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Molecular and Cellular Biology, October 2000, p. 7490-7504, Vol. 20, No. 20
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Increased Rates of Genomic Deletions Generated by Mutations in
the Yeast Gene Encoding DNA Polymerase 
or by Decreases in
the Cellular Levels of DNA Polymerase
Robert J.
Kokoska,
Lela
Stefanovic,
Jeremy
DeMai, and
Thomas D.
Petes*
Department of Biology, Curriculum in Genetics
and Molecular Biology, University of North Carolina, Chapel Hill,
North Carolina 27599-3280
Received 5 June 2000/Returned for modification 30 June
2000/Accepted 20 July 2000
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ABSTRACT |
In Saccharomyces cerevisiae, POL3 encodes
the catalytic subunit of DNA polymerase 
. While yeast
POL3 mutant strains that lack the proofreading
exonuclease activity of the polymerase have a strong mutator phenotype,
little is known regarding the role of other Pol3p domains in mutation
avoidance. We identified a number of pol3 mutations in
regions outside of the exonuclease domain that have a mutator
phenotype, substantially elevating the frequency of deletions. These
deletions appear to reflect an increased frequency of DNA
polymerase slippage. In addition, we demonstrate that reduction in
the level of wild-type DNA polymerase results in a similar mutator
phenotype. Lowered levels of DNA polymerase also result in increased
sensitivity to the DNA-damaging agent methyl methane sulfonate. We
conclude that both the quantity and the quality of DNA polymerase is important in ensuring genome stability.
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INTRODUCTION |
The low mutation rate observed in
wild-type cells reflects both the accuracy of DNA polymerases and the
existence of DNA repair systems that remove misincorporated bases.
Mutations affecting components of either of these systems can result in
a mutator phenotype, a global elevation in mutation frequencies
throughout the genome (27). In the yeast Saccharomyces
cerevisiae, a mutator phenotype has been associated with certain
mutations of POL3 and POL2, encoding the
replicative DNA polymerases and 
, respectively (12, 22, 37,
55); these alleles can reside in either a DNA-proofreading
exonuclease domain of POL3 (Exo I domain [Fig. 1]) or near domains required for
nucleotide binding (domains II and VI). Certain mutant substitutions of
POL30, encoding the DNA polymerase processivity factor PCNA,
also have a strong mutator phenotype (8, 20, 23, 56). In
addition, null mutations of RAD27 (encoding an Okazaki
fragment-processing enzyme) or certain alleles of RPA1
(encoding the large subunit of a single-stranded DNA binding protein)
substantially elevate mutation rates (7, 19, 22, 54). A
number of mutants in Schizosaccharomyces pombe, including
those affecting DNA polymerases 
and and DNA ligase, also
exhibit increased rates of mutation (33).
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FIG. 1.
Arrangement of conserved domains in yeast Pol3p
(16) and locations of amino acid alterations in
POL3 mutants. POL3 encodes a polypeptide of 124 kDa (1,097 amino acids). The coding sequence includes three regions
corresponding to the exonuclease proofreading active site (Exo I, II,
and III; Exo II is contained within domain IV). Other conserved domains
include a catalytic center (domain I), regions thought to be involved
in nucleotide binding (domains II, III, and V), and a putative zinc
finger DNA binding domain (Zn) (58). A putative
PCNA-interacting domain (PCNA) is located in the N-terminal region of
the protein (61). The locations of the mutator alleles
pol3-01 (37) and pol3-t
(12) are shown in the upper half of the diagram. The amino
acid substitutions of the unique POL3 missense and nonsense
alleles characterized in this paper are indicated in the lower half of
the diagram.
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In addition to mutations affecting DNA replication genes, mutations of
the DNA mismatch repair genes have a mutator phenotype. Most mismatch
repair in yeast involves two complexes, although other complexes have
minor roles (24). Base-base mismatches are corrected by a
heterotetramer involving Msh2p, Msh6p, Pms1p, and Mlh1p. Small DNA
loops, resulting from DNA polymerase slippage events on simple
repetitive DNA sequences (microsatellites) (Fig. 2A), are repaired by a complex that
includes Msh2p, Msh3p, Pms1p, and Mlh1p. Failure to repair base-base
mismatches results in an elevated frequency of single-base-pair
substitutions, whereas failure to repair DNA loops results in an
elevated frequency of deletions or insertions (47). Genetic
and biochemical data indicate that the Msh2p-Msh3p Pms1p-Mlh1p complex
can correct DNA loops up to 14 bases in size but is incapable of
correcting loops that are 16 bases or greater (13, 35, 48, 49, 51,
52).
