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Copyright  2000 by the Genetics Society of America
A DNA Polymerase ε Mutant That Specifically Causes ⫹1 Frameshift Mutations
Within Homonucleotide Runs in Yeast
J. M. Kirchner,1 H. Tran2 and M. A. Resnick
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health,
Research Triangle Park, North Carolina 27709
Manuscript received January 31, 2000
Accepted for publication April 20, 2000
ABSTRACT
The DNA polymerases ␦ and ε are the major replicative polymerases in the yeast Saccharomyces cerevisiae
that possess 3⬘ → 5⬘ exonuclease proofreading activity. Many errors arising during replication are corrected
by these exonuclease activities. We have investigated the contributions of regions of Polε other than the
proofreading motifs to replication accuracy. An allele, pol2-C1089Y, was identified in a screen of Polε
mutants that in combination with an exonuclease I (exo1) mutation could cause a synergistic increase in
mutations within homonucleotide runs. In contrast to other polymerase mutators, this allele specifically
results in insertion frameshifts. When pol2-C1089Y was combined with deletions of EXO1 or RAD27 (homologue of human FEN1), mutation rates were increased for ⫹1 frameshifts while there was almost no effect
on ⫺1 frameshifts. On the basis of genetic analysis, the pol2-C1089Y mutation did not cause a defect in
proofreading. In combination with a deletion of the mismatch repair gene MSH2, the ⫹1 frameshift
mutation rate for a short homonucleotide run was increased nearly 100-fold whereas the ⫺1 frameshift
rate was unchanged. This suggests that the Pol2-C1089Y protein makes ⫹1 frameshift errors during
replication of homonucleotide runs and that these errors can be corrected by either mismatch repair
(MMR) or proofreading (in short runs). This is the first report of a ⫹1-specific mutator for homonucleotide
runs in vivo. The pol2-C1089Y mutation defines a functionally important residue in Polε.
E
RROR avoidance and correction are essential for
reducing a species’ mutational load. DNA polymerases are intrinsically accurate during replicative synthesis due to both base selectivity and opportunities to
correct errors through proofreading (Kunkel 1992). In
addition, mismatch repair (MMR) systems provide for
postreplicational monitoring and correction of errors.
In all eukaryotes examined nuclear DNA polymerases
Polε and -␦ possess 3⬘ → 5⬘ exonuclease activity (Morrison et al. 1991). Genetic studies in the yeast Saccharomyces cerevisiae have demonstrated that the 3⬘ → 5⬘ exonuclease activities of these polymerases are responsible
for proofreading of newly replicated DNA (Morrison
et al. 1991; Simon et al. 1991; Morrison and Sugino
1994) and that proofreading mutants are frameshift and
base substitution mutators (Morrison et al. 1991; Simon
et al. 1991). Proofreading by other replicative polymerases can decrease DNA replication error rates up to two
orders of magnitude in vitro (Kunkel 1992) and in vivo
(Schaaper 1993). Proofreading activity is provided by
Corresponding author: Michael A. Resnick, National Institute of Environmental Health Sciences (NIEHS), Mail Drop D3-01, 111 T.W.
Alexander Dr., P.O. Box 12233, Research Triangle Park, NC 27709.
E-mail: [email protected]
1
Present address: Department of Chemistry and Biochemistry, 601
University Dr., Southwest Texas State University, San Marcos, TX
78666.
2
Present address: LifeSensors Inc., Malvern, PA 19355.
Genetics 155: 1623–1632 (August 2000)
the conserved amino acid motifs ExoI, ExoII, and ExoIII
found at the amino-terminal end of B family polymerases (Morrison et al. 1991; Simon et al. 1991; see
Figure 1).
Proofreading by either Pol␦ or Polε is ineffective at
correcting frameshift errors in long homonucleotide
runs during in vitro or in vivo replication (Kroutil et
al. 1996; Tran et al. 1997). Such runs are especially
prone to frameshift mutations, which are generally acknowledged to arise by replication slippage (reviewed in
Gordenin and Resnick 1998). Two models for slippage
have been proposed. One model proposes that slippage
occurs through disassociation of the polymerase from
the template and incorrect reannealing of the template
and nascent strands (Streisinger et al. 1966; Kunkel
and Soni 1988). Recently an alternative model for slippage leading to insertion frameshifts has been proposed. In this model frameshifts occur during reannealing of the DNA strands after partitioning of the nascent
strand between the polymerization and proofreading
domains (Fujii et al. 1999). The in vitro correction of
mispaired bases by proofreading is limited to approximately five nucleotides behind the 3⬘ terminus of the
primer (Lam et al. 1999). Genetic studies have revealed
similar limitations on proofreading in vivo (Tran et al.
1997).
The 5⬘ → 3⬘ exonuclease encoded by EXO1 is involved
in recombination, resistance to UV damage, and MMR
(Fiorentini et al. 1997; Tishkoff et al. 1997a; Johnson
1624
J. M. Kirchner, H. Tran and M. A. Resnick
et al. 1998; Qiu et al. 1998; Tran et al. 1999b) and
complements many defects in rad27 strains (Tishkoff
et al. 1997b; Parenteau and Wellinger 1999). Rad27,
the yeast homologue of human flap endonuclease
(FEN1), is a 5⬘ → 3⬘ exo/endonuclease responsible for
the maturation of Okazaki fragments during lagging
strand DNA synthesis and removal of 5⬘ flaps (reviewed
in Lieber 1997). Rad27 has also been proposed to function in MMR (Johnson et al. 1995). However, its role
in MMR has been questioned by the finding that in
forward mutation assays the spectra of ⌬rad27 and
⌬msh2 mutants are very different (Tishkoff et al.
1997b). The absence of RAD27 can result in increased
mutation rates including expansion of repeat sequences
and large duplications (Tishkoff et al. 1997b; Freudenreich et al. 1998; Kokoska et al. 1998; Maurer et al.
