Reversal of a Mutator Activity by a Nearby Fidelity-Neutral Substitution in the RB69 DNA Polymerase Binding Pocket

Abstract

Phage RB69 B-family DNA polymerase is responsible for the overall high fidelity of RB69 DNA synthesis. Fidelity is compromised when conserved Tyr567, one of the residues that form the nascent polymerase base-pair binding pocket, is replaced by alanine. The Y567A mutator mutant has an enlarged binding pocket and can incorporate and extend mispairs efficiently. Ser565 is a nearby conserved residue that also contributes to the binding pocket, but a S565G replacement has only a small impact on DNA replication fidelity. When Y567A and S565G replacements were combined, mutator activity was strongly decreased compared to that with Y567A replacement alone. Analyses conducted both in vivo and in vitro revealed that, compared to Y567A replacement alone, the double mutant mainly reduced base substitution mutations and, to a lesser extent, frameshift mutations. The decrease in mutation rates was not due to increased exonuclease activity. Based on measurements of DNA binding affinity, mismatch insertion, and mismatch extension, we propose that the recovered fidelity of the double mutant may result, in part, from an increased dissociation of the enzyme from DNA, followed by the binding of the same or another polymerase molecule in either exonuclease mode or polymerase mode. An additional antimutagenic factor may be a structural alteration in the polymerase binding pocket described in this article.

Full text

Reversal of a Mutator Activity by a Nearby
Fidelity-Neutral Substitution in the RB69 DNA
Polymerase Binding Pocket
Anna Trzemecka
1
, Agata Jacewicz
1
, Geraldine T. Carver
2
,
John W. Drake
2
and Anna Bebenek
1
⁎
1
Department of Molecular Biology, Institute of Biochemistry and Biophysics, Polish Academy of Sciences,
Pawinskiego 5a, 02-104 Warsaw, Poland
2
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park,
NC 27709, USA
Received 29 July 2010;
received in revised form
27 September 2010;
accepted 28 September 2010
Available online
13 October 2010
Edited by M. Gottesman
Keywords:
Exo;
Pol
Phage RB69 B-family DNA polymerase is responsible for the overall high
fidelity of RB69 DNA synthesis. Fidelity is compromised when conserved
Tyr567, one of the residues that form the nascent polymerase base-pair
binding pocket, is replaced by alanine. The Y567A mutator mutant has an
enlarged binding pocket and can incorporate and extend mispairs
efficiently. Ser565 is a nearby conserved residue that also contributes to
the binding pocket, but a S565G replacement has only a small impact on
DNA replication fidelity. When Y567A and S565G replacements were
combined, mutator activity was strongly decreased compared to that with
Y567A replacement alone. Analyses conducted both in vivo and in vitro
revealed that, compared to Y567A replacement alone, the double mutant
mainly reduced base substitution mutations and, to a lesser extent,
frameshift mutations. The decrease in mutation rates was not due to
increased exonuclease activity. Based on measurements of DNA binding
affinity, mismatch insertion, and mismatch extension, we propose that the
recovered fidelity of the double mutant may result, in part, from an
increased dissociation of the enzyme from DNA, followed by the binding of
the same or another polymerase molecule in either exonuclease mode or
polymerase mode. An additional antimutagenic factor may be a structural
alteration in the polymerase binding pocket described in this article.
© 2010 Elsevier Ltd. All rights reserved.
Introduction
The DNA polymerases (gp43 s encoded by gene 43)
of the related bacteriophages T4 and RB69 synthesize
DNA with high fidelity, inserting one wrong nucle-
otide per 10
5
replicated bases.
1
These polymerases
belong to the B-family—the same family that is
populated by the eukaryotic replicative polymerases
α,δ,andɛ. For RB69 DNA polymerase, crystal
structures are available for the apo enzyme,
2
abinary
editing complex with DNA bound in the exonuclease
domain,
3
and ternary complexes (replicating com-
plex) with DNA and dNTP bound in the polymerase
site.
4
Because of the availability of crystal structures in
so many conformations, RB69 polymerase is a good
structural model for studying the mechanisms re-
sponsible for high base selectivity.
RB69 DNA polymerase achieves its fidelity by the
coordinated action of two activities: a polymerase
(Pol) activity that is responsible for correct
*Corresponding author. E-mail address:
aniab@ibb.waw.pl
Abbreviations used: dsDNA, double-stranded DNA;
ssDNA, single-stranded DNA.
doi:10.1016/j.jmb.2010.09.058 J. Mol. Biol. (2010) 404, 778–793
Contents lists available at www.sciencedirect.com
Journal of Molecular Biology
journal homepage: http://ees.elsevier.com.jmb
0022-2836/$ - see front matter © 2010 Elsevier Ltd. All rights reserved.

