Mutation Research 510 (2002) 4554

Review
The proofreading 3
5
exonuclease activity of DNA
polymerases: a kinetic barrier to translesion DNA synthesis
Vineeta Khare, Kristin A. Eckert
Department of Pathology, Gittlen Cancer Research Institute, Penn State University College of Medicine,
500 University Drive, Hershey, PA 17033, USA
Received 5 July 2002; received in revised form 13 September 2002; accepted 16 September 2002

Abstract
The 3
5
exonuclease activity intrinsic to several DNA polymerases plays a primary role in genetic stability; it acts as
a first line of defense in correcting DNA polymerase errors. A mismatched basepair at the primer terminus is the preferred
substrate for the exonuclease activity over a correct basepair. The efficiency of the exonuclease as a proofreading activity
for mispairs containing a DNA lesion varies, however, being dependent upon both the DNA polymerase/exonuclease and the
type of DNA lesion. The exonuclease activities intrinsic to the T4 polymerase (family B) and DNA polymerase (family A)
proofread DNA mispairs opposite endogenous DNA lesions, including alkylation, oxidation, and abasic adducts. However,
the exonuclease of the Klenow polymerase cannot discriminate between correct and incorrect bases opposite alkylation
and oxidative lesions. DNA damage alters the dynamics of the intramolecular partitioning of DNA substrates between the
3
5
exonuclease and polymerase activities. Enzymatic idling at lesions occurs when an exonuclease activity efficiently
removes the same base that is preferentially incorporated by the DNA polymerase activity. Thus, the exonuclease activity
can also act as a kinetic barrier to translesion synthesis (TLS) by preventing the stable incorporation of bases opposite DNA
lesions. Understanding the downstream consequences of exonuclease activity at DNA lesions is necessary for elucidating the
mechanisms of translesion synthesis and damage-induced cytotoxicity.
2002 Elsevier Science B.V. All rights reserved.
Keywords: Exonuclease activity; Polymerase; Translesion

1. Introduction predisposition to neoplasia. The post-replication mis-

Numerous studies have examined cellular DNA
repair activities and their role in genetic stability and
Abbreviations: m6G, O6 -methylguanine; MMR, mismatch
repair; dNTP, deoxyribonucleotide triphosphate; dNMP, deoxyribo-
nucleotide monophosphate; HSV-tk, herpes simplex virus thymi-
dine kinase; MNU, N-methyl-N-nitrosourea; ENU, N-ethyl-N-
nitrosourea; TLS, translesion synthesis
Corresponding author. Tel.: +1-717-531-4065;
fax: +1-717-531-5634.
E-mail address: kae4@psu.edu (K.A. Eckert).
match repair (MMR) system is such a mechanism, and
loss of this repair pathway has been implicated in re-
sistance to cellular toxicity towards chemotherapeutic
agents as well as in the development of a mutator phe-
notype [1]. The primary function of MMR is to remove
errors formed by the DNA polymerase during replica-
tion or recombination [2]. The polymerase-associated
3
5
proofreading exonucleases are also involved
in avoidance of mutation, acting as the first line of
defense against polymerase-induced errors (see [3]
for a recent review). The proofreading exonuclease

0027-5107/02/$ see front matter 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 0 2 7 - 5 1 0 7 ( 0 2 ) 0 0 2 5 1 - 8
46 V. Khare, K.A. Eckert / Mutation Research 510 (2002) 4554
and MMR proteins act in series to limit mutations [4],
and the combined deficiency in both error avoidance
pathways can lead to cell death, possibly by the exces-
sive accumulation of mutations [5]. However, unlike
MMR, the contribution of the polymerase proofread-
ing function to the avoidance of damage-induced
mutations and the biological consequences of proof-
reading on damaged DNA templates have yet to be
elucidated. In this review, we will focus on lesion
processing by DNA polymerases containing an in-
trinsic 3
5
exonuclease activity and discuss the
cellular implications of proofreading action at the site
of DNA lesions.
2. DNA polymerases with intrinsic 3
5