1998). Furthermore, a structure with mispaired nucleotides in close association with a 5⬘ flap is processed by
Fen to remove both the flap and the mispaired nucleotides (Rumbaugh et al. 1999). Mutants lacking both
Exo1 and Rad27 are inviable (Tishkoff et al. 1997b;
Gary et al. 1999). Overexpression of Exo1 in a ⌬rad27
mutant complements the temperature sensitivity and
partially complements the mutator phenotype of the
mutants (Tishkoff et al. 1997b; Parenteau and Wellinger 1999) suggesting that Exo1 may also be able to
function directly in processing of flaps found in lagging
strand replication intermediates.
Error avoidance in long homonucleotide runs is accomplished predominantly by MMR (Kolodner 1996;
Tran et al. 1997). The Escherichia coli methyl-directed
mismatch repair system has provided a model for the
understanding of MMR in eukaryotes. The basic steps
of these MMR systems are similar, namely (1) mismatch
recognition, (2) incision of the newly replicated DNA
strand, (3) nuclease-mediated deletion of the mismatch,
and (4) gap filling and ligation. Similar to the E. coli
system, in vitro results with human extracts have shown
that mismatch excision can occur from either side of a
mismatch. In yeast the combination of a 3⬘ → 5⬘ Pol␦
or Polε exonuclease deficiency with a defect in either
the EXO1 or the RAD27/FEN1 5⬘ → 3⬘ exonucleases
caused a synergistic increase in mutations in long homonucleotide runs (Gary et al. 1999; Tran et al. 1999b). No
synergy was found between mutations in other mismatch
repair genes (deletion of MSH2, MSH3, MSH6, or
PMS1) and deficiencies in proofreading (Pol␦ or Polε)
for mutations occurring in long homonucleotide runs.
On the basis of these results it was proposed that the
exonuclease activities of Exo1, Rad27, and Polε or Pol␦
participate in and substitute for each other at the excision step of MMR (Tran et al. 1999b).
Relatively little is known about the regions of eukaryotic DNA polymerases that determine DNA replication
accuracy in vivo. One approach is to create mutations
within the polymerases and relate structural changes to
specific mutation-generating characteristics in vitro. We
have taken a similar in vivo approach in the generation
of DNA Polε mutations. In addition to functioning in
cellular DNA replication and proofreading, Polε has
been implicated in various types of DNA repair including nucleotide excision repair (NER), base excision repair (BER), and recombination (Budd and Campbell
1997; Burgers 1998). We have developed a novel mutation detection system, based on interactions with exo1
mutants, to identify functions of Polε other than proofreading that influence genome stability. We have identified a unique DNA polymerase mutator with defects
that are not due to a change in proofreading capacity or
the ability to function in MMR. Unlike other mutators, it
specifically causes ⫹1 frameshift mutations in homonucleotide runs.
MATERIALS AND METHODS
General genetic and molecular methods: Standard yeast
media and yeast extract-peptone-dextrose (YPD) media with
G418 have been described previously (Rose et al. 1990). Yeast
cells were grown at 30⬚ unless otherwise stated. Yeast transformations were performed by the method of Gietz and
Schiestl (1991). Preparation of bacterial growth media and
molecular methods have been described previously (Tran et
al. 1995, 1996).
Strains and plasmids: A series of isogenic strains was constructed from CG379 (Mat␣ ade5-1 his7-2 leu2-3,112 trp1-289
ura3-52) containing deletions of various DNA metabolism
genes. Insertion of the InsE element with a homonucleotide
run containing varying lengths of A within LYS2 has been
described (Tran et al. 1999b). The plasmid used to integrate
mutations within POL2, p173, was constructed by subcloning
the BamHI-BspEI fragment of POL2 from YCpPol2 (Morrison
et al. 1990) into the BamHI-AvaI sites of pFL34*. pFL34* is
identical to pFL34 (Bonneaud et al. 1991) except that the
URA3 marker is in the opposite orientation after Bgl II digestion (K. Lobachev, personal communication). Strains containing pol2-C1089Y were constructed using a site-directed mutant of p173. Site-directed mutants were made using the
QuickChange mutagenesis kit from Stratagene (La Jolla, CA)
according to the manufacturer’s instructions. Primers to make
the Cys to Tyr change and to add an RsaI restriction site were
5⬘-GGTAAAAGATAAAGGTCTACAGTACAAATATATTATT
AGTCAAAACC and 5⬘-GGTTTTGAACTAATAATATATTTG
TACTGTAGACCTTTATCTTTTACC.
Mutagenesis of plasmid DNA: One tube containing plasmid
p173 (pFL34* ⫹ POL2) was treated with 1 m hydroxylamine
in 50 mm pyrophosphate, 100 mm NaCl, pH 7.0, for 1 hr at
70⬚. After stopping the reaction on ice, the DNA (3 ␮g) was
dialyzed against TE and transformed into DH5␣ cells. DNA
was isolated using a QIAGEN (Chatsworth, CA) column as
described by the manufacturer. Since cells were grown for
multiple generations mutations at the same sites cannot be
considered independent.
Screen for A12 homonucleotide run mutators: The mutagenized plasmid DNA was digested with AgeI or PimAI and transformed into cells, which were plated to uracil drop-out media.
After 3 days of growth at 30⬚ colonies were replica plated to
uracil and to lysine drop-out media. After 3 days of growth,
colonies that exhibited four or more papillae on the lysine
drop-out medium were picked from the uracil drop-out plate
and streaked for single colonies. Mutator colonies were grown
Polε Insertional Frameshift Mutator
on 5-FOA and those that had lost the URA3 gene and were
still mutators were used for further study.
