The DNA Polymerase Replication Error

[CANCER RESEARCH 62, 3271–3275, June 1, 2002]
The DNA Polymerase ␤ Replication Error Spectrum in the Adenomatous Polyposis
Coli Gene Contains Human Colon Tumor Mutational Hotspots1
Brindha P. Muniappan and William G. Thilly2
Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
We have found a significant concordance between the in vitro replication errors of human DNA polymerase ␤ and in vivo point mutations of
the adenomatous polyposis coli (APC) gene that leads to colon cancer. We
determined the error spectrum of DNA polymerase ␤ in the human APC
gene under PCR conditions and compared it with the set of mutations
reported in human colon tumors. Polymerase ␤ created seven hotspot
mutations within 141 target bp analyzed in APC exon 15. Three of these
polymerase ␤ hotspots, 2 frameshifts and a bp substitution mutation, were
concordant with 3 of 13 APC hotspots detected in human colon cancers in
the same DNA sequences. These 3 concordant hotspots accounted for some
54% of reported in vivo APC hotspot mutations. Using the assumption of
a hypergeometric distribution of hotspot mutations among bp of the
scanned sequences, the probability of this concordance occurring by
chance is <4 ⴛ 10ⴚ4. These data support the hypothesis that DNA
polymerase ␤ errors are an important fraction of cancer-causing APC
Mutations in humans may be caused by exogenous mutagens (1) or
endogenous processes, such as reaction with metabolites (2), DNA
hydrolysis (3, 4), errors in DNA synthesis (5), or possibly through
errors of repair processes acting on undamaged DNA (6). We set out
to discover if a significant fraction of human nuclear point mutations
occurring in vivo could be attributed to a particular source. A potential
cause of point mutations in a defined DNA sequence can be tested by
determining if its point mutational spectrum in vitro is a statistically
significant subset of in vivo mutations (7–9). An overlap, or concordance, of hotspots between the two sets of mutations can identify a
contribution from a hypothesized mutagenic source to mutations that
cause human disease. If there were no contributions of the hypothesized mutagenic source to in vivo mutations, then a degree of concordance between the two mutational sets should not rise above what
is expected by chance. The null hypothesis may be tested using the
hypergeometric distribution in which it is assumed that mutational
hotspots are randomly distributed across the target sequence for both
the error spectrum of the hypothesized mutagenic source and the in
vivo mutational spectrum.
We selected the APC3 tumor suppressor gene for our study because
a large and diverse set of APC mutations are known to initiate most
somatic colon cancers (10, 11). Within 8532 bp of the APC gene
coding region, we compiled ⬎700 human colon tumor mutations from
38 separate reports to estimate the relative frequencies and distribution of the most frequently observed APC mutations (12). On the basis
of these data, we selected two APC gene sequences comprising 141 bp
(3896 –3970 and 4355– 4420) that contain the most frequently observed APC mutations in human colon tumors. Within these 141 bp,
51 distinct mutations had been recorded in 198 separate tumors. The
quantitative distribution of these events, which constitute the APC in
vivo mutational spectrum, is recorded in Fig. 1A.
In taking a broad view of the possible sources of in vivo point
mutations, we noted that one pathway, DNA polymerization, had not
been examined using mutational spectrometry methods to study primary or repair DNA replication errors. DNA polymerases, however,
have in vitro replication error rates that could account for a significant
fraction of in vivo human mutations (13–15). DNA polymerase ␤ in
particular, the smallest known human polymerase, is reported to have
a fidelity rate (measured in specific DNA sequences using phenotypic
selection systems) of 2 ⫻ 10⫺3 to 5 ⫻ 10⫺4 errors/base incorporation
(13, 16, 17). In vivo, polymerase ␤ is responsible for filling in gaps of
one to six nucleotides after the excision of sequences containing DNA
damage (18, 19). Because every day, human cells may repair up to one
million DNA lesions involving excision repair and filling of the
ensuing gap by polymerase ␤ (20), the summation of repair replication
errors created by polymerase ␤ could plausibly account for common
somatic nuclear mutations in humans. To test this hypothesis, we
sought to compare two quantitative point mutational spectra for the
same DNA sequence: (a) the human DNA polymerase ␤ in vitro error
spectrum; and (b) the set of APC gene mutations observed in human
colon tumors.