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FIG. 2.
Deletions involving repetitive DNA elements. (A)
Deletions generated within simple repetitive tracts by DNA polymerase
slippage. The top strand of each pair represents the primer strand, and
the bottom strand represents the complementary template. The horizontal
arrows indicate the 3' ends of the template strands, and the triangles
show the 3' ends of the primer strands. The rectangles illustrate the
repeat units within the tracts. As replication proceeds through this
repeat region, polymerase dissociation can allow strand separation
(step 1) followed by misaligned reannealing (step 2), resulting in a
loop of unpaired bases on the template strand consisting of an integral
number of repeat units. Uncorrected loops would result in a deletion of
a repeat unit(s) following the next round of replication (step 3).
Insertions can also be generated if the unrepaired loop appears on the
primer strand. (B) Deletions between nontandem direct repeats generated
by DNA polymerase slippage. The rectangles represent two small repeats
of identical sequence. Following replication of one of these units,
polymerase dissociation and strand separation (step 1) can be followed
by reannealing of the 3' end of the primer to the complementary region
of the downstream repeat unit. The resulting intermediate contains a
loop on the template strand consisting of one of the repeat units and
the unique sequence between the repeats (step 2). Failure to correct
this loop results in a large deletion (step 3). (C) Deletions between
nontandem direct repeats generated by SSA (40). The bottom
and top strand of each structure represents the template strand and two
adjacent Okazaki fragments, respectively, with DNA synthesis proceeding
from left to right. The rectangles represent two small units of
identical sequence. Prolonged exposure of single-stranded DNA on the
template strand can result in cleavage within this region, forming a
double-strand break. The 5' ends present at the break can be resected
by an exonuclease activity (step 1). Exposure of the complementary
nontandem-repeat units allow these short tracts to anneal (step 2).
This annealing may be promoted by Rad52p (shown as shaded circles).
Processing of the 3' noncomplementary tails (step 3) results in a
deletion.
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Although some mutators affect only DNA replication or DNA mismatch
repair, other mutators are likely to affect both processes. For
example, some pol30 alleles both reduce the efficiency of DNA mismatch repair and increase the rate of DNA polymerase slippage (8, 20, 56).
Mutators differ in two ways: the degree to which they elevate mutation
rates and the types of mutations that are elevated. For purposes of
this discussion, we will classify mutations as single-base-pair
substitutions, frameshifts (additions or deletions of repeat units to a
repetitive tract of DNA), or additions and deletions involving
nonrepetitive DNA sequences. Mutant substitutions affecting the
proofreading exonuclease domain of DNA polymerase (pol3-01 and pol2-4) greatly increase the
frequency of base pair substitutions but have a modest effect on the
stability of long microsatellites (37, 51). A different
allele of POL3 (pol3-t) and several of the
POL30 and RPA1 alleles greatly increase the rate
of deletions involving nonrepetitive DNA sequences (7, 8, 12), whereas mutations in RAD27 increase the
frequency of duplications (19, 22, 54). Mutations that
affect DNA mismatch repair enzymes elevate both single-base-pair
substitutions and frameshifts, but the elevation observed for
frameshifts is much greater (47).
Since only two mutator alleles of POL3 have been described,
we decided to do a genetic screen for new mutator alleles of this gene.
Following random mutagenesis of the entire POL3 gene, we isolated and characterized three new missense mutants. We
unexpectedly also isolated a number of other mutator alleles that
were nonsense mutations within POL3. As discussed
below, our analysis of these mutants and strains in which the level of
DNA polymerase was reduced demonstrated a novel mechanism for
producing a mutator phenotype.
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MATERIALS AND METHODS |
Media.
Standard enriched (YPD) or minimal (SD) growth medium
for yeast was utilized (46). Media for the growth of strains
containing POL3 under the control of the GAL1/10
promoter consisted of 1% Bacto Yeast Extract, 2% Bacto Peptone, 3%
raffinose (YPR), and various concentrations of galactose as indicated.