Gene replacement and disruptions: We used p173-rsa, described above, for replacement of the wild-type POL2 gene
with the C1089Y allele. The plasmid was digested with AgeI or
PimAI and transformed into cells with selection on uracil dropout media. Transformants were screened for the presence of
the site-directed change by a two-step procedure. After PCR
amplification the fragment was digested with RsaI to determine if the change was present. Strains that had the mutation
were grown on 5-FOA to select for loss of the URA3 gene. Other
strains containing DNA repair/replication mutants were made
from previously described strains (Tran et al. 1997, 1999b).
Mapping and DNA sequence analysis of mutants: The locations of the new POL2 mutations were determined by mapping
using gap repair with deleted versions of p173. After determining that the mutations were between the AspI and SpeI sites
of plasmid p173, DNA sequence analysis was performed on
genomic DNA from this area. Six DNA fragments were made
by PCR amplification using primers pairs (numbered from
the ATG with ⫹ indicating 5⬘ → 3⬘ strand and ⫺ indicating
the 3⬘ → 5⬘ strand; all 20 nucleotides): 1, pol2⫹1949 and
pol2⫺3300; 2, pol2⫹2948 and pol2⫺3800; 3, pol2⫹3220 and
pol2⫺4030; 4, pol2⫹3520 and pol2⫺4342; 5, pol2⫹3826 and
pol2⫺5000; 6, pol2⫹4100 and pol2⫺5000. Sequencing of the
PCR fragments was done using an ABI model 373A DNA
sequencer. Sequencing of Lys⫹ revertants was done by PCR
amplification of a fragment of the LYS2 gene that included
the InsE insertion that contained the homonucleotide run.
The primers used to amplify and sequence this fragment and
the his7-2 homonucleotide run have been described (Shcherbakova and Kunkel 1999; Tran et al. 1999b).
Measurement of mutation rates: Mutation rates were determined by a fluctuation test using the method of the median
(Lea and Coulson 1949) on at least 12 independent cultures
as described (Tran et al. 1999b).
RESULTS
Isolation of Polε mutators: Proofreading is required
for accurate replication by Polε. Little is known about
the contributions of other regions of this polymerase
to replication fidelity. We investigated the ability of randomly generated Polε mutations, external to the known
proofreading domain, to increase the mutation rate in
the long A12 homonucleotide run of the lys2::InsE-A12
allele. Such runs are highly sensitive to subtle changes
in proteins that can affect the appearance of mutations
(Tran et al. 1997, 1999a,b; Clark et al. 1999; Drotschmann et al. 1999). The lys2::InsE-A12 allele was used previously in the characterization of various DNA metabolic
mutants for their ability to influence ⫹1 frameshift mutations (Tran et al. 1999a,b).
The Polε mutants were examined in a strain containing a deletion of EXO1. Use of such a genetically
sensitized background (Perkins et al. 1999) can increase
the probability of finding mutants when screening large
numbers of random isolates. On the basis of the results
with other double mutants (Tran et al. 1999b), we predicted that mutant identification in the ⌬exo1 strain
would be more efficient than in the wild-type strain
because of anticipated synergistic interactions between
Polε mutations and the deletion allele of EXO1.
1625
Figure 1.—(A) Domain structure of the protein coded for
by DNA Pol2 cDNA and location of the changed amino acid in
the mutator allele. Regions of Polε including the exonuclease
domains, the polymerization domain, and the zinc finger domain are shown relative to the sequenced mutation. Shown
underneath is the region that was mutagenized in this study.
(B) Multispecies alignment of the region containing amino
acid C1089. The Cys residue at amino acid 1089 of S. cerevisiae
is in a 17-amino acid block that is highly conserved. When
four or more species have an identical amino acid in a given
location this position is considered conserved and it is highlighted in this figure.
Screening of ⬎5000 Ura⫹ transformants yielded 3
with significantly higher Lys⫹ reversion rates. Their mutator phenotype was complemented by the plasmid
YCpPol2 containing the wild-type POL2 gene. Isolates
37a and 45f were characterized further (the third mutant is under study). The role of the EXO1 defect in
revealing the POL2 mutator phenotype was determined
by introducing EXO1 into cells on the 2␮ plasmid
pRDK480. The relative mutation rates of both of the
pol2 exo1 mutants compared to wild type were increased
33-fold. Addition of the pRDK480 plasmid reduced the
relative rates to 2- and 3-fold over wild type, respectively.
This established that the pol2 mutants isolated are at best
weak lys2::InsE-A12 mutators on their own and require a
deficiency in the EXO1 gene for their strong mutator
phenotype in this assay.
Identification of the alterations within Polε: The Polε
mutator alleles were mapped initially by gap repair.
Mutations 37a and 45f were localized to the same 1 kb
of DNA. DNA sequencing revealed that both clones had
a G-to-A change at base 3266 (C1089Y) of the coding
region, suggesting a common origin for this mutation
in these strains (Figure 1A). Alignment of Polε homologues from S. cerevisiae, Schizosaccharomyces pombe, Mus
musculus, Caenorhabditis elegans, Emericella nidulans, Arabidopsis thaliana, and Homo sapiens identified a common
cysteine residue in a 17-amino acid block of homology
(Figure 1B) that is between 60 and 100% identical in
these organisms designated as C-2 (Huang et al.
1999a,b). The cysteine to tyrosine change at amino acid
1626
J. M. Kirchner, H. Tran and M. A. Resnick
1089 is located 83 amino acids downstream from the
last of the polymerase domains (Figure 1A) in a region
that has been proposed to be involved in subunit interactions (Kesti et al. 1993).
To demonstrate that the C1089Y substitution is the
only alteration in POLε required for the mutator phenotype, we made two site-directed mutant constructs of
plasmid p173. The first construct had only the change
found in the mutator allele. The second construct also
contained silent changes giving rise to an RsaI restriction site. Either plasmid was sufficient to yield the mutator phenotype observed in the original mutant (data not
shown). In our subsequent analysis we utilized strains
containing the p173-Rsa construct; this allele is referred
to as pol2-C1089Y. Plating efficiencies or growth rates
at 30⬚ or 37⬚ were not altered by this allele alone or in
combination with ⌬exo1 (data not shown).