nucleotide selection and an exonuclease (Exo)
activity that removes mismatched nucleotides from
the primer terminus. As with all polymerases that
replicate DNA with high fidelity, binding the correct
incoming dNTP introduces conformational changes
in both the enzyme and the DNA. The fingers
subdomain undergoes the largest conformational
change, moving toward the palm subdomain to
enclose the incoming dNTP and the complementary
template nucleotide and thus to generate a binding
pocket for the nascent base pair.
4–7
In the RB69 polymerase, the active Pol binding site
is formed by several highly conserved residues
located in the fingers and palm. Arg482 and Lys560
in the fingers are involved in hydrogen bonding to
the phosphate groups of the incoming dNTP.
Hydrophobic interactions with the nascent base
pair are formed by Leu415 and Tyr416 on the minor
groove side and by Tyr567 and Gly568 on the
template side. Asn564 and Ser565 form the rear wall
of the binding pocket and ensure a coplanar base
arrangement of the nascent base pair (M. Wang et al.,
unpublished results).
1,4,8
All of these residues play important roles in RB69
DNA replication fidelity. Leu415 and Tyr567 are
critical for substrate discrimination. L415F/G mutants
have elevated rates for a variety of base substitution
errors and single-base deletions in repetitive and
nonrepetitive sequences.
8
Replacing Tyr567 by Ala,
Ser, or Thr confers a strong mutator phenotype,
particularly for base substitution errors.
1
Tyr416 is
responsible for discriminating between ribose moiety
and deoxyribose moiety in incoming nucleotides,
9
while Leu561 discriminates sterically against mis-
matches in the nascent base-pair binding pocket.
10
Thus, the size and shape of the polymerase binding
pocket play crucial roles in replication fidelity.
Insertion of a wrong nucleotide compromises the
rate of primer extension. The polymerase stalls,
altering the balance between extension by the poly-
merase and excision by the mismatch-editing exonu-
clease. Newly incorporated mismatches reduce the
efficiency of subsequent nucleotide insertions and
extensions by 10
2
-fold to 10
6
-fold,
6,7,11,12
with the
magnitude depending on the mismatch and the
polymerase.
Polymerases with compromised fidelity due to
alterations at the Pol site may display reduced
partitioning to the Exo site and thus favor mismatch
extension over proofreading, notably with the T4
Pol
L412M
and RB69 Pol
L415F/G
mutants.
8,13
The same
mechanism probably applies to the RB69 Pol
Y567A
mutant, as previously proposed.
1
Conversely, T4
polymerase mutants (such as T4
A737V
) that display
an antimutator phenotype, mainly reducing
A·T→G·C rates, tend to display a decreased ability
to translocate after nucleotide incorporation, thus
favoring the formation of exonuclease complexes
and increasing the efficacy of proofreading.
14,15
In
preliminary screens, we observed that the RB69
S565G replacement had only weak impacts on
fidelity; however, surprisingly, when the Y567A
and S565G replacements were combined within the
highly conserved B-motif KX
3
NSXYG, the powerful
mutator activity of the Pol
Y567A
mutation was
strongly reduced. To better understand the role of
Ser565 in fidelity, we characterized some of the
biochemical properties of RB69 polymerase mutants
containing S565G replacement, Y567A replacement,
or both replacements. We also investigated the
impact of these replacements using fidelity assays
both in vivo and in vitro. Our results led us to
propose that introducing the S565G replacement
lowers the DNA binding affinity of the polymerase
and increases its ability to dissociate from the
primer/template, thus increasing the opportunity
for proofreading by the same or another polymerase
molecule (proofreading in trans) and contributing to
the antimutagenic impact of S565G on Y567A.
Results
Bacteriophage RB69 gp43 Y567 and S565 are
highly conserved residues within the B-family of
DNA polymerases and are located in the region
involved in dNTP binding. We showed previously
that Y567 plays a crucial role in base selection:
when Y567 was replaced with A, S, or T, the
mutant polymerase displayed severely decreased
fidelities both in vivo and in vitro.
1
To study the role
of S565, we replaced it with G in both Exo
+
and
Exo
−
backgrounds. We also examined the combi-
natorial effect of the double replacement Y567A/
S565G. Most of the assays were performed using
Exo
+
derivatives because of the surprising anti-
mutator effect of the double replacement in this
background.
Polymerase activity in vivo and specific activity
in vitro
In assays in vivo, we often used a hybrid system in
which T4, whose own gp43 is inactivated by amber
mutations at codons 202 and 386 (T4 43amam), is
replicated by wild-type or mutant RB69 gp43 s
expressed from plasmids.
1
Polymerase activity in
vivo was determined only for Exo
+
derivatives.
DNA synthesis by T4 gp43 was totally blocked by a
43amam mutation and performed instead by one of
the RB69 DNA polymerases expressed from a
plasmid. DNA synthesis was measured in infected
Escherichia coli cells at a time when host DNA
synthesis had ceased completely.
1
DNA synthesis
was moderate in the presence of S565G either alone
(30% of wild-type activity) or in combination with
Y567A (40% of wild-type activity), while the Y567A
mutant retained 50% activity.
779Suppression of a Mutator DNA Polymerase

We also measured polymerase-specific activity in
vitro for the RB69 DNA polymerases using activated
DNA as substrate. The specific activities for the Exo
+
versions of Pol
Y567A
, Pol
Y567A/S565G
, and Pol
S565G
were 80%, 72%, and 47%, respectively, of the Pol
+
Exo
+
value.
Fidelity in vivo
In rII reversion assays (Table 1), RB69 Pol
S565G
has
almost negligible impact on replication fidelity for
both base substitutions and frameshifts; the average
change in mutation rates was a 2-fold increase
compared to the wild-type enzyme. Similarly, in rI
forward mutation assays (Table 2), Pol
S565G
in Exo
+
background had a mutation rate elevated by a factor
of 5 compared to the wild-type enzyme; in Exo
−
background, no significant difference (rate decreased
0.8-fold) was observed between parental enzymes
and mutant enzymes. Surprisingly, the double
mutant (Pol
S565G/Y567A
Exo
+
) had strongly decreased
mutator activities compared to the Pol
Y567A
Exo
+
mutant in rII reversion assays: the second replace-
ment (at S565) suppressed most of the mutator traits
of the Y567A replacement. The most striking results
were the 90-fold decrease in base substitution
mutation rates at the UV375 A·T site and the 60-fold
decrease in base substitution mutation rates at the
UV256 G·C→A·T site, while −1 and +1 frameshift
mutations were reduced by 50-fold and 6-fold,
respectively, at other sites (Table 1). In rI forward
mutation assays, the Pol
S565G/Y567A
double mutant
decreased mutation rates by 40-fold compared to the
single Pol
Y567A
mutant (Table 2). In Exo
−
background,
the Pol
S565G/Y567A
mutant did not support the
production of viable T4 43amam progeny, perhaps
because of a lethally high mutation rate from the
combination of the mutator effects of the Y567A
replacement and the Exo deficiency
1,16
—acombina-
tion that the S565G mutation may reduce insufficiently
to restore viability.
We sequenced independent rI mutants from experi-
ments in which T4 43amam phage was replicated by
the RB69 polymerase mutants Pol
S565G
Exo
+
and
Pol
S565G/Y567A
Exo
+
, respectively. The kinds of muta-
tions made by these polymerases are shown in
Table 3, together with the kinds obtained previous-
ly for the Pol
+
and Pol
Y567A
enzymes.
1,16
Mutations
produced by the Pol
S565G
Exo
+
polymerase were
very similar to those produced by the Pol
+
Exo
+
polymerase; the complex mutations were almost all
GCG→CTA at the 146–148 hot spot.
16–18
There
were no strong differences between the kinds of
mutations produced by Pol
S565G/Y567A
Exo
+
poly-
merases and the kinds of mutations produced by
Pol
Y567A
Exo
+
polymerases, each making predom-
inantly base substitutions. G→A predominated
among transition mutations, with as much as 70%
localized at a hot spot at rI base 247. C→Tand
C→A mutations at another hot spot at rI base 202
were also present in the spectra produced by both
Table 1. Mutation rates for RB69 Exo
+
DNA polymerase mutants in rII reversion assays
rIItester mutation
tester mutation
Mutation
scored
Reversion rate× 10
8
Pol
+
[17] Pol
S565G
[6–7] Pol
Y567A
[7] Pol
S565G/Y567A
[13–14]
131 +1 9.0 (1) 4.3 (0.5) 37 (4.0) 5.6 (0.6)
UV232 −1 1.0 (1) 3.3 (3.3) 210 (210) 4.4 (4.5)
UV256 G·C→A·T 6.4 (1) 15 (2.4) 7200 (1100) 130 (20)
UV375 A·T→any 1.6 (1) 0.5 (0.3) 2600 (1700) 28 (18)
Values in brackets are the numbers of stocks providing the median rate. Values in parentheses below each rate are relative to those for
Pol
+
. The entries for Pol
+
and Pol
Y567A
were adapted from a previous article.
1
Table 2. Forward mutation rates of RB69 DNA
polymerase mutants in vivo
Polymerase
Number of
stocks
Mutation
rate× 10
5
Relative
rate
Pol
+
Exo
+
7 0.43 1
Pol
S565G
Exo
+
7 2.1 5
Pol
Y567A
Exo
+
7 210 490
Pol
S565G/Y567A
Exo
+
11 5.5 13
Pol
+
Exo
−
7 220 510
Pol
S565G
Exo
−
7 180 430
The entries for Pol
+
Exo
+
, Pol
+
Exo
−
, and Pol
Y567A
Exo
+
were
adapted from previous articles.
1,14
Table 3. Classes of rI mutations generated by Exo
+
RB69
DNA polymerase mutants
Polymerase
Pol
+
Pol
S565G
Pol
Y567A
Pol
S565G/Y567A
Number of mutations 103 53 79 45
A·T→G·C 7 0 25 5
G·C→A·T 22 6 43 27
Transitions 29 6 68 32
A·T→T·A 0 2 0 0
A·T→C·G 2 0 2 3
G·C→T·A 34 22 8 7
Transversions 36 24 10 10
±1 10 6 1 2
≥±2 7 2 0 0
Complex
GCG→CTA
21 15 0 1
The Pol
+
and Pol
Y567A
entries were adapted from previous
articles.
1,17
780 Suppression of a Mutator DNA Polymerase