exonuclease activity
Among the four families of eukaryotic DNA poly-
merases, two contain enzymes in which both the
polymerase and the 3
5
exonuclease activities
are present within the same polypeptide [6]. These
are the family A polymerases which include the mi-
tochondrial DNA polymerase , and the family B
polymerases, which include the nuclear DNA poly-
merases and . The amino terminus of each of these
polymerases encodes a discrete 3
5
exonuclease
domain that contains conserved amino acid residues
involved in metal binding, DNA binding and catalysis
[7]. Mutagenesis studies have confirmed the impor-
tance of critical amino acid residues within the exonu-
clease domains [8]. Enzyme derivatives that display
compromised exonuclease activity concomitant with
full polymerase activity emphasize the independence
of the polymerase and exonuclease active sites [9].
2.1. Proofreading activity of the 3
5

exonuclease
The polymerase-associated 3
5
exonucleases
utilize both ssDNA and melted dsDNA templates.
They catalyze the removal of nucleotides from the
nascent (primer) strand, producing deoxyribonu-
cleotide monophosphates (dNMPs). The enhance-
ment of polymerase accuracy, or proofreading, comes
from the intrinsic rate differences of the exonuclease
for correctly basepaired and mismatched basepairs.
A mismatched basepair at the primer terminus (the
3
OH) is the preferred substrate for the exonucle-
ase activity over a correct basepair. In contrast, a
WatsonCrick basepair at the primer terminus is
the preferred substrate for the polymerase activity
over a mispaired terminus. The slow rate of mispair
extension by the DNA polymerase activity serves
to enhance proofreading by kinetically partitioning
the DNA substrate to the exonuclease [10] (Fig. 1).
The outcome of the catalytic competition between
nucleotide incorporation by the polymerase activity
and removal of mispairs by the exonuclease activity
reflects the efficiency of error discrimination by a
DNA polymerase. Factors governing this competition
which reduce the efficiency of exonuclease proofread-
ing include a high deoxyribonucleotide triphosphate
(dNTP) concentration [11], which acts to drive the
polymerase reaction, and end-product inhibition of
the exonuclease by excess dNMPs. Because DNA
lesions alter both the structure (thermostability) of
DNA and the rate of basepair extension by the poly-
merase activity, it is reasonable to predict that the
natural kinetic partitioning (Fig. 1) of DNA substrates
between the polymerase and exonuclease active sites
will be disrupted by DNA damage.
2.2. Exonuclease active site switching
X-ray crystallographic studies of the Klenow frag-
ment of Escherichia coli Pol I (a family A polymerase)
[12] and bacteriophage RB69 DNA polymerase
Fig. 1. Kinetic partitioning between polymerase and exonuclease
activities while processing lesioncontaining templates. Kinetic
partitioning on natural DNA templates contributes to polymerase
fidelity [10]. Endogenous lesions that are substrates for the 3
5

exonuclease are shown (see text for details). kexo , exonuclease rate
constant; kpol , polymerase rate constant; koff , DNA dissociation
constant.
 V. Khare, K.A. Eckert / Mutation Research 510 (2002) 4554
(a family B polymerase) [13], in both editing and
polymerizing modes, have helped to elucidate the
mechanism by which the two competitive biochem-
ical activities are coordinated. The exonuclease is a
discrete domain of the enzymes, separated from the
polymerase active site by 30 (Klenow) or 40
(RB69). The structural principles governing the poly-
merase/exonuclease transition are shared between
these two representative enzymes [13]. First, the
primer terminus must be shuttled between the two ac-
tive sites, whereas the previously-synthesized duplex
DNA remains in approximately the same physical
position. Second, the only interactions that remain
constant during the active site switching are between
the tip of the polymerase thumb subdomain and the
backbone of the nascent (primer) DNA. This trans-
fer may occur by an intramolecular reaction, without
dissociation of the polymerase from the DNA, as has
been observed for the T4 DNA polymerase [14] and
polymerase [15] on natural DNA templates. How-
ever, the Klenow fragment performs proofreading by a
predominantly intermolecular process involving poly-
merase dissocation from a mispaired template and
reassociation of the DNA in the exonuclease site of
another Klenow enzyme molecule [16]. The effects of
DNA lesions on the transfer reaction and the extent to
which dissociation from lesion-containing substrates
(Fig. 1, koff ) occurs during active site switching have
not been examined.
3. Translesion synthesis and the 3
5