Impact of the Polε mutator in combination with other
mutations: The Polε defect was examined on its own
and in combination with mutations in other genes that
impact on the maintenance of homonucleotide runs.
Isogenic strains that contained A10, A12, and A14 runs
within the LYS2 gene or an A7 run in the HIS7 gene
(i.e., his7-2) were used. Revertants of the lys2 alleles
can occur only in a 79-bp window (Tran et al. 1997);
reversion of the his7-2 allele occurs in a 43-bp window
(Shcherbakova and Kunkel 1999). Defects in MMR
result in all (A10, A12, and A14) or nearly all (his7-2)
reversions occurring in these runs (Tran et al. 1997,
1999b; Shcherbakova and Kunkel 1999). Revertants
associated with the A12 and A7 runs are due to ⫹1
frameshifts and A14 and A10 revertants arise by ⫺1
frameshifts. As shown in Table 1 the pol2-C1089Y mutation itself does not increase mutation rates in the
lys2::InsE-A12, A7, A10, and A14 homonucleotide runs. In
contrast, the weak mutator phenotype for forward mutation rate at the CAN1 locus is increased relative to that
in POL⫹ strains. The mutator effect of pol2-C1089Y at
CAN1 is greater than that observed for the proofreading
mutant pol2-4 (Morrison and Sugino 1994; Tran et al.
1999b; Table 1).
When combined with other DNA replication/repair
defects, the pol2-C1089Y mutation was similar to pol2-4
for frameshifts in the A12 run. Both the Exo1 and Rad27
5⬘ → 3⬘ exonucleases have been implicated in mismatch
repair (Johnson et al. 1995) although this is controversial (Tishkoff et al. 1997b). The pol2-4 rad27 and pol2-4
exo1 double mutants exhibit synergistic increases in mutation rates for homonucleotide runs, as well as at the
CAN1 locus, relative to the single mutants (Tishkoff et
al. 1997b; Tran et al. 1999b). Results for double mutants
with pol2-C1089Y are presented in Table 1. Double mutants containing pol2-C1089Y and either ⌬exo1 or ⌬rad27
also led to synergistic (15- to 20-fold) increases in the
mutation rates in the lys2::InsE-A12 homonucleotide run.
There was no significant enhancement of the lys2::InsEA12 mutation rate when pol2-4 or pol2-C1089Y was com-
bined with a mutation in the MMR gene MSH2, possibly
due to the already high mutation rate of the msh2 mutant in this assay. The effect of the pol2-C1089Y mutation
does not appear to extend to recombination since there
was no effect in a plasmid-based recombination assay
(J. M. Kirchner and M. A. Resnick, unpublished observations).
The POL2-C1089Y protein causes ⫹1 frameshift mutations in long homonucleotide runs: As shown in Table
1, the combination of pol2-C1089Y with ⌬exo1 or ⌬rad27
caused a ⬎25-fold increase in reversion of the his7-2
allele. The proofreading defect pol2-4 leads to similar
synergistic increases in ⫺1 frameshifts within long homonucleotide runs (Tran et al. 1999b; Table 1). We
therefore examined combinations of ⌬exo1 or ⌬rad27
with pol2-C1089Y for their effects on reversion of lys2
alleles containing A10 or A14 runs. Surprisingly, pol2C1089Y allele did not lead to increased mutation rates in
these ⫺1 frameshift mutation detection assays, whereas
pol2-4 resulted in ⵑ34- and 4-fold increases in mutation
rates, respectively, when combined with ⌬exo1 or ⌬rad27
in the lys2::InsE-A14 reversion assay (Table 1).
To determine if the mutations scored in the lys2::InsEA12 assay were actually ⫹1 frameshifts within the homonucleotide run, we sequenced Lys⫹ revertants of the
pol2-C1089Y ⌬exo1 strain. As expected, all revertants
(12/12) of the pol2-C1089Y ⌬exo1 strain in the lys2::InsEA12 assay were due to ⫹1 frameshifts. Similarly, all mutations that occurred in the his7-2 homonucleotide run
in the pol2-C1089Y ⌬exo1 strain were associated with ⫹1
mutations (10/10 sequenced). Thus, when assayed for
frameshift mutator activity in long homonucleotide
runs, the pol2-C1089Y allele leads to a specific increase
in ⫹1 mutations.
The pol2-C1089Y mutator is not due to a proofreading
defect: The 3⬘ → 5⬘ proofreading exonuclease activity
of Polε (as well as Pol␦) can greatly reduce the potential
for mutations. To determine if the pol2-C1089Y allele
alters proofreading, even though the substituted amino
acid is outside the known exonuclease domains, this
mutant was examined for several phenotypic characteristics common to proofreading mutants. In comparison
with the single mutants, the double mutant pol2-4 msh2
exhibits a synergistic increase in CAN1 forward mutation
rates (Morrison and Sugino 1994; Tran et al. 1999b).
In pol2-C1089Y msh2 strains the mutation rate in the
CAN1 forward mutation assay is not significantly different from the msh2 single mutant (Table 1). The combinations pol2-4 exo1 or pol2-4 rad27 also result in synergistic increases in CAN1 mutation rates; however, no
synergy is observed for the corresponding pol2-C1089Y
double mutants (Table 1).