Pol
Y567A
Exo
+
and Pol
S565G/Y567A
Exo
+
polymerases
(Fig. 1). Only A→G mutations were underrepre-
sented in this spectrum, with only one appearing,
while 11 were found in the Pol
Y567A
Exo
+
spectrum
(Table 3). Thus, the decreased mutation rates in the
double mutant hardly affected specificity but
merely lowered the overall mutation rate.
Fidelity in vitro
To learn more about the role of Ser565 in fidelity, we
conducted studies in vitro using the lacZαDNA
template in gapped M13mp2 DNA (Table 4). The
lacZαmutant frequencies for the Pol
+
Exo
+
and
Pol
S565G
Exo
+
mutants were 8× 10
−4
and 4×10
−4
,
respectively, with the values indistinguishable from
each other, similar to the 5× 10
−4
to 7× 10
−4
frequen-
cies typically obtained with gapped DNA,
16,19
and
representing only the mutations that accumulated
during the growth of the M13 phage stock. The lacZα
mutant frequency for the Pol
Y567A
Exo
+
mutant in
earlier studies was 55× 10
−4
, and we observed a 5-fold
decrease in mutation frequency when the DNA was
copied by the Pol
S565G/Y567A
Exo
+
polymerase. This
frequency (10× 10
−4
) was close to that of the Pol
+
Exo
+
polymerase. These results were fully consistent
with those obtained in our fidelity studies conducted
in vivo.
The lacZαmutant frequency for the Pol
S565G
Exo
−
polymerase was 14×10
−4
, modestly lower that the
29× 10
−4
frequency obtained previously with the Pol
+
Exo
−
enzyme. The Pol
S565G/Y567A
Exo
−
polymerase
was more proficient in proofreading some errors than
was the Pol
Y567A
Exo
−
polymerase (Table 4;89×10
−4
versus 164× 10
−4
). However, the Pol
S565G/Y567A
Exo
−
polymerase retained a high mutation frequency,
which probably resulted in lethally mutated progeny
during growth in vivo.
The contribution of S565G to the fidelity of
Pol
S565G/Y567A
As previously reported,
16
the RB69 Pol
Y567A
Exo
+
polymerase is a strong base substitution mutator in
vitro, mostly for transition mutations (especially for
T→C produced by T·G mispairs) but also for
frameshift mutations in homopolymeric runs,
while frameshift mutations in runs were almost
absent in vivo.ThePol
S565G/Y567A
Exo
+
double
mutant also produced base substitutions, again
PolS565G/Y567A Exo+
T A T A
ATGGCCTTAAAAGCAACAGCACTTTTTGCCATGCTAGGATTGTCATTTGTTTTATCTCCATCGATTGAAGCGAATGTCGATCCTCATTTTGATAAATTTA 100
PolY567A C T + C T C G
Exo+ C
A
A +
T T A + T CTA C
TGGAATCTGGTATTAGGCACGTTTATATGCTTTTTGAAAATAAAAGCGTAGAATCGTCTGAACAATTCTATAGTTTTATGAGAACGACCTATAAAAATGA 200
C AG A C G C A C
C A G C
C
AAAAA
T AAA
AT AAA
AT C A
AT C A A
ATAC A AT G
CCCGTGCTCTTCTGATTTTGAATGTATAGAGCGAGGCGCGGAGATGGCACAATCATACGCTAGAATTATGAACATTAAATTGGAGACTGAATGA 294
ATACA A G T C A AT T G G G
AT C G T AT T G
T C A G
T C AAAAA
TT AAAAA
TTT AAAAA
AAAAAA
Fig. 1. rI mutational spectra for the RB69 Pol
S565G/Y567A
Exo
+
and Pol
Y567A
Exo
+
polymerases. The sequence
represents the wild-type rI strand complementary to the coding strand, with every 10th base in boldface. Upper-case
letters indicate base substitutions. The addition of a single base is indicated by “+.”The underlined three-base
mutation at residues 146–148 is a hot spot for complex mutations. The entries for Pol
Y567A
were adapted from a
previous article.
1
Table 4. Fidelities of RB69 polymerases in lacZαforward
mutation tests in vitro
Pol Exo MF× 10
4
Mutations
Base
substitutions −1 Other
Pol
+
+ 8.4 22 20 2 —
Pol
S565G
+ 3.6 ————
Pol
Y567A
+ 55 147 110 37 —
Pol
S565G/
Y567A
+ 10.4 100 55 31 14
Pol
+
−29 159 93 40 26
Pol
S565G
−14 48 24 15 9
Pol
Y567A
−164 154 120 28 6
Pol
S565G/
Y567A
−89 103 75 25 3
MF, mutation frequency. Mutations are numbers detected by
sequencing; mutants from the Pol
S565G
Exo
+
polymerase were not
sequenced because the mutant frequency was at the historical
background for uncopied DNA. “Other”mutations consisted of
larger deletions and a few complex mutations.
The data for Pol
+
and Pol
Y567A
were adapted from a previous
article.
17
781Suppression of a Mutator DNA Polymerase