The replication error spectra of any DNA polymerase can be determined by
analysis of the mutations created when it is used in DNA amplification (21,
22). Mutant sequences generated during replication can then be physically
separated from wild-type sequences and from each other using CDCE (23) or
denaturing gradient gel electrophoresis (24). Individual mutants may be directly collected, isolated, and identified by sequencing (25–27).
High-Fidelity PCR Conditions. For this study, the target APC sequences
(bp 3896 –3970 and 4355– 4420) were first made suitable for CDCE analysis
by the attachment of a thermostable oligonucleotide (“clamp”) through DNA
amplification with Pfu polymerase. APC bp 3876 –3988 were amplified with
primers 3 (5⬘ AATAGGATGT AATCAGACGA) and 5⬘ fluorescein-labeled
GAACTTCGCT CACAGGAT). APC bp 4335– 4440 were amplified with
GCAGCTTGCT) with a 5⬘ fluorescein label. PCR mixtures consisted of 0.2
␮M each primer, 150 ␮M each deoxynucleotide triphosphate (Pharmacia), 1.5
units of cloned Pfu polymerase (Stratagene), 100 mg/ml BSA (New England
Biolabs), and cloned Pfu buffer [20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM
(NH4)2SO4, 2 mM Mg2SO4, 0.1% Triton X-100, and 0.1 mg/ml BSA]. PCR
reactions within sealed glass capillaries in an Air Thermocycler (Idaho TechReceived 11/27/01; accepted 4/2/02.
nology) began with a denaturation step at 94°C for 2 min, then cycling at 95°C
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
for 10 s, 50°C or 57°C for 30 s, and 72°C for 30 s.
18 U.S.C. Section 1734 solely to indicate this fact.
Purification of CDCE-suitable Target Sequences. The target APC se1
Supported by The National Institute of Environmental Health Sciences through
quences used as substrates for DNA replication experiments were in the form
Grants T32-ES0702 (to B. P. M.) and P01-ES07168-06 (to W. G. T.), which provided
of highly purified wild-type homoduplexes. This purification step is essential
additional support for this project.
To whom requests for reprints should be addressed, at Biological Engineering
for subsequent determination of the error spectrum of any DNA polymerase.
Division, Massachusetts Institute of Technology, 21 Ames Street, 16-743, Cambridge,
First the desired sequences were amplified with Pfu DNA polymerase, and
MA 02139. Phone: (617) 253-6221; Fax: (617) 258-5424; E-mail: [email protected]
then CDCE was used to separate the wild-type molecules from Pfu-induced
The abbreviations used are: APC, adenomatous polyposis coli; Pfu, Pyrococcus
mutant sequences. PCR products (10 –50 ␮l volumes, 1010⫺5 ⫻ 1010 copies/
furiosis; CDCE, constant denaturant capillary electrophoresis.
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incubated at 37°C for 5 min for each cycle. Final polymerase concentrations
between 50 and 150 nM were found suitable for amplification of the target APC
sequences. 108 product copies were removed and further amplified when
greater than eight manual PCR cycles were required. The products of DNA
amplification with DNA polymerase ␤ were amplified for 14 additional
template doublings with Pfu DNA polymerase. This degree of amplification
with Pfu polymerase did not create mutations that interfered with observations
of the DNA polymerase ␤ spectrum, as observed in comparison of Fig. 2, A or
B with Fig. 2C.
High-Fidelity PCR and CDCE. The mutant sequences created by polymerase ␤ were collected in mutant:wild-type heteroduplex form from a first
CDCE run to enrich the mutants relative to wild-type homoduplexes. Highfidelity PCR was performed on these enriched heteroduplexes before a second
round of CDCE in which mutant homoduplexes were observed, collected
through electroelution from the capillary column, checked for purity, and
sequenced (22, 26, 27).