Inactivating the 3⬘ → 5⬘ exonuclease activity of Polε
does not increase mutation rates in long homonucleotide runs (Kroutil et al. 1996; Tran et al. 1999b; Table
1). This suggests that Polε acts together with other
nucleases, such as Exo1 and Rad27, to reduce mutations
TABLE 1
Interaction of pol 2-C1089Y with mutations affecting error avoidance: increases in mutation rates for reversion in homonucleotide runs
(A10, A12, A14 of lys2::InsE; A7 of his7-2) and forward mutation in CAN1
Mutation rates (⫻ 108)a
A12 (⫹1)b
Wild type
pol2–4
pol2–C1089Y
⌬exo1
⫹pol2-4
⫹pol2-C1089Y
⌬rad27
⫹pol2-4
⫹pol2-C1089Y
⌬msh2
⫹pol2-4
⫹pol2-C1089Y
A14 (⫺1)
Mut.
rate
Rel.
ratec
Mut.
rate
Rel.
rate
14 (12–21)d
15 (8–19)
31 (24–41)
28 (17–30)
1.6K (1K–2.3K)f
463 (360–550)
390 (292–448)
3.0K (2K–5.2K)
6.3K (3.7K–9K)
10.0K (6.5K–32K)
28.5K (25K–53K)
50.0K (16K–75K)
1
1
2
2
114
33
28
214
452
714
2035
3570
2.3 (1.6–3.2)
nde
3.7 (3.2–6.7)
9 (7.2–16)
nd
362 (180–450)
38.6 (32–58)
nd
1.0K (630–1.6K)
450 (380–750)
13.9K (9.7K–36K)
3.9K (2.3K–10K)
1
nd
2
4
nd
158
17
nd
435
195
6035
1696
Mut.
rate
19
40.4
50
500
17.2K
746
950
3.5K
667
220K
435K
450K
(15–44)
(24–71)
(31–83)
(326–875)
(10K–24K)
(570–930)
(710–2K)
(2K–5.7K)
(470–890)
(150K–340K)
(330K–770K)
(260K–1250K)
A10 (⫺1)
Rel.
rate
1
2
3
26
905
39
50
184
35
11000
23000
24000
Mut.
rate
13.9
25
13
89
54.5
162
156
35.7K
17.3K
10.7K
(7.9–21)
(14–50)
(11–19)
(76–130)
nd
(38–75)
(140–240)
nd
(115–240)
(22K–48K)
(16.6K–45K)
(4.9K–13K)
CAN1
Rel.
rate
1
2
1
6
nd
4
12
nd
12
2570
1245
770
Mut.
rate
24
30
99
160
890
257
240
3.6K
556
616
11.4K
1.9K
(6.1–27)
(15–83)
(70–110)
(110–270)
(620–1.7K)
(170–390)
(160–410)
(2850–5740)
(460–690)
(423–925)
(7.4K–32K)
(690–2150)
Rel.
rate
1
1
4
7
37
11
10
150
23
25
475
79
Polε Insertional Frameshift Mutator
Genotype
A7 (⫹1)
a
The mutation rate of the strain designated was calculated by the method of the median (Lea and Coulson 1949).
⫹1 indicates mutations that occur by 1 base insertion, ⫺1 indicates mutations that occur by 1 base deletion.
c
Rel. rate indicates the relative mutation rate compared to the wild-type strain for the given homonucleotide run.
d
The numbers in parentheses are the 95% confidence intervals for the mutation rate data.
e
nd indicates that the rate was not determined.
f
Large numbers are given with K to represent 1000s.
b
1627
1628
J. M. Kirchner, H. Tran and M. A. Resnick
TABLE 2
a
Mutation rates in short homonucleotide runs (A4 and A5 of lys2::InsE)
Mutation rates ⫻ 108
A4 (⫺1)b
A5 (⫹1)
Genotype
Mut.
rate
Ratioc
Rate in
rund
Rel.
ratee
Mut.
rate
Ratio
Rate in
run
Rel.
rate
Wild type
pol2-4
pol2-C1089Y
⌬msh2
⌬msh2 pol2-4
⌬msh2 pol2-C1089Y
0.5
0.7
1.6
4.6
166.0
9.4
(4/55)
(7/20)
(1/8)
(20/30)
(26/29)
(8/10)
0.04
0.23
0.20
3.10
144.80
7.54
1
6
⬍5
78
3620
190
0.3
1.6
3.9
7.4
1190.0
770.0
(7/21)
(13/27)
(8/18)
(16/30)
(30/30)
(12/12)
0.1
0.8
1.7
3.9
1190.0
770.0
1
8
17
39
11900
7700
a
Mutation rates for strains involving pol2-C1089Y were obtained in this study. All others were from Tran et
al. 1999a,b. However, mutation rates for all strains except msh2 pol2-4 were repeated and found to be in
agreement with the previous publications.
b
⫺1 indicates reversions that occurred by a ⫺1 frameshift; ⫹1 are reversions that occurred by a ⫹1 frameshift.
c
Ratio is the number of mutations occurring in a run divided by the total number of mutants sequenced.
d
The mutation rate within the homonucleotide run.
e
Rel. rate is the rate in a run for a mutant vs. the wild type.
in long homonucleotide runs so that double mutants
exhibit greatly enhanced mutation rates for both ⫹1
and ⫺1 frameshifts (Gary et al. 1999; Tran et al. 1999b;
Table 1). The pol2-C1089Y mutation differs from pol2-4
in that only ⫹1 frameshifts are increased. Haploid pol2-4
pol3-01 double mutants, lacking both proofreading activities, are inviable, presumably due to a catastrophic increase in mutations (Morrison et al. 1993; Morrison
and Sugino 1994). We therefore transformed a strain
containing the Pol␦ proofreading mutation pol3-01 and
a plasmid expressing wild-type POL2 with the pol2C1089Y integrating plasmid. Following selection for the
presence of only pol2-C1089Y and loss of the POL2 plasmid, viable double mutants could be isolated that exhibited normal growth rates. The mutation rate of the double mutant pol3-01 pol2-C1089Y at lys2::InsE-A12 was
comparable to that of the pol3-01 mutant alone (8.7 ⫻
10⫺6 vs. 4.0 ⫻ 10⫺6). These results lead us to conclude
that the pol2-C1089Y allele’s defect is unlikely to result
from a deficiency in proofreading.