exonuclease
The 3
5
exonuclease activity has been shown
to modulate the processing of DNA lesions by DNA
polymerases [17,18]. Biochemically, the effect of
DNA lesions on the polymerase reaction can be ei-
ther inhibitory, leading to nascent chain termination,
or non-inhibitory, leading to translesion synthesis
(TLS). Genetically, TLS can be either non-mutagenic
or mutagenic, depending on whether a correct or in-
correct base is incorporated opposite the lesion. In
general, exonuclease-deficient polymerases perform
TLS more frequently than exonuclease-proficient
polymerases, suggesting that the exonuclease con-
tributes to DNA synthesis inhibition. Comparisons
of exonuclease-proficient and exonuclease-deficient
47
DNA polymerase forms have been used to understand
the independent significance of polymerizing and
exonucleolytic activities of DNA polymerases. The
ability of the exonuclease to remove lesion-induced,
premutational mispairs depends not only on the DNA
lesion, but also on the specific polymerase.
3.1. Genetic analyses of proofreading and
DNA lesions
Our laboratory has developed and utilized an in vitro
forward polymerase assay to study damage-induced
polymerase errors at the herpes simplex virus type
1 thymidine kinase (HSV-tk) gene [17,1922]. Our
method ensures the exclusive analysis of polymerase
mutations derived from the DNA strand produced
during in vitro synthesis. In the absence of DNA
damage, the 3
5
exonuclease proofreading func-
tion enhances polymerase accuracy six-fold for the
Klenow polymerase and eight-fold for the T4 poly-
merase (Table 1). The in vitro HSV-tk mutation
frequencies of the exonuclease-deficient derivatives
of Klenow and T4 polymerases are similar to those
of DNA polymerase -primase and polymerase
(20 104 ), enzymes which do not possess an
intrinsic proofreading activity (Table 1).
We have studied the effect of 3
5
exonuclease
activity on the mutagenic processing of alkylation
damage by the Klenow and T4 DNA polymerases,
model enzymes for family A and family B poly-
merases, respectively. Using the Klenow polymerase
in vitro, we observed no significant reduction in mu-
tation frequency when either N-methyl-N-nitrosourea
(MNU) or N-ethyl-N-nitrosourea (ENU) treated DNA
was used as a template for the exonuclease-proficient
Klenow polymerase, as compared to the exonuclease-
deficient variant (Table 1) [17]. In addition, the fre-
quency of ENU-induced G A transition mutations
also did not differ between the exonuclease-proficient
and deficient forms of Klenow polymerase. We con-
clude that the Klenow 3
5
exonuclease activity
cannot discriminate between correct and incorrect
bases that are incorporated opposite DNA alkyl le-
sions, including O6 -alkylguanine lesions. Hence, no
selective removal of premutagenic intermediates is
performed by the Klenow exonuclease, and no re-
duction in mutation frequency is observed when the
exonuclease is present. A similar lack of proofread-
48 V. Khare, K.A. Eckert / Mutation Research 510 (2002) 4554

Table 1
DNA polymerase HSV-tk in vitro mutation frequencies in the absence and presence of alkylation damage to the templating strand
Polymerase (family) 3
5
exo HSV-tk mutation frequencya 104 Reference