Yeast strains and plasmids.
The yeast strains used in this
study are listed in Table 1. All of the
strains are isogenic with AMY125 ( ade5-1 leu2-3 trp1-289
ura3-52 his7-2 [51]) except for changes
introduced by transformation. EAS63a (provided by E. Sia) was a
derivative of MS71 in which the mating type was switched, the
ade5-1 mutation was reverted, and a poly(GT) tract 29 bp in
length was inserted into the ADE2 gene
(ade2-100). RJK48 is a leu2 derivative of EAS63a constructed by targeted disruption of the LEU2 gene with
BglII-treated pNKY85 (1).
The plasmid pBL304 contains the wild-type
POL3 gene cloned
into YCp50. The plasmid pBK100, in which the
URA3 of pBL304
was
replaced with
LEU2, was generated in vivo by
transforming the
strain RJK67-5 (RJK48 plus pBL304) with a PCR fragment
containing
the
LEU2 gene flanked by 40 bp of
URA3
homology; this fragment
was generated by PCR using
SalI/
HindIII-digested pRS315
(
LEU2)
as a template and the primers
5'-AACCCTTGGCAGAACATATCCATCGCGTCCGCCATCTCCAtcaattgtcctgtacttcc
and
5'-GTGATTCAT TCTGCTAACCAGTAAGGCAACCCCGCCAGCCtaaggccgtttctgacaga (the
bases
in lowercase indicate
LEU2-specific
sequences).
To delete the chromosomal copy of
POL3, we transformed
RJK67-5 with a PCR-generated fragment in which the
kanMX
gene is flanked
by DNA sequences derived from the regions upstream and
downstream
of
POL3. The oligonucleotides used for the
amplification were
5'-TTGCTATTAAGCATTAATCTTTATACATATACGCACAGCAcgtacgctgcaggtcgac
and (downstream)
5'-CCTTTCTTAATCCTAATATGATGTGCCACCCTATCGTTTTatcgatgaattcgagctcg
(the lowercase letters correspond to
kanMX sequences);
the substrate
for the amplification reaction was a plasmid containing
Tn
903 (
57). The resulting strain was RJK122-3.
Strains similar to
RJK122-3 with plasmid-borne copies of
POL3 or mutant
pol3 genes
were isolated by the
plasmid shuffle technique described below.
To create strains bearing
rad52
and/or
msh3 in combination with
the
plasmid-borne
POL3 genes, we formed diploids by crossing
appropriate
haploid strains, sporulated the diploids, and identified
spore
colonies that contained the desired
genotypes.
The strain RJK341 contains a version of
POL3 in which an
epitope (3 × HA) is inserted at the N terminus of
POL3
and the promoter
is replaced with the
GAL1/10 promoter; a
kanMX cassette is immediately
upstream of the
GAL1/10 promoter. This strain was constructed
by
transforming MS71 with a DNA fragment generated by PCR amplification
of
pFA6a-kanMX6-PGAL1-3HA as a template (
34) using the primers
5'-TATTGAGCACTTGCTATTAAGCATTAATCTTTATACATATgaattcgagctcgtttaaac
and
5'-TCTTCACATCAACCATGGGAAGGGATCTTTTTTCACTCATgcactgagcagcgtaatctg.
The uppercase sequences correspond to nucleotides
49 to
10
and
nucleotides +40 to +1 relative to the
POL3-initiating AUG. The
construction replaces the 9 nucleotides just upstream of the
POL3-initiating
AUG with
the
kanMX cassette, the
GAL1/10 promoter, and
the 3×HA
tag. The epitope tag is present with its own initiating codon
downstream of the
GAL1/10 promoter and is in frame and
directly
upstream of the entire
POL3 coding
sequence.