Fig. 1. Comparison of in vivo colon tumor hotspot mutations and polymerase ␤
replication error hotspots within 141 bp of the human APC gene. Concordant hotspots
within APC bp 3896 –3970 and bp 4355– 4420 are shown in gray; discordant hotspots are
in black. Mutational spectra are shown as a function of bp position in the APC mRNA
(GenBank accession no. M74088) and number of independent tumors carrying a particular
mutation (A) or mutant fractions (average of duplicate independent trials) after five
template doublings (B). ---- in the top panel, the minimum number of observations (2) that
define an in vivo hotspot. Fifty-one different kinds of in vivo mutations have been
identified within the 141 APC gene bp scanned: 39 mutations were reported once, 7 were
reported twice, and 6 other mutations were reported 4, 5, 6, 24, 32, and 80 times. In some
instances, ⱖ2 types of mutations observed in vivo were reported at the same bp. For clarity
in depiction, the magnitude of the bars in this figure represent the most frequently
occurring mutation at each site that contains ⬎1 type of mutation.
␮l) were dialyzed on a 0.25 ␮M filter (Millipore) against MilliQ water for
30 – 40 min and then speed vacuumed to reduce volumes to roughly 2 ␮l.
Samples of 1–2 ␮l were loaded into a 1-cm length of Teflon tubing (inner
diameter ⫽ 254 ␮m; outer diameter ⫽ 1588 ␮m) and electroinjected into a
capillary (inner diameter ⫽ 250 ␮m; outer diameter ⫽ 350 ␮m) at 40 ␮A for
2 min. Wild-type homoduplexes were separated from wild-type:mutant heteroduplexes within a 5-cm denaturation zone at 72°C. Wild-type DNA moleFig. 2. Distinguishing true PCR-induced mutations from mutations arising from
cules were collected into 4 ␮l of collection buffer [45 mM Tris-borate, 0.5 mM preexisting DNA damage. Mutations created from bypass of preexisting template damage
EDTA (pH 8), and 0.3 mg/ml BSA] and stored at ⫺20°C.
will remain constant in mutant fraction as template doublings increase, whereas PCRAmplification with Recombinant Human DNA Polymerase ␤. Human induced mutations will increase in mutant fraction with each PCR cycle. PCR with
DNA polymerase ␤ was provided in recombinant form by Drs. S. Wilson, R. recombinant human DNA polymerase ␤ was performed on purified copies of the target
sequence until an appropriate number of template doublings was achieved. Individual
Prasad, and W. Copeland at the National Institute of Environmental Health mutants that increased linearly in fraction with PCR cycles were recognized as putative
Sciences (28, 29). 10 molecules of CDCE-suitable, purified, wild-type APC DNA polymerase errors. Fig. 2 displays the mutations created in APC target sequence bp
templates were used for successive cycles of replication by human polymerase 3896 –3970. A and B, negative control samples amplified only by Pfu DNA polymerase.
peaks observed after 5 doublings with DNA polymerase ␤; D, peaks detected after 10
␤. The purified double-strand template DNA was mixed with 0.2 ␮M each C,
template doublings; E, peaks detected after 15 doublings. Peak 1, a five base deletion
primer, 450 ␮M each deoxynucleotide triphosphate, 20 mM Tris-HCl (pH 8), 7 within bp 3939 –3949; peak 2, a G:C ⬎ C:G transversion at bp 3970; peak 3, a G:C ⬎ T:A
mM MgCl2, 100 mM NaCl, 1.5 mM DTT, 2% glycerol, and 0.2 mg/ml BSA. transversion at bp 3943; peak 4, a G:C ⬎ A:T transition at bp 3914. All of the seven
Manual PCR cycling in 20-␮l reaction volumes consisted of denaturation in polymerase ␤ replication errors we identified in the APC gene increased linearly with PCR
boiling water for 30 s, then primer-template annealing at 50°C or 53°C for 5 doublings, indicating none arose from preexisting template damage. The mutant peaks
detected between peak 1 and the wild-type peak do not increase with template doublings
min. Recombinant human polymerase ␤ in 50 mM Tris-HCl (pH 6.5), 100 mM
and, therefore, have not been discussed here, although we have isolated and identified
NaCl, 1 mM EDTA (pH 8), and 20% glycerol, was added, mixed, and these mutants.