Untreated 5 mM MNU 20 mM ENU

Klenow (A) + 4.2 120 26 [17,22]

24 85 27
D355A, E357A
T4 (B) + 2.4 110 46 [21]b
21 760 84
D324A
Pol -primase (B) 22 N.D.c 61 [17,22]
Pol (X) 24 650 130 [17,22]d
Single-stranded HSV-tk-containing DNA primed for DNA synthesis was used as a template by the indicated polymerase. The ssDNA was
either untreated or treated with an alkylating agent prior to DNA synthesis. The polymerase reaction products were rescued by hybridization
to a gapped duplex molecule, as described in [22]. The resulting heteroduplex plasmids were used to electroporate E. coli for mutational
analyses. Plating bacteria in the presence of chloramphenicol selects cells harboring plasmids that are progeny of the polymerase-synthesized
strand, while plating in the presence of 5-fluoro-2
-deoxyuridine (FUdR) selects for bacteria harboring HSV-tk gene mutations. Thus, the
HSV-tk mutant frequency is defined as the number of FUdR-resistant + chloramphenicol-resistant colonies divided by the total number of
chloramphenicol-resistant colonies.
a Values represent the mean of two or more independent determinations.
b K. Eckert, unpublished observations.
c N.D.: not determined.
d K. Eckert and S. Hamid, unpublished observations.

ing by the Klenow polymerase was observed by an approximately four-fold reduction in A C

others using an M13 in vitro assay and polymerase-
induced errors by 8-oxo-dGTP [23]. However, using
UV-irradiated DNA templates, a lower mutation
frequency was reported after DNA synthesis by
exonuclease-proficient Klenow polymerase, relative
to the exonuclease-deficient form [24].
The T4 DNA replication proteins are widely used
as a model for eukaryotic DNA replication, and the
T4 DNA polymerase is a model for the replicative
DNA polymerase [25]. Using the native T4 DNA
polymerase and exonuclease-deficient variants, we
observed a significant difference in the frequency of
MNU [17,21] and ENU-induced HSV-tk mutations
(Table 1). The alkylation-induced mutation frequency
for the exonuclease-deficient T4 polymerase was
similar to that observed for polymerase and poly-
merase (Table 1). The frequency of MNU-induced
G A transitions is decreased three-fold while other
alkyl-induced mutations are decreased up to 200-fold
by the presence of the T4 exonuclease activity [21].
These results suggest that the T4 exonuclease does
contribute to the avoidance of alkylation mutations.
Using 8-oxo-dGTP substrates, Pavlov et al. observed
a similar enhancement of T4 polymerase accuracy
by the presence of the T4 exonuclease activity and
transversions [23]. Overall, these studies demon-
strate that DNA lesions are subject to exonucleolytic
proofreading, but the efficiency is dependent on the
lesion as well as the polymerase-exonuclease studied.
Factors likely to be involved in exonuclease effi-
ciency include neighboring sequence context of the
lesion, stacking interactions, and thermostability of
the primer terminus. In addition, the inherent ability
of the polymerase to extend a lesion-containing base-
pair will also affect kinetic partitioning (Fig. 1) and
therefore the efficiency of the exonuclease activity.
3.2. Biochemical analyses of proofreading and
DNA lesions
The ability of an intrinsic exonuclease activity to
remove a base opposite a DNA lesion also has been
analyzed using biochemical assays. In this approach,
proofreading is defined as a differential rate of exonu-
cleolytic removal of incorrect and correct terminal
basepairs in the absence of dNTP substrates. We mea-
sured the exonuclease activity of T4 DNA polymerase
on various templates and demonstrated that O6 -
methylguanine (m6G) and abasic lesions-containing
DNA templates are substrates for the 3
5
exonu-
 V. Khare, K.A. Eckert / Mutation Research 510 (2002) 4554
clease activity of T4 DNA polymerase ([21]; Khare
and Eckert, manuscript in preparation). On control
templates, the 3
5
exonuclease activity of T4
DNA polymerase exhibits proofreading activity by
degrading terminal mispaired substrates (e.g. GT) at a
greater rate than correctly paired (e.g. GC) substrates.
We observed that the exonuclease activity degrades
both the m6GC and m6GT substrates at rates similar
to a GT mispaired substrate [21]. Moreover, the non-
mutagenic m6GC basepair and mutagenic m6GT mi-
spair were degraded similarly. These results show that
although m6G-containing templates are substrates for
the T4 exonuclease activity, this activity may not con-
tribute a significant proofreading function to DNA
polymerase errors, because both m6GC and m6GT
substrates are excised at a similar rate. On the other
hand, a report on DNA polymerase exonuclease
activity demonstrated the preferential removal of the
8-oxoguanine (8-oxoG)A mispair over the 8-oxoGC
basepair, consistent with a proofreading function. We
have examined the processing of templates containing
a defined abasic lesion, and found that the T4 ex-
onuclease preferentially removed dAMP and dGMP
opposite the lesion (Khare and Eckert, manuscript in
preparation). Similarly, the preferential removal of
purines (G > A) over pyrimidines opposite an abasic
lesion was reported for polymerase [26]. However,
studies of the Klenow polymerase failed to detect
any differences in the relative efficiency of exonu-
cleolytic removal of dAMP, dGMP, dTMP or dCMP
opposite a defined abasic lesion [27]. Together, the
genetic and biochemical studies demonstrate that the
T4 polymerase and polymerase associated 3
5