The strain JED213-30 has a 3×HA epitope tag inserted into a wild-type
POL3 gene. This strain was constructed by transforming
the
strain MS71-
pol3-t (which has a temperature-sensitive
pol3 allele) with a PCR fragment designed to simultaneously
revert
the
pol3-t allele and insert the 3×HA epitope tag at
the beginning
of the
POL3 coding sequence. This PCR fragment
was generated using
genomic DNA from strain RJK341 as a template with
the following
primers: upstream,
5'-AAATAGATATTGAGCACTTGCTATTAAGCATTAATCTTTATACATATACGCACAGCAatgtctttaattaacatcttt,
and downstream, 5'-CAATAGAAACCAAGGAACAGGAATC. The
sequence shown
in uppercase on the first primer corresponds to
nucleotides
57
to
1 relative to the
POL3 start codon,
and the lowercase letters
correspond to the first 21 nucleotides of the
3×HA epitope tag
coding sequence. The downstream primer consists of
nucleotides
2545 to 2521 of
POL3. Transformants were
selected by growth at
37°C.
PEP4 was deleted by replacing it with
URA3. The
DNA fragment used for this replacement was generated by PCR using
pRS306 (
URA3)
as the template and the following two primers
(
PEP4 sequences
are capitalized): upstream,
5'-GTA TTTAATCCAAATAAAATTCAAACAAAAACCAAAACTAACcgcttttcaattca
attc,
and downstream,
5'-GCAGAAAAGGATAGGGCGGAGAAGTAAGAAAAGTTTAGCcagggtaataactgatataa.
To evaluate the effects of various mutations on dinucleotide repeat
instability, we transformed appropriate strains with pSH44
[
CEN
TRP1] (
15). This plasmid contains a 33-bp poly(GT)
tract
inserted in frame within the
URA3 coding
sequence.
POL3 mutagenesis and mutant isolation.
The
POL3 gene was randomly mutagenized by treating pBK100 with
hydroxylamine (50). Following mutagenesis of pBK100,
POL3 mutants were isolated by the plasmid shuffle technique
(50). Yeast strain RJK122-3 was transformed with 1 µg of
the mutagenized pBK100, plated on solid minimal medium lacking leucine,
and incubated at 22°C for 5 days. The Leu+ colonies were
replica plated onto two plates containing minimal medium lacking
leucine and containing 5-fluoroorotate (5-FOA). This selection
(4) allows for growth of cells that have lost the pBL304
plasmid and retained the mutagenized plasmid. Isolates containing
potential temperature-sensitive lethal POL3 mutants were
distinguished by growth on 5-FOA medium at 22°C and loss of growth on
this medium at 37°C.
Assays for cell viability and sensitivity to DNA damage.
For
the experiments that tested for cell viability upon loss of
[PSI+], strains were grown at 22°C on solid
YPD medium to single colonies. The colonies were replica plated onto
YPD medium with or without 5 mM guanidine hydrochloride (GuHCl) and
grown overnight at 22°C. The replica plating was repeated twice more
onto identical media. To test the damage sensitivity of the
temperature-sensitive POL3 mutants, we pregrew yeast strains
on solid YPD medium for 2 days at 22°C. For the similar experiments
that utilized the strain bearing the GAL1-POL3 construct,
strains were pregrown on solid YPR medium containing 0.05% galactose
for 2 days at 30°C. Fivefold serial dilutions of each strain were
spotted onto appropriate media. To test for UV sensitivity, we treated
patches of cells of various serial dilutions with UV light from a
germicidal lamp. MMS (methyl methane sulfonate) sensitivity was
examined by spotting serial dilutions on appropriate solid medium
containing 0.025% MMS. All plates were incubated for 3 to 4 days in
the dark at 30°C.
Determination of spontaneous mutation rates and mutational
spectra.
The forward mutation rate at the CAN1 1ocus
was determined by standard methods (48), using at least 12 independent cultures for each rate estimate. Rates were calculated from
the frequencies of canavanine-resistant mutants by using the method of
the median (28). For strains bearing the GAL1/10
promoter upstream of POL3, the cells were pregrown in YPR
medium containing appropriate concentrations of galactose.
The mutant substitutions in
CAN1 were analyzed by PCR
amplification of the 1.8-kb
CAN1 gene. Primers located at
three positions
within the gene were used in the sequence analysis. For
some experiments,
the 1.8-kb
CAN1 gene was treated with
SspI and the resulting fragments
(with sizes of 515, 652, and 718 bp) were examined by electrophoresis
using 2% Metaphor (FMC
Corp.) agarose gels; this method can detect
deletions as small as 8 bp.