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Table 1 In vivo hotspot mutations detected within APC gene bp 3896 –3970 and bp 4355– 4420
Hotspots (observations ⱖ2) were defined by assuming a Poisson distribution and applying the Bonferroni inequality to establish an overall confidence of 95%. As defined, within
141 APC bp, 13 hotspots were found in human colon tumors.
Surrounding APC sequence
G:C ⬎ A:T
G:C ⬎ T:A
TAAA del
A ins
G:C ⬎ T:A
GAAA del
G del
G:C ⬎ A:T
A del
AA del
AG del
AGAG del
Gln ⬎ Stop
Glu ⬎ Stop
Glu ⬎ Stop
Arg ⬎ Stop
Comparison between two mutational spectra has the greatest statistical power when all of the elements of each spectrum are nonrandom events, denoted as “hotspots.” We selected the target APC
sequences (bp 3896 –3970 and 4355– 4420) because they contained
the most frequently observed mutations or hotspots in human colon
tumors. Within these two sequences, 198 separate colon tumors were
reported to carry 51 separate point mutations (12). Of these mutations,
13 were reported two or more times and were denoted as nonrandom
events (hotspots) using ␣ ⫽ 0.05 and a Bonferroni correction to
account for the fact that hotspots could arise at any position in the
scanned target sequences of 141 bp. These data are summarized as a
function of APC position in Fig. 1A. The identity and frequency of
each of the 13 hotspots within the target APC sequences are provided
in Table 1.
To determine the human DNA polymerase ␤ replication error
spectrum, we used the polymerase to copy the two target APC
sequences in a PCR-like process (21, 30). The products of the PCRlike reactions were analyzed using CDCE coupled to high-fidelity
PCR (22, 23). Under the conditions used, polymerase ␤ created 7
mutational hotspots, each occurring at ⬎0.5% of all polymerase
␤-induced mutants within each target APC sequence. Of the identified
polymerase ␤ replication hotspots, four were deletion mutations, and
three were bp substitutions. These data are summarized in Fig. 1B.
Independent experiments eliminated the important possibilities that
these mutations arose from bypass of preexisting damage to the
template DNA or bypass of heat-induced damage (Figs. 2 and 3,
respectively). We calculated the fidelity of polymerase ␤ during
replication of the target APC sequences and found that polymerase ␤
has an average error rate of 5.2 ⫻ 10⫺6/bp/doubling in bp 3896 –3970
and an average error rate of 9.7 ⫻ 10⫺5/bp/doubling in bp 4355–
A comparison of the human polymerase ␤ replication errors in APC
bp 3896 –3970 with those of commercially available polymerases Pfu
and Thermus aquaticus (Taq) revealed that there were no hotspots in
common with either the in vivo APC tumor spectrum or the polymerase ␤ spectrum. Pfu, a highly accurate DNA polymerase, created
G:C ⬎ T:A transversions, whereas Taq, an error-prone polymerase
that, like polymerase ␤, lacks 3⬘ ⬎ 5⬘ exonuclease activity, created
A:T ⬎ G:C transition mutations (data not shown). Not one of the Pfu
or Taq DNA polymerase replication hotspots was concordant with any
of the polymerase ␤ replication errors.
Three polymerase ␤ replication errors were found among the 13 in
vivo hotspot mutations of the APC tumor spectrum. It was necessary
to test the null hypothesis that this degree of concordance (the number
B. P. Muniappan and W. G. Thilly, manuscript in preparation
of hotspots shared by the two spectra) could have arisen by chance.