mostly transition mutations. This polymerase also
produced frameshift mutations and large deletions
not seen in the Pol
Y567A
Exo
+
spectrum (Table 5).
Among 12 large deletions, 11 (of 27–371 nucleotides)
occurred between direct repeats. Most of the
frameshift mutations occurred at runs of two or
more repeated bases.
The roster of mutational classes showed a
significant decrease in T →C transitions with the
double replacement compared to that with
Pol
Y567A
alone. There were at least five sequence
contexts where T →C mutations either were absent
or appeared only once in the double-mutant
spectrum but were strongly represented in the
single-mutant spectrum (Fig. 2a). Overall, base
substitution rates decreased by 14-fold in the
double mutant, with the decrease in T·dGMP
rates being as much as 35-fold compared to the
Pol
Y567A
polymerase (Table 5). The other transition
mutation rates decreased 7-fold for C·dATP, 8-fold
for G·dTMP, and 5-fold for A·dCMP. Transversion
mutations were less frequent in the spectra of both
polymerases. A·dAMP rates decreased 14-fold,
G·dGMP rates decreased 7-fold, and C·dTMP rates
decreased 6-fold; for the remaining mismatches, the
mutation rates decreased 3-fold to 1.5-fold (Table 5).
Frameshift mutation rates decreased about 8-fold in
thedoublemutantcomparedtothesinglemutant.
We did not sequence lacZαmutants from the
Pol
S565G
Exo
+
polymerase background because the
mutation frequency (4× 10
−4
) for this polymerase was
at the level of the historical background frequency
(5.7×10
−4
to 6.2×10
−4
) for unfilled template DNA.
This low mutation frequency probably recorded the
intrinsic mutation frequency resulting from phage
M13 replication and made further analysis of the
Pol
S565G
Exo
+
polymerase unreliable.
The contribution of proofreading to the fidelity of
the Pol
S565G/Y567A
Exo
+
polymerase
The lacZαmutation frequency for the Pol
S565G/Y567A
Exo
−
polymerase (89× 10
−4
) was 6-fold higher that
that for the Pol
S565G
Exo
−
polymerase (14× 10
−4
)and
about 2-fold lower than that for the Pol
Y567A
Exo
−
mutant (164× 10
−4
). The Pol
S565G/Y567A
Exo
−
poly-
merase had reduced nucleotide selectivity compared
with the parental Pol
+
Exo
−
polymerase, but was less
error prone than the Pol
Y567A
Exo
−
polymerase.
Proofreading improved fidelity 8-fold in the
Pol
S565G/Y567A
Exo
+
polymerase but only 3-fold in
the Pol
Y567A
Exo
+
polymerase (Table 4). Proofreading
in the Pol
S565G/Y567A
double mutant removed both
base mismatches and frameshift mutations. The error
rates for all 12 possible base mismatches over all
detectable sites in the lacZαtemplate and for Exo
+
and
Exo
−
single and double Pol mutants are presented in
Table 5.InExo
−
background, the spectra for the single
and double mutants showed no striking differences,
and the distribution of mutations along the lacZα
sequence revealed no significant differences between
the two enzymes (Fig. 2b and c).
Table 5. RB69 DNA polymerase mutation specificities in lacZαforward mutation tests in vitro
Mutations
Pol
Y567A
Exo
+
Pol
S565G/Y567A
Exo
+
Pol
+
Exo
−
Pol
S565G
Exo
−
Pol
Y567A
Exo
−
Pol
S565G/Y567A
Exo
−
147 100 159 48 154 103
nμnμnμnμnμnμ
Base substitutions 110 47 55 3.4 93 17 24 4.4 120 160 75 78
A→G (A·dCMP) 2 6 3 1.3 1 1 2 2.5 0 ≤97 ≤7
G→A (G·dTMP) 7 18 6 2.2 8 9 2 2.1 54 420 20 122
C→T (C·dAMP) 17 38 17 5.4 45 42 9 8.5 12 82 1 37
T→C (T·dGMP) 76 150 15 4.3 13 11 4 3.4 37 230 33 158
Transitions 102 60 41 3.5 67 17 17 4.3 103 187 61 85
A→C (A·dGMP) 0 ≤3 2 0.9 1 1 0 ≤1.4 1 10 1 8
A→T (A·dAMP) 2 5 0 ≤0.35 1 1 1 1.2 2 15 2 12
T→G (T·dCMP) 0 ≤20≤0.35 0 ≤10≤10≤75 29
T→A (T·dTMP) 2 7 5 2.5 0 ≤10≤1.5 8 85 4 33
G→T (G·dAMP) 1 2 4 1.3 15 14 4 3.8 2 14 1 5
G→C (G·dGMP) 2 6 2 0.8 7 8 1 1.2 4 34 1 6
C→A (C·dTMP) 1 3 1 0.47 1 1 0 ≤1.4 0 ≤10 1 7
C→G (C·dCMP) 0 ≤5.8 0 ≤0.9 1 3 1 2.6 0 ≤19 0 ≤15
Transversions 8 2.9 14 0.7 26 4.1 7 1.1 17 19 15 13
± 1 37 10 31 1.3 40 5 15 1.8 28 24 25 17
−(residues 2–436) 0 ≤55 12 96 23 540 8 189 5 850 3 401
Other 0 ≤55 2 16 3 71 1 27 1 170 0 ≤134
Mutation rates (μ) are incorporated per 10
6
nucleotides. “≤”values were calculated as if one mutant had been detected. Mutation rates for
the Pol
+
Exo
+
and Pol
S565G
Exo
+
polymerases are not displayed because of the lack of signals significantly above the historical
background. Values for Pol
Y567A
Exo
+
, Pol
+
Exo
−
, and Pol
Y567A
Exo
−
were adapted from a previous article.
16
The number of detectable
sites is not defined for deletions N1 nt, and their mutation rates are therefore not normalized to the number of opportunities and thus only
appear to be larger than other normalized values.
782 Suppression of a Mutator DNA Polymerase

Exonuclease activities of single and double Pol
mutants
One possible explanation for the observed de-
crease in mutation rates in the Pol
S565G/Y567A
mutant
would be increased exonuclease activity. We there-
fore conducted exonuclease assays on substrates
with either a correct base pair or a T·dGMP
mismatch at the 3′terminus to compare the various
polymerase variants. However, both Pol
S565G
and
Pol
S565G/Y567A
polymerases were less efficient than
the wild-type and Pol
Y567A
polymerases in
Fig. 2. lacZαmutational spectra. The 5′→3′sequence of the viral template strand of the lacZαsequence in M13mp2 is
shown from position −84 through position + 197, where + 1 is the first transcribed base and every 10th base is underlined.
Upper-case letters indicate base substitutions. The deletion of a single base is indicated by “Δ,”whereas the addition of a
single base is indicated by “+.”Large deletions and complex mutations are not shown. (a) Spectra for Pol
Y567A
Exo
+
(above the lacZαsequence) and Pol
S565G/Y567A
Exo
+
(below the lacZαsequence). The five lacZαbase triplets in red are
positions where significant differences were observed between the two spectra. (b) Spectra for Pol
Y567A
Exo
−
(above) and
Pol
S565G/Y567A
Exo
−
(below). (c) Spectra for Pol
S565G/567A
Exo
+
(above) and Pol
S565G/Y567A
Exo
−
(below). The spectra for
Pol
Y567A
were adapted from a previous article.
17
783Suppression of a Mutator DNA Polymerase