exonuclease activities are able to remove an incorrect
base incorporated opposite a template lesion. How-
ever, the contribution of this activity to the overall
accuracy of the enzyme depends upon the lesion, the
discrimination properties of the exonuclease, and the
relative rates of the competing polymerase activity
(Fig. 1). The consistent failure to detect proofreading
of lesions by the Klenow polymerase [17,23,27] may
be related to differences in the kinetic behavior and/or
the processivity of the Klenow exonuclease, relative
to that of the T4 exonuclease [28].
The exonuclease and polymerase domains uti-
lize distinct mechanisms of substrate discrimination.
DNA polymerase accuracy is determined, in part, by
the geometric discrimination of basepairs within the
49
active site. Physical determinations have shown that
the m6GC basepair is in an altered wobble geome-
try, while the mutagenic m6GT basepair simulates
WatsonCrick geometry [29], a fact that has been used
to explain the preference of polymerases for contin-
ued synthesis from the premutagenic m6GT interme-
diate [30]. In contrast, the exonuclease likely uses a
thermodynamic mechanism of substrate recognition.
The m6GC and m6GT basepairs are of equivalent
stability, and both are less stable than correct GC
[29]. Thus, the significant thermodynamic difference
for correctly paired and mispaired natural bases is
eliminated by formation of the m6G lesion, consistent
with our observation that proofreading does not oc-
cur by the T4 polymerase. In summary, DNA lesions
alter structural parameters of the DNA primer tem-
plate thereby affecting both the rate of exonucleolytic
excision and the rate of polymerization.
4. Kinetic partitioning on lesion-containing
templates
The earlier studies demonstrate that the exonu-
clease activity, in isolation (e.g. in the absence of
dNTP substrates), recognizes and removes lesion-
containing mispairs. Likewise, several studies of
DNA polymerase activity, in isolation (e.g. using
exonuclease-deficient polymerase derivatives), have
demonstrated that translesion synthesis occurs with
some probability. While these biochemical approaches
have provided insight into the molecular mechanisms
of lesion recognition and bypass, they are not rep-
resentative of the physiological state of polymerases
with intrinsic 3
5
exonuclease activity. In cells,
dNTP substrates are readily available to a fully func-
tional polymerase; thus, the polymerase and exonu-
clease activities will be functioning simultaneously
on the same lesion-containing DNA substrate. In this
situation, the important question is, who will win the
competition depicted in Fig. 1?.
To address this question, we have used a bio-
chemical approach that allows us to monitor kinetic
partitioning of the exonuclease-proficient T4 DNA
polymerase in the presence of all four dNTP sub-
strates [21]. Control reactions using DNA substrates
with natural base mispairs demonstrate that efficient,
full-length polymerase extension occurs only after
50 V. Khare, K.A. Eckert / Mutation Research 510 (2002) 4554