The extents of deletions that removed
one or both ends of
CAN1 were determined using 14 primer pairs
to amplify
sequences centromere-distal to
CAN1 (
7). The
sequence
of deletions followed by telomeric additions was determined by
a PCR procedure utilizing a series of degenerate primers
(
7).
Analysis of plasmid-borne simple-repeat instability.
Yeast
strains were transformed with pSH44 to determine the rate of
instability within a 33-bp poly(GT) tract by methods described previously (15). Alterations within the repetitive tract
were determined by PCR amplification of the repetitive region and
analysis by gel electrophoresis as described previously
(48).
Western blot analysis.
Liquid cultures for RJK368-4 were
grown in YPR medium containing various concentrations of galactose and
those for RJK366-5 and RJK394 were grown in YPD medium to mid-log
phase. Protein extracts from spheroplasts of these strains were
prepared as described previously (2). Proteins were
fractionated on a sodium dodecyl sulfate-7.5% polyacrylamide gel,
transferred to nitrocellulose, and blocked by standard procedures
(43). Membranes were probed with mouse antihemagglutinin
(anti-HA) monoclonal antibody (clone 12CA5) (Boehringer Mannheim).
Detection of antibody binding was performed using horseradish
peroxidase-linked anti-mouse immunoglobulin G (Amersham). Quantitation
of protein bands was performed on a scanning densitometer (Molecular
Dynamics). Quantitation was based on the intensity of a band specific
to Pol3p normalized by the intensity of a band representing a protein
that is nonspecifically recognized by the HA antibody.
Statistical methods.
Calculation of 95% confidence
intervals was done as described previously (22). Fisher
exact tests were performed using the InStat version 1.12 program.
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RESULTS |
Isolation and phenotypes of yeast POL3 missense
mutants.
In order to identify new mutator alleles of DNA
polymerase , we isolated mutants of the yeast POL3 gene
by the plasmid shuffle technique (50). The plasmid
pBK100 (YCp POL3 LEU2) was randomly mutagenized by
treating the plasmid DNA with hydroxylamine. This mutagenized plasmid
was transformed into a yeast strain containing pBL304 (YCp
POL3 URA3) and a deletion of the POL3 chromosomal locus. Cells derived from each transformant that lost pBL304 were detected on medium containing 5-FOA, which selects against
Ura+ cells (4). In order to increase the
probability of identifying strains with a mutagenized POL3
gene, we screened the transformants containing mutagenized pBK100
(7,500 strains examined) for temperature sensitivity (loss of growth at
37°C) on minimal growth medium containing 5-FOA; POL3 is
an essential gene in yeast, and temperature-sensitive alleles of
POL3 were previously isolated (14). Ten
independent strains with a temperature-sensitive pol3
mutation were identified. DNA sequence analysis of these mutant alleles
showed that four (pol3-ts1, pol3-ts11,
pol3-ts18, and pol3-ts26), representing three
different alterations (pol3-ts1 is equivalent to
pol3-ts18), contained single missense mutations within the
POL3 gene. The positions and amino acid alterations of these
mutations are shown in Fig. 1.
These
POL3 missense mutants were examined for abnormal cell
growth at the semipermissive temperature (30°C) and were screened
for
sensitivity to DNA damage. Many of the mutant cells had buds
approximately the same size as the mother cell. The percentages
of
cells with this morphology at 30°C for the wild type,
pol3-ts1,
pol3-ts11, and
pol3-ts26
were 7, 39, 18, and 63%, respectively
(500 cells were counted). After
the incubation of cells at 37°C
for 20 h, the percentages of
cells with this morphology for the
wild-type,
pol3-ts1,
pol3-ts11, and
pol3-ts26 were 6, 98, 95,
and
95%, respectively (500 cells were counted). The accumulation
of
large-budded cells at 37°C suggests that the growth defect
associated
with these mutant strains is due to a defect in DNA
replication that
does not allow for efficient completion of S
phase, since a similar
position of cell cycle arrest is observed
for other types of mutations
affecting DNA synthesis (
29). Each
POL3 mutant
also exhibited moderate to high sensitivity to MMS
but no sensitivity
to UV radiation, suggesting that the mutant
polymerases confer specific
defects in the repair of damaged DNA.