degrading double-stranded DNA (dsDNA) with
either a correct primer terminus base pair or an
incorrect primer terminus base pair (Fig. 3a and b).
The exonuclease activity on single-stranded DNA
(ssDNA) was indistinguishable among the Pol
+
,
Pol
Y567A
, and Pol
S565G/Y567A
polymerases, but was
slightly reduced in the Pol
S565G
polymerase (Fig. 3c).
DNA binding activity
Decreased exonuclease activity may result from a
change in the dynamic equilibrium between the
formation of complexes at the Pol active site and the
formation of complexes at the Exo active site.
13
A
mismatch at the 3′terminus usually increases Exo
activity by weakening Pol binding to the substrate.
7
Using gel retardation assays, we measured the
strength of dsDNA binding for Pol
+
,Pol
Y567A
,
Pol
S565G
, and Pol
S565G/Y567A
polymerases in Exo
+
backgrounds with either a normal terminal base pair
or a terminal T·dGMP mismatch using the same
substrates as for the exonuclease assays (Fig. 4aand
b). K
d(DNA)
values were then determined from
reciprocal plots of enzyme concentration (Table 6).
The Pol
S565G
polymerase had the highest dissociation
constants when binding correctly paired DNA
(K
d(DNA)
=38.9 nM) or the mismatched substrate (K
d
(DNA)
=132 nM). These dissociation constants were
about 6-fold higher than for the Pol
+
polymerase and
3-fold higher than for the Pol
Y567A
polymerase on
normal dsDNA, and 3-fold higher for the Pol
+
polymerase and 5-fold higher for the Pol
Y567A
polymerase on dsDNA with the mismatch. The
Pol
S565G/Y567A
polymerase also bound less strongly
to both substrates compared to the Pol
+
and Pol
Y567A
polymerases (K
d(DNA)
=29.9 nM for normal dsDNA
and K
d(DNA)
=79.9 nM for mismatched dsDNA,
compared with K
d(DNA)
=6.9 nM and K
d(DNA)
=
13.2 nM for Pol
+
and Pol
Y567A
polymerases on
correctly paired dsDNA, and K
d(DNA)
=38.2 nM and
K
d(DNA)
=25.7 nM for Pol
+
and Pol
Y567A
polymerases
on mismatched dsDNA, respectively).
Weaker DNA binding in the Pol site does not
correlate with decreased dsDNA exonuclease activ-
ity for the Pol
S565G
or Pol
S565G/Y567A
polymerase.
Thus, both enzymes probably dissociate from either
normal or mismatched primer/template substrates
more often that do either the Pol
+
polymerase or the
Pol
Y567A
polymerase.
Primer-extension activities
The error rate of the Pol
S565G/Y567A
Exo
+
polymer-
ase is decreased for almost all mispairs compared to
the Pol
Y567A
Exo
+
polymerase. These differences are
also observed in Exo
−
background and thus may not
simply reflect partitioning ratios between the Pol sites
and the Exo sites. Analysis of mutation spectra for the
Pol
S565G/Y567A
Exo
+
and Pol
Y567A
Exo
+
polymerases
showed that, among transition mutations, the biggest
difference was in extending T·dGMP mismatches
(Table 5). Therefore, we used standing-start and
running-start assays to measure the abilities of the
Pol
+
,Pol
Y567A
, and Pol
S565G/Y567A
polymerases (all in
Exo
+
backgrounds) to insert dGTP opposite T and to
extend the T·dGMP mismatch.
The Pol
+
and Pol
S565G
polymerases misinsert
dGTP inefficiently opposite a template T (Fig. 5)
and also extend a T·dGMP mismatch inefficiently
(Fig. 6). The Pol
Y567A
polymerase both generates
and extends T·dGMP mismatches more efficiently
(Figs. 5 and 6). The Pol
S565G/Y567A
polymerase is
Fig. 2 (continued)
784 Suppression of a Mutator DNA Polymerase

much less efficient in both insertion and extension
(Figs. 5 and 6). The same pattern was observed in
running-start assays: the Pol
+
and Pol
S565G
poly-
merases were unable to detectably insert dGTP
opposite template T. The Pol
Y567A
polymerase both
inserted and extended a mismatch, while the
Pol
S565G/Y567A
polymerase was again much less
efficient in both steps (Fig. 7). The Exo
−
variants of
the Pol
+
, Pol
S565G
, and Pol
S565G/Y567A
polymerases
were similarly less efficient in both activities, in
contrast to the Pol
Y567A
polymerase, which inserted
and extended the T·dGMP mismatch efficiently
(data not shown).
The Pol
S565G/Y567A
Exo
−
polymerase supports
the replication of T4 phage in vivo
It was reported previously that the Pol
Y567A
Exo
−
polymerase was unable to support the in vivo growth
of either T4 43amam or T4 43
+1
. This dominant-lethal
phenotype was ascribed to the low fidelity of DNA
synthesis conducted by this enzyme, resulting in
Fig. 3. Exonuclease activities of RB69 DNA polymerases on ssDNA using a
32
P-labeled 20-mer, on normal dsDNA
using a
32
P-labeled 20-mer/35-mer, and on dsDNA containing a terminal G·T mismatch using a
32
P-labeled 20-mer/35-
mer. Reactions were incubated at 37 °C for 10 s, 20 s, 40 s, 80 s, and 300 s. Products of degradation were analyzed by
electrophoresis on a 15% polyacrylamide gel with 7 M urea, followed by autoradiography. Exonuclease assays on (a)
normal dsDNA, (b) dsDNA with a terminal G·T mismatch, and (c) ssDNA.
785Suppression of a Mutator DNA Polymerase

lethally mutated progeny phage. In the complemen-
tation assay, the Pol
S565G/Y567A
Exo
−
polymerase is
also unable to support the growth of T4 43amam but
can support the growth of T4 43
+
(Fig. 8), suggesting
that Pol
+
can compete sufficiently with Pol
S565G/Y567A
to produce some viable progeny whose plaques are,
however, small compared to T4 plaques growing on
BB cells harboring a plasmid expressing Pol
+
Exo
−
or
Pol
S565G
Exo
−
.
Discussion
Reversal of a powerful mutator activity by a
nearby fidelity-neutral substitution
The phage RB69 DNA polymerase mutant Pol
Y567A
displays a powerful mutator activity that can exceed
10
3
-fold at some sites.
1,16
In the crystalstructure of the
RB69 DNA polymerase ternary complex, Y567 dis-
plays an unfavorable rotamer conformation that is
maintained by hydrogen-bonding interactions with
two nearby residues.
20
These interactions are essential
for forming a nucleotide binding pocket. The aromatic
phenyl ring of Y567 plays an important role in base
discrimination, forming a hydrogen bond with the
minor-groove edge of the DNA duplex at the primer
terminus and thus helping to check Watson–Crick
base-pair geometry. Removing the γ-hydroxyl from
Y567 (Y567F) disrupts the hydrogen-bonding net-
work, allows Y567F to adopt a more favorable
rotamer conformation that distorts the nucleotide
binding pocket, reduces the affinity of incoming
dNTP, and blocks DNA replication.
20
On the other
hand, replacing the phenolic side chain with a methyl
group (Y567A) produces a vigorous polymerase that
is a powerful base substitution mutator both in vivo
and in vitro and a moderate mutator for frameshift
mutations in vitro. The Pol
Y567A
mutant polymerase is
better able to accommodate noncanonical base-pair-
ings, and the Y→A replacement does not interfere
with the geometry of correctly paired bases.
1,16
Thus,
Y567 plays a major role in base discrimination by
providing a steric gate to check pairing (Fig. 9).
The role of Ser565 in maintaining fidelity has come
under scrutiny only recently. Because Ser565 con-
tributes to the rear wall of the binding pocket, we
expected the S565G replacement to enlarge the size of
the pocket, thus more readily accepting base mispairs
and displaying mutator activity. However, the
Pol
S565G
polymerase displays, at most, very weak
mutator and antimutator activities both in vivo and in
vitro (Tables 1–5). We also anticipated that the double
mutant Pol
S565G/Y567A
polymerase would contain a
further expanded nucleotide binding pocket com-
pared with the Pol
Y567A
polymerase and would
therefore display further increased mutation rates.
Surprisingly, however, the Pol
S565G/Y567A
polymerase
displayed sharply decreased mutation rates both in
vivo and in vitro compared to the Pol
Y567A
polymerase
(Tables 1–5). On average, Pol
S565G/Y567A
was 50-fold
more accurate than Pol
Y567A
in rII reversion assay and
40-fold more accurate in rI forward mutation assay.
L561 is another residue that contributes to the
geometry of the Pol pocket and, when reduced in
bulk by the L561A replacement, displays modest
mutator activities in mutation tests in vivo and in
kinetic assays in vitro.
10
In contrast to the antimuta-
genic action of S565G in the Pol
S565G/Y567A
context,
we observed no antimutagenic effect of L561A in the
Pol
L561A/S565G
context (data not shown). Combining
the L561A and Y567A replacements produced a
Fig. 3 (continued)
786 Suppression of a Mutator DNA Polymerase