Fig. 2. Biochemical outcomes of kinetic partitioning using lesion-containing DNA templates. To biochemically examine kinetic partitioning,
lesion-containing DNA substrates (DNAn ) are incubated with DNA polymerase in the presence of dNTP substrates. The reaction products
are analyzed both quantitatively and qualitatively by polyacrylamide gel electrophoresis (right panels; lane a represents the starting DNA
substrate). Theoretically, three different outcomes are possible: (1) when kexo > kpol , DNA excision products will accumulate during the
DNA synthesis reaction (lane 1b); (2) when kexo and kpol are approximately equivalent, polymerase idling occurs, which is characterized
by no net DNA synthesis (lane 2b), in this situation, translesion synthesis is not observed; (3) when kpol > kexo , translesion synthesis
products accumulate during the reaction (lane 3b).

complete excision of the terminal mispair. Using
lesion-containing substrates, the sizes and amounts of
DNA products vary, depending on the relative rates
of the exonuclease and polymerase activities (Fig. 2).
4.1. DNA polymerase partitioning at
m6G lesions
Using m6G-containing substrates and T4 DNA
polymerase, we observed that excision products per-
sist throughout the course of the polymerase reaction
and predominate over extension products [21]. We
also observed the accumulation of DNA products
that represent idling, or repeated cycles of incorpo-
ration and excision of bases, at the lesion-containing
primer terminus (scenario 2, Fig. 2). However,
once translesion synthesis had occurred, we ob-
served no DNA polymerase idling at DNA templates
sites removed from the position of the lesion. The
continued partitioning of m6G substrates between
the exonuclease and polymerase active sites resulted
in dramatically less full-length translesion synthesis
product, relative to natural substrates, indicating that
the 3
5
exonuclease contributes to DNA synthesis
inhibition by alkylating agents.
These results may have biological significance for
the behavior of proofreading-proficient polymerases
at sites of m6G lesions during long patch DNA re-
pair synthesis. The m6G lesion has been suggested to
be cytotoxic when used as a substrate by MMR en-
zymes, in what has been termed a futile cycle [31].
In this model, DNA repair of the strand opposite the
lesion will produce an m6G-containing gapped DNA
substrate for either pol or pol in eukaryotic cells,
both of which contain an intrinsic 3
5
exonucle-
ase activity. The presence of the m6G lesion in this
gap will severely inhibit the efficiency of DNA repair
synthesis, consistent with our observation that <10%
of m6G-containing templates give rise to full-length
product by the exonuclease-proficient T4 polymerase.
Our observations provide a biochemical explanation
for how persistent gaps arise in genomic DNA from
methylated substrates.
4.2. DNA polymerase partitioning at
abasic lesions
We and others have observed that the T4 DNA
polymerase-associated and polymerase -associated
3
5
exonuclease activities preferentially remove
 V. Khare, K.A. Eckert / Mutation Research 510 (2002) 4554
purines opposite an abasic site. Interestingly, the same
nucleotide preference has been reported for the T4
polymerase activity, in which purines (A > G) are
inserted with greater efficiency than pyrimidines op-
posite abasic lesions [32]. Therefore, the exonuclease
is removing what the polymerase is preferentially
incorporating, demonstrating that the proofreading
exonuclease can counteract the polymerase bias for in-
corporation of nucleotides opposite a non-informatio-
nal lesion. However, the continued partitioning of the
enzyme between the polymerase and exonuclease ac-
tive sites, while providing a mechanism of mutation
avoidance, also prevents stable incorporation of nu-
cleotides opposite the lesion. DNA lesions that have