An example of these phenotypes
for the
pol3-ts1 mutants is shown
in Fig.
3. Strains with
rad1
(conferring sensitivity to UV) or
rad52 (conferring
sensitivity to MMS) mutations are shown as
controls.
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FIG. 3.
Sensitivity of pol3 mutant strains to DNA
damage. Fivefold serial dilutions of each strain were spotted onto YPD
plates in the presence of MMS or subjected to UV irradiation. The
strains examined were RJK160-1 (wild-type [WT]; top rows), RJK158-3
(missense mutant pol3-ts1; second rows), RJK185-1 (nonsense
mutant pol3-ts8, third rows), EAS102 (rad1;
fourth rows), and HM6 (rad52, bottom rows). The results
were similar for all POL3 missense and nonsense mutants
identified in this study. The rad1 strain was used as a
control for UV sensitivity, and the rad52 strain was the
control for MMS sensitivity.
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POL3 missense mutants exhibit mutator phenotypes
and dissimilarities in mutational spectra.
To determine if
the strains bearing the POL3 missense mutations
conferred a mutator phenotype, we assayed for the forward mutation rate
at the CAN1 locus in vivo. Wild-type cells are sensitive to
the arginine analog canavanine, and any mutation that inactivates the
arginine permease encoded by CAN1 results in canavanine
resistance. We found that strains bearing any of the three
POL3 alleles exhibit four- to sixfold-elevated rates of
mutation at CAN1 (Table 2).
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TABLE 2.
Forward mutation rates at CAN1 in strains with
POL3 missense mutations and/or mutations affecting DNA
repair or recombination
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Although all of these strains are mutators, they do not display the
same spectrum of mutations. The mutational spectrum for
each strain was
determined by sequencing the
CAN1 gene from approximately
20 independent canavanine-resistant (Can
r) isolates. As
shown in Table
3,
over one-half of the mutations
generated in the
pol3-ts1 and
-ts11 strains were deletions larger
than a few base pairs. The deletions within
CAN1 in the
pol3-ts1 strain ranged from 16 to 336 bp in length, while
the
pol3-ts11 mutation resulted in deletions of 16 to 864 bp. One distinguishing
characteristic of each of the sequenced
CAN1 deletions is the
presence of an imperfect direct repeat
flanking each deletion.
These repeat motifs range from 5 to 11 bp
(Table
4); with one
exception, each pair
of flanking repeats has a perfect match of
at least 75% of the bases.
These types of deletions have been
previously observed in strains
bearing the
rfa1-t29 allele of
S. cerevisiae
(
7) and several
pol3 alleles of
S. pombe (
33).
In addition, one canavanine-resistant
isolate in the
pol3-ts11 strain exhibited a complete
deletion of approximately 35,000 bp
of one end of chromosome V
(including the entire
CAN1 gene) and
the addition of a
telomeric sequence (repeats of TG
1-3);
the breakpoint of
this deletion mutation occurred in a GT-rich
region of the chromosome
(Table
3). Telomeric additions have
previously been observed in other
strains with mutations affecting
DNA repair or replication (
6,
7) or associated with repair
of an HO-induced DNA break
(
25). Only 20% of the mutations generated
within these
pol3 strains are base substitutions. Since 55% of
the
sequenced mutations in an isogenic wild-type strain are substitutions,
it appears that
pol3-ts1 and
-ts11 have a small
(or no) effect
on the accumulation of this class of mutation.
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TABLE 3.
Mutational spectra at CAN1 in strains with
POL3 missense mutations and/or mutations affecting DNA
repair or recombination
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In contrast to the results obtained with
pol3-ts1 and
-ts11, 70% of all Can
r isolates derived from
the
pol3-ts26 allele are single-base substitutions.
Fifteen
percent of the
CAN1 mutations in this strain background
were
deletions flanked by imperfect direct repeats; however, these
deletions are much smaller (8 to 27 bp) than those observed in
pol3-ts1 and
-ts11. Thus, it appears that this
mutation near the
C terminus of the protein primarily increases the
rate of base
substitutions.