For this purpose, we used the hypergeometric distribution to ask what
would be the probability of having any three mutations from the in
vivo observations overlap with any three of a different set of mutations
Fig. 3. Control for heat-induced mutations within the set of polymerase ␤ replication
errors. During PCR, the denaturation step (at 100°C in this case) can create potentially
mutagenic lesions (3, 4). Mutational spectra of polymerase ␤ errors determined after PCR
with 30-s denaturation steps were thus compared with spectra determined after increasing
heat exposure to 300 s to discover if any putative DNA polymerase error was created as
a thermal artifact. Fig. 3 displays the mutant peaks detected in APC target bp 3896 –3970
after five doublings with either 30-s (shown in duplicate in C and D) or 300-s (shown in
duplicate in E and F) denaturation steps. Negative control samples based on amplification
with Pfu DNA polymerase alone are shown in A and B. The areas of each polymerase ␤
replication mutation (peaks 1– 4, identified in Fig. 2) were determined through comparison
with the area of an internal standard. None of the seven identified polymerase ␤
replication errors were affected by the increased period of thermal denaturation, indicating
they did not arise from thermally induced DNA lesions during the PCR process.
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(the polymerase ␤ replication errors). We estimated that there are
ⱖ564 different phenotypically observable mutations that could occur
within the 141 target bp of the APC gene. These include 282 types of
frameshift mutations (additions or deletions) and 282 possible bp
substitutions that could result in stop codons, splice site errors, and
missense mutations that inactivate the APC gene (this latter estimate
was calculated by multiplying the 141 target bp by the 3 bp substitutions possible at any base and then subtracting the number of
wobble base positions). With ⱖ564 possible phenotypically observable mutational events, we calculate that the probability of observing
concordance of any three hotspot mutations from the set of seven
polymerase ␤ replication errors with the set of 13 in vivo APC
mutations is ⬍4 ⫻ 10⫺4. This concordance allows us to reject the null
hypothesis that the concordance occurred by chance with reasonable
The three tumor hotspots concordant with the human DNA polymerase ␤ replication error spectrum alone contain 87 of the 159 separate
APC mutational events that have been reported within the scanned
sequences or 54% of the in vivo hotspot mutations within 141 bp. The
most frequently observed APC mutation in human colon tumors,
representing ⬃15% of germline and 7% of somatic APC mutations
leading to colon cancer (12), was a prominent member of the polymerase ␤ error spectrum. This deletion of AAAAG or AAAGA within
bp 3939 –3949 occurs in a region containing a twice-repeated motif:
AAT(A)AAAGAAAAG(A)TTG. A second concordant hotspot mutation is a 1-bp deletion that occurs within the repeat sequence
CCTAAAAATAA (bp 4378 – 4382). These two mutations could have
resulted from a polymerase slippage error within repeated nucleotide
sites as hypothesized by Streisinger (31). The third concordant
hotspot is a G:C ⬎ T:A transversion mutation at bp 3943 (AAAGAAA) that changes glutamic acid to a stop codon. This mutation
might also have been formed during replication by a “slippage”
misalignment (32) of the daughter strand where bp 3940 –3942 of the
nascent strand is paired with bp 3935–3937 (AGAAATA) of the
template strand. Replication past the next bases and then realignment
of the daughter strand to the correct position would produce a G:A
mismatch at bp 3943 that is stabilized by the correct neighboring A:T
Four in vivo hotspots were not found in the human polymerase ␤
spectrum of APC replication errors. This was not unexpected because
polymerase ␤ would not be likely to mediate all human point mutations. It is of immediate interest to discover if other human DNA
polymerases, such as ␦ and ⑀, which are involved in primary DNA
replication and are implicated in proliferating cell nuclear antigenmediated base excision repair (19, 33), create any of the observed in
vivo APC mutations. Such measurements will require availability of
these polymerases in suitable form for use in similar DNA replication
To our knowledge, the four DNA polymerase ␤ hotspots that were
not found in the in vivo spectrum have not appeared as even single
occurrences among all APC mutations detected in colon tumors. One
of these hotspot mutations is a G:C ⬎ A:T transition at bp 3914 that
substitutes valine for alanine. This conservative amino acid substitution may not affect APC protein function and therefore not produce an
observable mutant phenotype (some 95% of all known APC somatic
or inherited disease-causing mutations create stop codons or frameshifts that would be expected to destroy gene function; Ref. 34). This
explanation might also account for the G:C ⬎ C:G transversion at bp
3970, although this mutation exchanges aspartic acid (an acidic amino
acid) for histidine (a basic amino acid). Two other discordant muta-
tions, a 35-base deletion within bp 4378 – 4423 and a 5-base deletion
within bp 4416 – 4420 would, however, be expected to inactivate the
APC gene product. The 35-base deletion could have been created
through a misalignment between the two AAGCA motifs that flank
this large deletion. However, in vivo, polymerase ␤ functions during
base excision repair and fills gaps of one to several bases (19).