POL2-C1089Y protein creates errors in short homonucleotide runs: Although proofreading is not efficient
at correcting frameshift errors in long homonucleotide
runs, both Pol␦ and Polε proofreading can greatly reduce errors in short runs (Tran et al. 1999b; Table 2).
We investigated the impact of the pol2-C1089Y mutation
on the appearance of frameshift mutations in short homonucleotide runs (A5 or A4) using the lys2::InsE-A run
system. A defect in MMR (⌬msh2) leads to 39- to 78fold increases in mutation rates in the A5 (⫹1
frameshifts) and A4 (⫺1 frameshifts) runs (Table 2;
Tran et al. 1997), respectively. There was a strong synergistic increase in mutation rates when msh2 and pol2C1089Y were combined in the lys2::InsE-A5 strain, as was
also the case for the pol2-4 msh2 strain. No synergistic
effect was observed for the double mutant pol2-C1089Y
⌬msh2 in a lys2::InsE-A4 strain. In contrast, a pol2-4,
⌬msh2 double mutant showed a strong synergistic increase in ⫺1 frameshifts. The pol2-4 mutation also shows
synergy with ⌬exo1 and ⌬rad27 for both the lys2::InsEA4 and the lys2::InsE-A5 mutation targets (Tran et al.
1999b; Table 2). In contrast to pol2-4, no increase in
mutation rate was observed for double mutants formed
between pol2-C1089Y and either ⌬exo1 or ⌬rad27 in the
⫹1 short homonucleotide run assay (A5) over that found
for either of the single mutants (data not shown). (Since
pol2-C1089Y has shown synergy only for ⫹1 frameshifts,
⫺1 frameshifts were not examined in these double mutants.) Synergy for mutation with ⌬msh2 in the double
mutant strain verified that pol2-C1089Y is not a MMR
mutant. Thus, the ⫹1 frameshift mutations generated
in the pol2-C1089Y mutant occur in both long and short
homonucleotide runs and are correctable by the postreplicational MMR systems. The specificity for ⫹1
frameshifts and the interaction between the Pol2C1089Y protein and nucleases affecting MMR are inconsistent with it being a proofreading defect. These data
lead us to suggest that pol2-C1089Y encodes a novel
error-prone DNA polymerase mutant.
The Pol2-C1089Y protein does not appear to affect
processing of Okazaki fragments: Since the pol2-C1089Y
allele does not lead to a MMR defect, the observation
that there is synergy with either the ⌬rad27 or the ⌬exo1
mutation in long homonucleotide runs suggests that
the pol2-C1089Y allele may cause a defect in the completion of lagging strand replication or it may generate
mutations. To rule out involvement of pol2-C1089Y in
Okazaki fragment processing, we have examined mutation rates in double and triple mutants of genes affecting
flap processing and MMR in combination with pol2-
Polε Insertional Frameshift Mutator
C1089Y. Previously, it was shown that Exo1 as well as
Rad27/FEN1 might have a role in removing flaps generated during processing of Okazaki fragments since a
⌬exo1 ⌬rad27 double mutant is inviable and overexpression of Exo1 can compensate for the temperature sensitivity and partially compensate for the mutator phenotype of a ⌬rad27 strain (Tishkoff et al. 1997b;
Parenteau and Wellinger 1999).
If the elevated mutation rates in the double mutants
formed with ⌬rad27 or ⌬exo1 are due to additional impairment of Okazaki fragment processing by the pol2C1089Y, then the triple mutants pol2-C1089Y ⌬exo1
⌬msh2 or pol2-C1089Y ⌬rad27 ⌬msh2 would be expected
to exhibit a substantial increase in mutation rate over
the pol2-C1089Y ⌬msh2 mutant (this has the highest
mutation rate of any of the double mutant combinations
between these genes). This was not the case. The triple
mutants and the double mutants exhibited comparable
mutation rates for the lys2::InsE-A12 allele: 1.0 ⫻ 103 and
1.1 ⫻ 103 for the triple mutants formed with exo1 and
rad27, respectively, compared to 0.5 ⫻ 10⫺3 for the pol2C1089Y, msh2 strain.
DISCUSSION
Isolation of Polε mutators: Assays based on long homonucleotide runs are highly sensitive systems for examining changes in mutation avoidance systems (reviewed
in Gordenin and Resnick 1998). Combinations of single gene defects, each having only a subtle phenotype
separately, can result in synergistic mutation responses
on these runs (Gary et al. 1999; Tran et al. 1999b).
Homonucleotide runs have been employed in screens
to identify mutators, to isolate mutants with subtle
changes in MMR, and to characterize the role of some
DNA metabolic genes in the mutation process. We initiated a study to identify Polε mutations, external to the
known proofreading domains, that could destabilize
long homonucleotide runs. Because this polymerase is
thought to participate in multiple repair processes (reviewed in Budd and Campbell 1997) as well as replication, such mutations could aid in determining structural
and functional roles of the protein. Using a genetically
sensitized exo1-deleted background to screen the mutants, we identified a novel Polε mutator that specifically
increases ⫹1 frameshift mutations.
Mutator effects of the pol2-C1089Y mutation: The
⌬exo1 mutation alone is not a mutator for A12 runs.
However the ⌬exo1 mutation was expected to greatly
enhance the impact of other mutators in a lys2::InsE-A12
reversion assay based on responses of double mutants
formed between ⌬exo1 and pol2-4 (Table 1) or pol3-01
(in a diploid strain; Tran et al. 1999a). The fragment
of POLε that was mutagenized in our screen excluded
the exonuclease domains. However, it is possible that
alterations in the amino acid sequence in regions distant
1629
from the proofreading motifs might physically interact
with these motifs and disrupt proofreading ability.