We also examined the effects of these polymerase mutations on the
instability of a 33-bp poly(GT) tract. This instability
is measured by
a frameshift assay (
15) utilizing a plasmid (pSH44)
containing a repetitive GT sequence fused upstream and in frame
to URA3. In yeast strains transformed with this plasmid, additions
or
deletions of repeat units within the poly(GT) tract that result
in an out-of-frame
URA3 sequence can be detected by growth
in
medium containing 5-FOA. This assay has previously been used to
demonstrate that mutations in genes required for DNA mismatch
repair
(
48,
51,
52) and DNA replication (
RAD27 and
POL30)
(
19,
20,
22,
23,
56) significantly enhance
the rate
of microsatellite instability. We found that
pol3-ts1 elevates
the frequency of tract instability about
twofold and that neither
pol3-ts11 nor
pol3-ts26
had any significant effect (Table
5).
Effect of msh3 mutation on accumulation of genomic
deletions in pol3-ts1 and -ts11 strains.
A
possible source of the deletions observed in the strains bearing
pol3-ts1 and -ts11 is DNA polymerase
slippage. This mechanism has commonly been invoked to explain the
instability of simple repetitive DNA (Fig. 2A) (53). A
deletion flanked by imperfect repeats may also occur by this mechanism
(Fig. 2B). Once replication proceeds through one homologous region,
reannealing of separated strands may occur between the newly
synthesized primer strand and a homologous region further
downstream on the template, resulting in formation of a DNA loop.
Failure to repair this loop would ultimately result in a deletion of
the type that is prevalent in pol3-ts1 and -ts11 strains.
It has previously been shown that the DNA mismatch repair system
efficiently corrects loops ranging from 1 to 14 nucleotides
in length
(
49,
60). All of the deletions in the "deletions
flanked
by direct repeats" class in the
pol3-ts1 and
-ts11 strains
were 16 bp or greater (Table
3). One
explanation of this observation
is that slippage events resulting in
DNA loops of less than 16
bp are not observed because such loops are
efficiently repaired
by the DNA mismatch repair system. This
interpretation predicts
that deletions of less than 16 bp would be
observed in the polymerase
mutant strains in the absence of DNA
mismatch repair. To test
this hypothesis, we combined
pol3-ts1 with a null mutation of
MSH3, a MutS
homolog that specifically functions in the correction
of DNA loops up
to 14 nucleotides in length (
48,
49).
We found that while the mutation rate at the
CAN1 locus was
unaffected by
msh3 in a wild-type strain, there was a
synergistic
effect between
pol3-ts1 and
msh3.
While the rate of mutation
for
pol3-ts1 was 6-fold greater
than that of the wild type, a
20-fold effect was observed for
pol3-ts1 msh3 (Table
2). Sequencing
of 39 Can
r isolates from the double-mutant strain reveals two
classes of
mutations rarely seen in the
pol3-ts1 single
mutant (Table
3).
One class consists of deletions in microsatellite
sequences within
CAN1 in which the size of the repeat unit
ranges from 1 to 6 bp.
Approximately one-half (19 of 39) of the
Can
r isolates in the
pol3-ts1 msh3 strain are of
this class, which
is significantly different from the frequency seen in
the
pol3-ts1 single mutant (
P = 0.001;
Fisher exact test). One common deletion
that appeared in 25% of the
independent isolates is an (AG)
4 to
(AG)
3
deletion at positions 254 to 261 of the
CAN1 open reading
frame. We also transformed the
pol3-ts1 msh3 strain with
pSH44
in order to assay the effects of these two mutations on the
instability
of a 33-bp poly(GT) tract (Table
5). We found that the
effects
of these two mutations are multiplicative. This strain
exhibited
a 150-fold enhancement in instability over the wild type for
this
dinucleotide repeat. Sequencing of the repeat tract from 10 independent
5-FOA
r isolates showed that 70% of the
mutations were frameshifts resulting
from 2-bp deletions within the
repeat tract. These results are
similar to the effects seen for these
two mutations on the rate
of deletion formation within
CAN1 microsatellites.
The second class of mutations consists of deletions flanked by
imperfect direct repeats in which the size of the deletion
is less than
16 bp. These deletions were observed in approximately
one-quarter (9 of
39) of the mutations in the
pol3-ts1 msh3 strain.
