA polymerase 39→59 exonuclease activity in proofreading newly
synthesized DNA. Continued biochemical characterization of antimutator DNA polymerases is providing further insights into the regulation of exonucleolytic proof-
reading activity and the mechanism of “active-siteswitching.” Perhaps an even more important benefit of
studies of antimutator DNA polymerases is an awareness
that errors in DNA replication can arise by a variety
of mechanisms in addition to nucleotide misinsertion.
Although the level of exonucleolytic proofreading detected for wild-type T4 DNA polymerase reduces DNA
replication errors by 100-fold or more, increased proofreading as detected for antimutator DNA polymerases
cannot further reduce the frequency of all DNA replication errors. Some types of replication errors are insensitive to exonucleolytic proofreading. The cost and limitations of DNA polymerase proofreading may be linked
to the development of mismatch-repair systems that correct replication errors that are missed by proofreading.
I thank current and former lab members for research and for
helpful comments on the manuscript. I also extend special thanks to
Jimin Wang and Tom Steitz for providing structural information and
to T.-C. Lin and W. Konigsberg for T4 DNA polymerase expression
vectors. Research in my lab has been supported by grants from the
Natural Sciences and Engineering Research Council of Canada and
the National Cancer Institute of Canada with funds from the Canadian
Cancer Society. L.R.-K. is a Scientist of the Alberta Heritage Foundation for Medical Research.
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