polymerase with exceptionally high mutation rates,
and the strong mutator phenotype was again
suppressed when S565G was added to the combi-
nation (data not shown).
Associated biochemical parameters
We searched for altered behaviors that might
explain the sharply contrasting fidelities of the
Pol
S565G/Y567A
and Pol
Y567A
polymerases. Polymer-
ase activities were modestly reduced both in vitro
and in vivo, but not in informative ways. Proofread-
ing estimated by comparing mutation rates in the
Exo
+
and Exo
−
versions of the mutant polymerases
(Table 4), and exonuclease activities on either
dsDNA or ssDNA (Fig. 3) were uninformative as
to the fidelity paradox. In mispair formation and
extension assays in vitro under both standing-start
and running-start conditions, the Pol
+
, Pol
S565G
, and
Pol
S565G/Y567A
polymerases were inefficient, while
the Pol
Y567A
polymerase was substantially more
efficient—results consistent with, but not explain-
ing, the mutation patterns. Pre-steady-state kinetic
data showed that the Pol
+
, Pol
S565G
, and Pol
S565G/
Y567A
polymerases display high K
d,app
values for
both G·dTMP and T·dGMP mispairs, while the
Pol
Y567A
polymerase has reduced K
d,app
values for
both mispairs.
22
Our present kinetic data for the
Pol
+
and Pol
Y567A
polymerases are in good agree-
ment with published data.
Fig. 4. RB69 DNA polymerase binding to dsDNA with normal base-pairing or a terminal G·T mismatch. The 5′-labeled
double-stranded substrates (5 nM) were incubated with indicated RB69 polymerases at 5 nM, 10 nM, 25 nM, 50 nM,
100 nM, and 200 nM. After gel electrophoresis, the bands corresponding to bound or free DNA were quantified by
autoradiography. (a) Binding to normal dsDNA. (b) Binding to mismatched dsDNA.
Table 6. DNA binding affinities of Exo
+
RB69 DNA
polymerase mutants
Polymerase
K
d(DNA)
(nM)
[dsDNA]
K
d(DNA)
(nM) [dsDNA, T·G
mismatch]
Pol
+
6.9± 2.1 38.2 ± 4.3
Pol
Y567A
13.2± 1.4 25.7 ± 3.2
Pol
S565G
38.9± 3.4 132.1 ± 5.7
Pol
S565G/
Y567A
29.9± 1.9 79.9 ± 4.8
787Suppression of a Mutator DNA Polymerase

An article (M. Wang, et al., unpublished results)
explores the kinetic parameters of mispair formation,
extension, and proofreading using templates that
mimic the sequences of either a mutational hot spot or
a region of apparently low mutation rates in the rI
mutation reporter used here. Although not addres-
sing the question on the antimutagenic properties of
the S565G replacement, the assays do largely repro-
duce in vitro the relative mutation rates observed by
us in vivo as a function of local sequence, the first such
demonstration known to us. Because spontaneous
mutation rates vary greatly from site to site (for
instance, by about 10
3
-fold in phage T4
23
), rII
reversion rates are expected to vary considerably
among sites, and mutation spectra typically show a
wide range of site-specific rates of forward mutation.
Thus, it is often desirable to use a diversity of
substrates for kinetics studies of polymerase accuracy.
Based on measurements of DNA binding using
gel retardation assays with correct or mispaired
termini, the Pol
S565G
and Pol
S565G/Y567A
poly-
merases displayed weaker binding compared to
the Pol
+
or Pol
Y567A
polymerases (Fig. 4 and Table 6).
Weaker DNA binding for both Pol
S565G
and
Pol
S565G/Y567A
polymerases compared to Pol
+
and
Pol
Y567A
polymerases may reflect less specific
polymerase–DNA interactions whose in vivo con-
sequences could be manifested in a less efficient
DNA synthesis, but direct extrapolation to replica-
tion in vivo is difficult and requires caution. Using a
Fig. 5. T·dGTP mismatch forma-
tion by RB69 DNA polymerases. (a)
A
32
P-labeled primer/template was
present at 50 nM in all reactions.
The sequence of a portion of the
substrate is shown on the left with
an incoming dGTP (“dG”). For each
polymerase, 0.05 nM enzyme and
1 mM dGTP were incubated for
1 min, 3 min, and 5 min. A band at
the +1 position indicates insertion
of dGMP, while bands below the
primer are degradation products.
(b) Percentages of mismatch inser-
tion and primer degradation are
plotted against reaction time for
each of the polymerases.
788 Suppression of a Mutator DNA Polymerase