a high rate of exonuclease activity but poor rate of
DNA polymerase extension, such as the abasic site,
will be strongly inhibitory, as DNA excision products
will predominate in the presence of dNTP substrates
(scenario 1, Fig. 2). An important unknown biochem-
ical factor in this scenario is the degree to which
accessory replication proteins affect kinetic partition-
ing and TLS by exonuclease-proficient enzymes. In
this regard, Avkin et al. [33] have recently reported
efficient translesion replication of abasic lesions in
human tumor cell lines by a replicative DNA poly-
merase, evidence that other cellular components have
a profound affect on TLS.
4.3. The 3
5
exonuclease and translesion
synthesis in E. coli
Intracellular TLS is best understood in the E. coli
model system. Unlike eukaryotic polymerases, the
3
5
exonuclease activity associated with the repli-
cative polymerase in E. coli is contained within a
separate polypeptide, the subunit. This polypeptide
is tightly associated with the (polymerase) and
subunits to form the Pol III core polymerase. Early
genetic and biochemical studies of UV lesion pro-
cessing in E. coli demonstrated that the exonuclease
activity affects TLS by the replicative Pol III [18,34].
The 3
5
exonuclease of Pol III can be inhibited
by the presence of RecA protein and single-strand
binding protein or by the presence of mutations in
the dnaQ gene which encodes the subunit [35].
Neither genetic nor biochemical inhibition of the
3
5
exonuclease is sufficient to increase Pol III
bypass of UV-induced photoproducts [36]. However,
51
efficient TLS past TT cyclobutane dimers and
N-2-acetylaminofluorene lesions can occur if both the
proofreading activity of and the umuDC proteins are
absent [37,38]. The gene products of umuD interact
with the C-terminal domain of , but the biological
consequences of this interaction are unknown [39].
In E. coli where TLS is stimulated by SOS induc-
tion, it is tempting to speculate that inhibition of the
exonuclease activity could be an important parameter
for any TLS. Nevertheless, it has also been suggested
that limited exonuclease activity is required to process
nascent lesion-containing DNA chains and generate a
proper DNA primer-terminus for TLS [40].
5. Perspective: biological consequences of
3
5
exonuclease activity at DNA lesions
The biochemistry of DNA lesion tolerance and
translesion DNA synthesis in eukaryotic cells has
yet to be elucidated, and significant gaps in our
knowledge include the following: (1) the biochemical
signal initiating checkpoint activation, (2) the extent
to which replicative polymerases perform TLS, and
(3) the mechanism of switching from a replicative
to a specialized DNA polymerase for TLS. The bio-
chemical idling of replicative polymerases at DNA
lesions due to the competition between the 3
5

exonuclease and the polymerase (Fig. 1) may be an
important factor in signaling cell cycle checkpoint
responses to DNA damage (Fig. 3). Inactivation or
mild inhibition of the proofreading exonuclease may
lead to replicative polymerase TLS, whereas an ex-
aggerated kinetic partitioning at sites of damage may
result in the recruitment of a specialized polymerase
for TLS (Fig. 3).
Genetic studies in Saccharomyces cerevisiae have
linked the C-terminal domain of the exonuclease-
proficient, replicative DNA polymerase to DNA
damage signaling [41,42]. Biochemical factors that are
likely to be disrupted during polymerase/exonuclease
idling at DNA lesions include local dNTP pools and
the coordination of leading and lagging strand repli-
cation. Stalling or collapse of the replication fork can
lead to DNA strand breaks [43,44]. Although inhibi-
tion of the exonuclease activity has been proposed as
a mechanism by which TLS by the E. coli replicative
Pol III holoenzyme can occur, this pathway has not
52 V. Khare, K.A. Eckert / Mutation Research 510 (2002) 4554