The screen, in a repair-compromised strain, identified the mutation pol2-C1089Y, which markedly increased mutation rates in the lys2::InsE-A12 run. The
increase in mutation rates in the new pol2 mutants was
dependent on loss of exo1 function. This mutator could
not have been revealed through traditional screens
since the pol2 single mutant had a barely detectable
mutator phenotype for long homonucleotide runs. The
lack of a strong phenotype of pol2-C1089Y alone on A7,
A10, A12, and A14 is not surprising if the errors generated
are corrected by the proofreading and/or the MMR
systems.
The approach of using a sensitized strain background
(i.e., ⌬exo1) could be used to isolate additional mutations of DNA polymerases ε or ␦. We utilized ⌬exo1, but
other nuclease defects that impact on error avoidance
could be considered. For example, the rad27 pol2C1089Y and exo1 pol2-C1089Y double mutants had similar phenotypes.
pol2-C1089Y is not a proofreading defect, but instead
induces mutations that are subject to MMR and proofreading: Several lines of evidence lead us to conclude
that pol2-C1089Y is not a proofreading mutation but
instead that the altered protein increases errors during
DNA synthesis. The combination of a proofreading mutation, in either Pol␦ or Polε with a deletion of either
exo1 or rad27, synergistically increases both ⫹1 and ⫺1
frameshift mutations in homonucleotide runs as well as
CAN1 forward mutations (Tran et al. 1999b; Table 1).
This is contrary to the results obtained with pol2-C1089Y
where only the rate of ⫹1 frameshift mutations was
increased and there was little increase in mutations to
CAN resistance (Tables 1 and 2). The pol3-01, pol2C1089Y combination was viable as a haploid and did
not exhibit increased mutation rates in long homonucleotide runs. Double mutants lacking polymerase ␦ and
polymerase ε proofreading exhibit extremely high mutation rates as diploids and they are inviable as haploids
(presumably due to error catastrophe; Morrison and
Sugino 1994). We propose that the pol2-C1089Y allele
leads to a change in the DNA polymerase that does
not involve error correction, but instead increases the
likelihood of errors during replication of homonucleotide runs.
Mutations arising in long homonucleotide runs are
subject to MMR (reviewed in Gordenin and Resnick
1998). Previously it was suggested that Polε and Pol␦
proofreading nucleases as well as Exo1 participate directly in MMR error excision (Tran et al. 1999b). The
observation of synergy between the msh2 and pol2C1089Y defects for reversion of his7-2 and lys2::InsE-A5
suggests that this pol2 mutation can cause a substantial
mutational burden, most of which is repaired by the
MMR and/or the proofreading system. Proofreading
could explain the relatively modest 9-fold mutation rate
1630
J. M. Kirchner, H. Tran and M. A. Resnick
increase in the ⌬msh2 pol2-C1089Y strain over the single
⌬msh2 strain for A7 runs. Errors in short homonucleotide runs (A4 and A5) are efficiently corrected by both
the MMR system and by proofreading (Kroutil et al.
1996; Tran et al. 1997). The 200-fold increased reversion
of lys2::InsE-A5 in the ⌬msh2 pol2-C1089Y strain relative to
msh2 alone supports the view that MMR corrects errors
generated by pol2-C1089Y. Results with reversion systems
that focus on changes in short homonucleotide runs
also indicate that Pol2-C1089Y generates errors that are
correctable by proofreading. Combining a mismatch
repair defect with a proofreading defect increases mutation rates in short homonucleotide runs 30- to 300-fold
over the rates in a MMR⫺ mutant (Tran et al. 1999b;
Table 2). A similar effect was found for ⫹1 frameshift
mutations in lys2::InsE-A5 when pol2-C1089Y was combined with ⌬msh2. However, ⫺1 frameshifts in lys2::InsEA4 were not increased. On the basis of these observations
we propose that the involvement of pol2-C1089Y in replication or repair results in the induction of mutations
and that these mutations are subject to both MMR and
proofreading. The mechanism of induction and the
reason for the ⫹1 mutator phenotype in homonucleotide runs may be due to differences in ability of the
nascent and template strands to form structures that
could result in replication slippage by this mutant polymerase (discussed below).
Consequences of the pol2-C1089Y mutation on Pol2
function: The increase in only ⫹1 frameshifts seen in
the pol2-C1089Y mutant is unusual in that most DNA
polymerases have higher levels of ⫺1 frameshifts when
examined for spontaneous mutations (Kunkel 1992).
The interaction of T7 DNA polymerase with thioredoxin
increases the processivity of the polymerase (Tabor et
al. 1987) and, in the absence of thioredoxin, there is
an increase in frameshift errors (Kunkel et al. 1994;
Kroutil et al. 1996). The ⫹1 frameshift mutation rate
is nearly 50-fold higher in the absence of thioredoxin
and there is a five-fold bias in ⫹1 vs. ⫺1 frameshifts.
This specificity is similar to the strong bias toward ⫹1
frameshifts with the pol2-C1089Y mutation.
PCNA is key to DNA polymerase processivity in eukaryotes. PCNA binds to DNA polymerases ␦ and ε in
vitro and stimulates processivity (Tsurimoto 1998).
PCNA can interact directly with the Polε protein (Loor
et al. 1997); however, this interaction could also be stabilized through subunit interactions. The region where
the Pol2-C1089Y substitution occurs has been proposed
to function in interactions of Polε with its subunits
(Kesti et al. 1993). Of the four identified subunits, DPB2
and DPB11 genes are essential, while DPB3 and DPB4
are not (reviewed in Sugino 1995). It is possible that
the C1089Y substitution interferes with some interaction
of the subunits and/or PCNA. This was tested by creating strains deleted for Dpb3 or Dpb4, or overexpressing
Dpb2 or Dpb11, and examining their genetic interactions with ⌬exo1 or ⌬rad27 in the presence or absence
of pol2-C1089Y. Neither deletion of the Dpb-3 or Dpb4 subunits nor overexpression of Dpb2 or Dpb11 altered
the mutation rate in the lys2::InsE-A12 assay. These data
suggest that the pol2-C1089Y defect was not due to altered subunit interactions (data not shown).