different assay and different interaction conditions,
no such difference were observed (M. Wang, et al.,
unpublished results).
Candidate mechanisms for the antimutagenic
activity of S565G
One possible explanation for the increased fidelity
of the Pol
S565G/Y567A
polymerase compared to
Pol
Y567A
is the increased partitioning of the mis-
matched primer-terminus to the Exo site. Diverse
changes in the Pol site can perturb the tight
coordination between the Pol cycle and the Exo
cycle,
6,24
and changing Y567 to alanine not only
enlarges the nucleotide binding pocket but probably
affects Pol/Exo partitioning in favor of the Pol site,
1,16
increasing mismatch extension and thus decreasing
proofreading. Conversely, proofreading may become
more proficient with any inhibition in polymeriza-
tion, as might be caused by mutations in the Pol site
that reduce the ability to form polymerizing com-
plexes and thus shift the balance in favor of Exo
complexes.
14,15
(A side effect is that increased
proofreading lowers discrimination between mis-
matched primer termini and matched primer-termini
and increases the degradation of newly synthesized
DNA.
25,26
) Because both Pol
Y567A
and Pol
S565G/Y567A
enzymes bind less tightly todsDNA substrates (Fig. 4
and Table 6), they are expected to form exonuclease
complexes more readily and, as a consequence, to
display increased exonuclease activities. However,
we observed no such increase in exonuclease activity.
One explanation for this failure would be increased
polymerase dissociation from the primer/template
DNA (whether or not mismatched), followed by
rebinding to DNA to form either an Exo complex in
Fig. 6. T-dGMP mismatch exten-
sion by RB69 DNA polymerases. (a)
A
32
P-labeled primer/template
with a terminal T-dGMP mismatch
was present at 50 nM in all reac-
tions. The sequence of a portion of
the substrate is shown on the left.
For each polymerase, 0.05 nM en-
zyme and 1 mM correct dGTP were
incubated for 1 min, 3 min, and
5 min. (b) Percentages of mismatch
extension and primer degradation
are plotted versus reaction time for
each of the polymerases.
789Suppression of a Mutator DNA Polymerase

the presence of a mismatch or a Pol complex and
continued replication. T4 DNA polymerase molecules
do exchange during DNA replication,
27
and RB69
wouldbeexpectedtodothesame.Frequent
dissociation will increase the opportunity to excise a
mismatch by either the original polymerase molecule
or another polymerase molecule, and thus may be
responsible for the increased fidelity of replication by
Pol
S565G/Y567A
compared to Pol
Y567A
.Thisexplana-
tion for the increased fidelity of Pol
S565G/Y567A
compared to Pol
Y567A
is supported by our observation
of decreased DNA binding by the Pol
Y567A
and
Pol
S565G/Y567A
enzymes, but is challenged by the
failure to observe such a difference (M. Wang, et al.,
unpublished results). However, the conditions used
Fig. 7. Running-start reactions for T·dG mismatch formation and extension for the indicated RB69 DNA Exo
+
polymerases. A
32
P-labeled primer/template was present at 50 nM in all reactions. A partial sequence of the substrate is
shown on the left. The reaction conditions were the same as for the standing-start reactions, and the reaction time was
5 min. dGTP was added at the indicated concentrations (μM). RB69 Pol
Y567A
not only inserts dGTP opposite template T
(band +3) but extends the mismatch, creating the next correct G·C base pair (band +4). Neither the Pol
+
enzymes nor the
Pol
S565G
enzymes can create the mismatch, but terminate synthesis at band + 2. The Pol
S565G/Y567A
double mutant has a
sharply reduced ability to insert, extend, and create a second mismatch compared to Pol
Y567A
.
Fig. 8. Complementation assay. Phage growth images
are spot tests in which 20,000 (left) or 100 (right) viable
particles of either T4 43am or 43
+
were spotted onto lawns
of cells carrying a plasmid producing the indicated Exo
−
RB69 gp43.
Fig. 9. A view of the major groove edge of the nascent
dTTP·dA base-pair binding pocket illustrating the posi-
tions of Ser565 and Tyr567 in α-helix P (wide gray ribbon).
“n”indicates the template dA, and “n−1”indicates the
nucleotide upstream of the templating nucleotide. The
active-site metals are represented as green spheres A and
B. The arrow indicates the upstream direction of the DNA
duplex. The narrow gray ribbon represents the duplex
DNA backbone (residues omitted for clarity). The image
was created in Chimera
21
from Protein Data Bank ID 1IG9.
790 Suppression of a Mutator DNA Polymerase

in the two measurements were different, so that
decreased binding remains a possibility in vivo.
Note also that a more frequent dissociation from
the primer/template would also tend to result in the
production of large deletions, as observed in vitro in
the absence of the gp45 processivity clamp (Table 5).
The inhibition of a large deletion mutagenesis
between direct repeats by accessory proteins in
vitro was observed previously for RB69 DNA
polymerase and yeast DNA polymerase δ.
16,28
Another explanation for the increased fidelity of the
Pol
S565G/Y567A
polymerase compared to Pol
Y567A
is
provided by the wealth of crystallographic data (M.
Wang, et al., unpublished results). One key observa-
tion is that the replacement of the bulky Y567 side
chain by alanine reduces the rigidity of the Pol pocket
in the Y567A single mutant by disrupting the
hydrogen-bonding network involving the OH groups
of Y567, Y391, and T587, so that the templating base
can be displaced downward to accommodate mis-
pairs. When the S565G replacement was introduced
into the Y567A mutant, base selectivity was increased
compared to Pol
Y567A
even though the volume of the
Pol pocket was somewhat greater in the Pol
Y567A/S565
double mutant than in Pol
Y567A
. This appeared to be
due to an increase in the hydrophobic van der Waals
interactions of G565 with the templating base, making
it more rigid.
The two candidate explanations for the antimuta-
genic impact of S565G on Y567A are not mutually
exclusive.
Materials and Methods
Construction, expression, and purification of RB69
DNA polymerase variants
Plasmids pCW19R and pCW.50R were generous gifts
from Jim Karam (Tulane University). Plasmid pCW19R
carries a wild-type RB69 gene 43 (encoding the gp43
polymerase) under the control of the T7 Φ10 promoter of
the cloning vector pSP72 (Promega). Plasmid pCW.50R
encodes a Pol
+
Exo
−
polymerase carrying the exonuclease-
inactivating replacements D222A and D327A. Site-direct-
ed mutagenesis to create the Pol
S565G
, Pol
Y567A
, and
Pol
S565G/Y567A
variants was carried out using the Strata-
gene QuikChange Site-Directed Mutagenesis protocol and
was confirmed by sequencing. Expression and purifica-
tion of the Pol
S565G
, Pol
Y567A
, and Pol
S565G/Y567A
gp43 s
were performed as previously described.
17
Mutation tests in vivo
Reversion tests using T4 rII mutations were performed
as described previously.
1
T4 43amam rII mutants were used
to infect E. coli BB cells carrying a plasmid expressing the
desired allele of RB69 gene 43.rII131 reverts by + 1
(A5→A6), rIIUV232 reverts by −1 (A3 →A2), rIIUV256
reverts by G·C→A·T transitions, and rIIUV375 reverts
probably by both transitions and transversions at three
adjacent A·T sites. Forward mutation rI assays were also
performed as described previously.
1
To determine the
kinds of rI mutations introduced in vivo by various RB69
polymerase mutants, we collected mutants of independent
origin, we resuspended mutant plaques in 40 μl of water,
and we amplified the rI gene by PCR and sequenced it as
described previously.
1
When sequencing was not per-
formed, a historical correction factor of 0.64 was used to
estimate the frequencies of rI mutants among all rmutants.
Mutation tests in vitro
Mutation tests in vitro were performed with phage
M13mp2 lacZα-gapped substrates prepared as described
previously.
19
The incubation mixture (25 μl) contained
25 mM Tris-acetate (pH 7.5), 10 mM Mg-acetate, 150 mM
K-acetate, 2 mM dithiothreitol, 150 ng of M13mp2 lacZα-
gapped substrate, 1 mM of each dNTP, and 7–10 pmol of
wild-type or mutant polymerase. Reactions were incubated
at 37 °C for 30 min, stopped by addition of ethylene-
diaminetetraacetic acid, and analyzed by agarose gel
electrophoresis. Products of reactions in which gap
filling was complete were introduced into MC1061
cells and plated on CSH50 cells to score M13 plaques
as either wild-type (dark blue) or mutant (white to less
dark blue). A representative set of mutants from most
collections was sequenced to determine the types of
errors and to adjust the mutation frequency for rare
phenotypic mutants without a mutation in the lacZα
reporter and for mutants with more than one detectable
mutation; a historical correction factor of 0.95 was
applied to mutant frequencies when sequencing was
not performed. Mutation rates (μ) were calculated as
described previously
19
by multiplying the net mutant
frequency [adjusted for the historical background
(6.2× 10−
4
) of uncopied lacZαDNA] by the proportion
of mutants in each class and dividing by 0.6 (a correction
factor for detecting errors in E. coli) and by the number of
detectable sites (opportunities) for each class of mutation.
DNA synthesis in vivo
T4 43amam was used to infect E. coli BB cells carrying a
plasmid expressing the desired allele of RB69 gene 43.At
22 min after infection, 20μCi/ml [
3
H]thymidine was
added (at a specific activity of 20 μCi/μg dT). After
15 min,
3
H labeling was terminated in an ice bath, and
trichloroacetic-acid-precipitable
3
H counts were deter-
mined. Measurements of DNA synthesis in vivo were
performed as previously described.
1
Polymerase-specific activity
Polymerase-specific activity was measured by the incor-
poration of [α-
32
P]dATP (Hartmann Analytic) into activated
calf thymus DNA (Sigma), as previously described.
8
DNA substrates
All nucleotides were purchased from the Laboratory of
DNA Sequencing and Oligonucleotide Synthesis (Institute
791Suppression of a Mutator DNA Polymerase