Consequently, it may be that there is no physical opportunity in vivo
for polymerase ␤ to create such a large deletion. However, the fourth
discordant polymerase ␤ mutation is a 5-base deletion (bp 4416 –
4420) that would be expected to be both molecularly possible and
phenotypically detectable. Plausible reasons for its absence in the set
of human tumor mutations could include absence by chance from the
limited number of in vivo mutational reports, absence of damageinduced repair at this sequence in vivo, or suppression by undefined
editing or repair functions in vivo.
The mutational hotspots created by human DNA polymerase ␤ in
APC bp 3986 –3970 were compared with mutations created by exonucleolytic Pfu polymerase and error-prone Taq polymerase in the
same target sequence. Each DNA polymerase created a distinct mutational spectrum of replication errors, with no concordant mutations.
Previous studies have demonstrated that different DNA polymerases
create nonidentical replication error spectra within the same DNA
sequence (13, 14, 21, 22). Although polymerase ␤ replicates DNA
patches of 1– 6 bases in a processive manner, replication of longer
sequences is performed in a distributive manner, so it might be
suggested that our results are biased. However, measurement of
polymerase ␤ error rates during synthesis of 1 or ⬎350 bases have
shown no significant differences in fidelity (17). Furthermore, simple
overexpression of polymerase ␤ in mammalian cells has been found to
have strong mutator effects (35). This observation and in vitro experiments demonstrating the ability of polymerase ␤ to compete with
primary DNA replication polymerases has led Servant et al. (36) to
hypothesize that extra polymerase ␤ molecules could become involved competitively with DNA replication. We would like to offer an
alternate hypothesis that excess polymerase ␤ molecules would have
a mutator effect by simply increasing the number of unforced excision
repair events (37). We further speculate that the number of unforced
repair events might well significantly outnumber sites of DNA damage requiring excision repair in an average cell day and thus leave the
mutational fingerprint of excision repair processes using polymerase
␤ throughout the genome of unstable somatic and germinal cells.
In summary, it is clear that the replication error spectrum of human
DNA polymerase ␤ contains a significant subset of the in vivo APC
hotspots found in human colorectal cancers. To our present knowledge, no other potential cause of human point mutation has been
found to account for a statistically significant set of specific point
mutations in the same nuclear gene sequence (the findings that UV
light induces tandem mutations in several experimental systems and
that similar tandem mutations are found in sunlight-irradiated human
skin strongly support an inference of causation, although these in vivo
and in vitro observations were made in different DNA sequences; Ref.
1). We believe our experiments provide a strong test of the specific
hypothesis that errors of human DNA polymerase ␤ are a significant
source of APC point mutations leading to colon cancer in humans. The
results give credence to a more general hypothesis that a significant
fraction of human somatic point mutations arise from DNA polymerase ␤ replication errors and point to the importance of discovering
the error spectra of other human DNA polymerases.
We thank Dr. Rajendra Prasad for graciously providing recombinant human
polymerase ␤ and discussing optimal activity conditions. We also thank Dr.
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Lynn S. Ripley for invaluable discussions of this research and Dr. Stephan
Morgenthaler for consultation on the determination of statistical significance.
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Downloaded from on June 16, 2017. © 2002 American Association for Cancer Research.
The DNA Polymerase β Replication Error Spectrum in the
Adenomatous Polyposis Coli Gene Contains Human Colon
Tumor Mutational Hotspots
Brindha P. Muniappan and William G. Thilly
Cancer Res 2002;62:3271-3275.
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