Crystal structure data for DNA polymerases have led
to a model where the enzyme resembles a partly open
right hand with three domains called fingers, palm, and
thumb forming a U shape ( Joyce and Steitz 1994).
Cocrystal structure data for DNA PolI and Taq DNA
polymerases with DNA indicate that the thumb subdomain has many interactions with the phosphate backbone of the DNA. Multiple interactions occur between
the thumb and the template-primer duplex DNA molecule (Joyce and Steitz 1994). The crystal structure of
the class B DNA polymerase from phage RB69 has been
determined recently (Wang et al. 1997). The replicative
polymerases ␦ and ε of S. cerevisiae belong to the class
B polymerase family. Many polymerases of this family
show good amino acid alignment with each other until
the start of the thumb subdomain, after which the sequences diverge (Wang et al. 1997). A method for estimating the location of the pol2-C1089Y mutation against
the known crystal structure is to start at the last location
of alignment, the end of the T region in the RB69
structure described by Wang et al. (1997), and to add
83 amino acids, corresponding to the distance from that
position to amino acid 1089 in the yeast protein. This
places the amino acid C1089 in the thumb subdomain.
Minnick et al. (1996) showed that a 24-amino acid
deletion at the tip of the thumb domain of the large
fragment of E. coli PolI (Klenow fragment) causes a
specific increase in ⫹1 frameshift mutations in vitro.
Their mutant, like pol2-C1089Y, did not increase base
substitutions or ⫺1 frameshift mutations. Characterization of the mutant PolI enzyme revealed a fourfold
decrease in processivity relative to the wild-type enzyme.
Since frameshift mutations in homonucleotide templates are caused by slippage of the template-primer
(Kunkel 1992), the processivity defect suggests that the
mutator phenotype of the polymerase might be due
to incorrect reannealing after disassociation. However,
frameshift mutations can also occur during highly processive DNA synthesis in vitro (Kroutil et al. 1998).
An alternative model for insertional frameshifts has
been proposed recently by Fujii et al. (1999). These
researchers suggest that partitioning of the nascent
strand between the polymerization site and the exonuclease site of the polymerase during proofreading
allows for incorrect reannealing of the nascent strand
and the specific generation of ⫹ frameshift mutations.
This model implicates proofreading activity itself in the
generation of frameshift errors. An attractive feature of
this model is that it does not require disassociation of
the polymerase from the template strand during the
frameshift generation process.
On the basis of the above observations, we suggest
Polε Insertional Frameshift Mutator
that the pol2-C1089Y mutation may affect processivity
either directly or due to an altered interaction with the
appropriate replication factor(s). Alternatively, more
slippage events may occur on the nascent strand compared to the template strand leading to synergy for ⫹1
frameshift mutants according to the model of Fujii et al.
(1999). Genetic and biochemical analyses of intragenic
pol2-4, C1089Y double mutants that may allow us to
distinguish between these two possibilities are underway.
Implications of the pol2-C1089Y mutator: While pol2C1089Y had a dramatic effect on mutation rates when
combined with defects in other genes, there was little
if any consequence on its own. Thus, it appears that a
neutral Polε mutation can have a dramatic effect in
combination with another subtle mutation. The strong
synergy for mutations in short homonucleotide runs
seen when pol2-C1089Y is combined with ⌬msh2 suggests
that this polymerase allele could also lead to genome
instability when combined with a partial defect in MMR.
There are now examples of synergistic interactions between mild mutations in DNA metabolic genes leading
to dramatic effects. Recently it was demonstrated that
a mutation in the PCNA binding site of Rad27 was genetically neutral. However, cells were inviable when this
mutation was combined with a subtle DNA polymerase
␦ proofreading defect in the heterozygous diploid pol301/POL3 (Gary et al. 1999).
The present results, in which we identify an apparently silent DNA polymerase ε mutation, have important
implications for human disease. Homonucleotide runs
are common in all organisms. MMR defects that destabilize homonucleotide runs have been identified in several tumor cell lines, particularly those from colon cancers. A recent report describes somatic instability
associated with the adenomatous polyposis coli (APC)
gene variant APC I1307K. This variant, which appears
in several examples of familial colorectal neoplasia
(Gryfe et al. 1998, 1999), results from a mutation that
creates an A8 homonucleotide run from separate A3 and
A4 runs. In 42% of carriers (who have the A8 run),
compared to 4% in the general population (who have
the A3XA4 run), a ⫹1 frameshift occurs in the run resulting in gene inactivation. The tumor predisposition
caused by the APC I1307K allele is not associated with
a MMR deficiency (Prior et al. 1999). The identification
of a Polε mutation that can cause a preponderance
of ⫹1 frameshift mutations in homonucleotide runs
suggests that altered replication could contribute to the
appearance of tumors even if the cells are MMR proficient. This example indicates that some disease susceptibilities resulting from genetic instability may not be due
to loss of a single function, such as MMR. These genetic
instabilities could arise by interactions between subtle
or silent mutations in genes that can impact on mutation
avoidance or mutation generation in at-risk motifs
(ARMs) such as homonucleotide runs.
1631
We are grateful to Drs. A. Greene and D. Gordenin for primers;
to Drs. R. Kolodner, H. Araki, and A. Sugino for plasmids; and to
Drs. W. Copeland, D. Gordenin, K. Lewis, K. Lobachev, K. A. Street,
and Y. Pavlov for critical comments on the manuscript, and especially
to Drs. Gordenin, Lewis, and Lobachev for important discussions
during the course of this work.
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Communicating editor: L. S. Symington