Fig. 3. Possible biological outcomes of exonuclease-mediated DNA polymerase idling. Processing of damaged DNA by proofreading 3
5

exonucleases may generate biochemical signals leading to either cytotoxicity (apoptosis) or cell survival (lesion tolerance). Translesion
synthesis (TLS) may occur by either the replicative polymerase or by a specialized polymerase (see text for details).

been investigated in eukaryotic cells. Physical studies
of the RB69 polymerase/exonuclease have revealed
that partitioning of the DNA substrate between the
two active sites is accomplished by the movement of a
specific domain of the protein [13] Conceivably, dis-
ruption of this movement by the association of an ac-
cessory protein could be a mechanism to alter kinetic
partitioning in favor of the polymerase activity (kpol ,
Fig. 1) and thus allow replicative polymerase TLS.
The fate of an idling polymerase that is asso-
ciated with accessory proteins within a replicative
complex is unknown. In particular, there are no ex-
perimental data as to whether the polymerase pas-
sively dissociates at a DNA lesion (koff , Fig. 1) or
can be actively dissociated by specialized factors.
If the lesion-containing substrate transfer between
the polymerase and exonuclease active sites occurs
by an intramolecular pathway, as has been demon-
strated for natural DNA substrates [14,15], the rate
of passive dissociation may be very slow. In this
case, an active dissociation of the replicative poly-
merase may be required in order for a specialized
translesion polymerase to gain access to the DNA
substrate. Replication fork collapse at DNA lesions
has been described in E. coli, where SOS regulated
gene products are recruited to restart the replication
fork [43,44].
6. Conclusion
The fidelity of DNA replication is an important
determinant of mutation accumulation during neo-
plastic progression, and the 3
5
exonuclease has
 V. Khare, K.A. Eckert / Mutation Research 510 (2002) 4554
a fundamental role in reducing polymerase errors.
Transgenic mice containing a polymerase allele
carrying a mutation in the exonuclease domain ex-
hibit an increased incidence of cancer, consistent with
the generation of a mutator phenotype [45]. Exonu-
cleolytic proofreading of polymerase errors requires
two steps. First, the enzyme must discriminate be-
tween correctly paired and incorrectly paired bases,
and second, the enzyme must remove the 3
-terminal
basepair. The ability to discriminate between sub-
strates is essential for a proofreading function. In the
absence of discrimination at sites of DNA damage,
non-specific exonucleolytic degradation results from
continued substrate switching between the polymerase
and exonuclease active sites. This scenario has been
observed for the T4 DNA polymerase at m6G lesions
[21]. The m6G lesion alters the physical parameters
used for proofreading discrimination, such as a slow
rate of mispair extension by the polymerase and dif-
ferential thermostabilities of basepairs and mispairs
at the primer terminus. Alternatively, the ability of
the exonuclease to discriminate correct and incorrect
lesion-containing basepairs can result in the avoidance
of lesion-induced DNA polymerase errors [21,23]. In
case of the abasic site, the preference of adenine or
purine nucleotides by both the exonuclease and poly-
merase activities prevents the stable incorporation of
a nucleotide opposite the lesion.
Mutations arise and accumulate in the presence of
mechanisms to maintain the integrity and fidelity of
the genome. The exonucleolytic proofreading path-
way is an important mechanism that acts as a first
line of defense against error accumulation once a cell
has entered into chromosomal DNA replication. The
exonuclease can repair a subset of the potential
mutagenic errors caused by DNA lesions, and also
can act as a barrier to translesion DNA synthesis.
The exonuclease thus contributes significantly to the
quality control of DNA replication and consequently
in the quality of life.
Acknowledgements
Work in the authors laboratory was supported by
The American Cancer Society (RPG 95-075-CNE),
the American Association for Cancer Research, and
the Pennsylvania State University Four Diamonds
53
Fund. We thank members of the Eckert laboratory for
critical reading of the manuscript.
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