of Biochemistry and Biophysics, Warsaw) and purified on
a 20% polyacrylamide gel with 7 M urea. All primers were
5′end labeled with [γ-
32
P]dATP (5000 Ci/mmol; Hart-
mann Analytic) using T4 DNA polynucleotide kinase
(Takara). Annealing of primer to template was performed
in 10 mM Tris–HCl (pH 7.5) at a primer/template molar
ratio of 1:1.3. The primer/template was annealed at 75 °C
for 5 min and allowed to cool slowly to 25 °C.
DNA gel retardation assay
The interactions of RB69 wild-type or mutant gp43 s
with dsDNA were assayed using 5′-end-labeled 20-mer
(5′-TTTGATGTATTATCAATTGT-3′)hybridizedtoa
1.3 molar excess of complementary 35-mer (5′-
TGCCTTCGTAATCTTACAATTGATAATACATCAAA-
3′). The interactions of RB69 polymerases with a mispaired
primer terminus were assayed using a 5′-end-labeled 20-
mer (5′-ATGTGCTGCAAGGCGATTAG-3′) hybridized to
a 30-mer (5′-GTTACCCAACTTAATCGCCTTGCAGCA-
CAT-3′). The 10-μl reaction mixture contained 10 mM
Hepes (pH 8.0), 0.5 mM dithiothreitol, 1 μg of bovine
serum albumin, 10 mM ethylenediaminetetraacetic acid
(EDTA), 5 nM DNA substrate, and increasing concentra-
tions of gp43. After incubation for 5 min at 4° C, products
were separated on a precooled 6% polyacrylamide
nondenaturing gel and quantitated on a Fuji Phosphor-
Imager. To determine K
d(DNA)
, we plotted the reciprocal of
the fraction of DNA shifted as a function of enzyme
concentration.
Exonuclease activity
Assays of dsDNA and ssDNA 3′→5′exonuclease
activities were performed as previously described.
17
Exo
activity on mismatched DNA was assayed under the same
conditions using 5 nM gp43 and 50 nM
32
P5′-end-labeled
20-mer (5′-ATGTGCTGCAAGGCGATTAG-3′) annealed
to 30-mer (5′-GTTACCCAACTTAATCGCCTTGCAGCA-
CAT-3′).
Extension of a primer-terminus mismatch
The assay for the extension of a terminal T·dGMP
mismatch contained 50 nM
32
P5′-end-labeled 20-mer (5′-
ATGTGCTGCAAGGCGATTAG-3′) annealed to 30-mer
(5′-GTTACCCAACTTAATCGCCTTGCAGCACAT-3′).
The 10-μl reaction mixture contained 25 mM Tris-acetate
(pH 7.5), 10 mM Mg-acetate, 2 mM DTT, 0.05 nM gp43,
and 1 mM dGTP. Reactions were incubated at 37 °C for
1 min, 3 min, and 5 min, and then quenched by addition of
stop-dye solution. Product bands were resolved by
electrophoresis on a 16% polyacrylamide gel with 7 M
urea, analyzed by autoradiography, and quantified by
densitometry.
Misinsertion assay
The substrate for the misinsertion of dGMP opposite
template T under standing-start conditions was
32
P5′-
end-labeled 20-mer (5′-ATGTGCTGCAAGGCGATTAC-
3′) annealed to a complementary 27-mer (5′-GTAAGATG-
TAATCGCCTTGCAGCACAT-3′). The reaction mixture
contained 25 mM Tris-acetate (pH 7.5), 10 mM Mg-acetate,
2 mM DTT, 50 nM DNA substrate, 0.05 nM gp43, and
1 mM dGTP. Reactions were incubated at 37 °C for 1 min,
3 min, and 5 min, and then quenched by addition of stop-
dye solution.
Running-start assays were conduced in the same
reaction buffer. The 10-μl reaction mixture contained
50 nM
32
P5′-end-labeled 20-mer (5′-ATGTGCTG-
CAAGGCGATTAC-3′) annealed to a complementary 27-
mer (5′-GAACTCCGTAATCGCCTTGCAGCACAT-3′),
0.05 nM gp43, and increasing concentrations of dGTP
(250 μM, 500 μM, and 1000 μM). Reactions were incubated
at 37 °C for 5 min and quenched by the addition of 5 μlof
stop-dye solution. Product bands were resolved by
electrophoresis on a 16% polyacrylamide gel with 7 M
urea, analyzed by autoradiography, and quantified by
densitometry.
Acknowledgements
We thank Bill Beard for providing Fig. 9 and
both Bill Beard and Mike Murray for critical
readings of the manuscript and Bill Konigsberg
for advice on the implications of his structural
models. This research was supported, in part, by
funds allocated to project number Z01ES061054 of
the Intramural Research Program of the National
Institutes of Health, National Institute of Environ-
mental Health Sciences, USA, and by grant
N301014433 from the Polish Ministry of Science
and Higher Education (to A.B.).
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793Suppression of a Mutator DNA Polymerase