This
distribution is significantly different from that seen in
the
pol3-ts1 strain, in which this deletion class is entirely
absent (0 of 20) (
P = 0.02). In addition to these two
unique classes
of mutations, larger deletions flanked by direct repeats
persist
in the
pol3-ts1 msh3 strain, but they are less
frequent than in
the
pol3-ts1 strain (5 of 39). In addition,
the sizes of these
deletions in the double-mutant strain appear to be
generally smaller
(16 to 38 bp) than the deletions in the
pol3-ts1 strain. We did
a more limited analysis of an
additional 60 independent Can
r isolates in the
pol3-ts1 msh3 strain. Rather than sequencing
all of these
isolates, only those that exhibited apparent deletions
by a
PCR-restriction digest screen were chosen for sequencing.
This
screening allowed for detection of deletions as small as
8 bp by gel
electrophoresis. Of these 60 isolates, 11 were deletions
ranging from 8 to 14 bp and 9 were larger deletions (18 to 135
bp). The frequencies at
which these classes of deletions were
present were similar to those
that were observed in the original
39 Can
r isolates.
High rate of deletion formation in the pol3-ts1 strain
depends on Rad52p.
To investigate the role of Rad52p in the
generation of deletions in the pol3-ts1 strain, we combined
rad52 with the polymerase mutation. This double-mutant
strain grew much more slowly than either of the two single-mutant
strains. We also found that the effect of these two mutations on the
overall mutation rate at CAN1 was additive (Table 2). In
addition, loss of Rad52p activity eliminated all of the deletions
between direct repeats that are generated in the pol3-ts1
strain (Table 3). A similar effect was previously observed when the
rfal-29 allele was combined with rad52
(7). These data indicate that Rad52p is required for the
formation of deletions in the polymerase mutant strain.
When
rad52 was combined with
pol3-ts1 msh3, we
found that for two independently derived isolates, the overall rate of
mutation
was identical to that of
pol3-ts1 msh3 (Table
2).
In the triple-mutant
strain, however, there is a significant reduction
in the frequency
at which frameshifts within microsatellites and
deletions flanked
by direct repeats are generated relative to a
pol3-ts1 msh3 strain
(
P < 0.0001 for
comparison to RJK363 and
P = 0.0017 for comparison
to
RJK381) (Table
3). In addition, the high rate of poly(GT)
instability
seen in the
pol3-ts1 msh3 strain decreased by a factor
of 3 in the absence of Rad52p (Table
5).
Although
rad52 in combination with
pol3-ts1
has its most striking effect on the rate of deletions, loss of Rad52p
also substantially
increased the rate at which base substitutions are
generated in
pol3-ts1. Most (85%) of the mutations in
pol3-ts1 rad52 were base
substitutions, while only 20% of
the mutations in
pol3-ts1 were
of this class. Based on the
overall rates of mutation and the
fraction of mutations that are base
substitutions for each strain,
we found that the rates at which base
substitutions are generated
are 1.3 × 10
7 (wild
type; relative rate of 1×), 2.8 × 10
7
(
pol3-ts1; 2×), 2.2 × 10
6
(
rad52; 17×), and 5.4 × 10
6
(
pol3-ts1 rad52; 42×) per cell division. This result
suggests
that the effects of the mutant polymerase and the loss of
Rad52p
activity operate synergistically to generate a high rate of
point
mutations.
POL3 nonsense mutations exhibit a mutator phenotype and
are viable as a consequence of PSI-mediated nonsense
suppression.
In addition to the missense mutants described above,
we also identified five mutants (pol3-ts8, -tsl5,
-ts27, -ts28, and -ts29) representing
four unique nonsense mutants of POL3 (pol3-ts27
is equivalent to pol3-ts29). The positions of these
mutations are shown in Fig. 1. One mutant (pol3-17), which
was not investigated further, had three mutant substitutions (silent
mutation at Y336, L781F, and Q963UAA). All four nonsense mutants
examined were temperature sensitive and MMS sensitive (Fig. 3). In
addition, a large fraction of large budded cells were observed at both
30 and 37°C. The percentages of cells with this morphology at 30°C
for the wild type and pol3-ts8, -ts15,
-ts28, and -ts29 were 7, 37, 35, 42, and
39%, respectively (500 cells were counted). After incubation of
the cells at 37°C for 20 h, the percentages of cells with this
morphology for the wild type and pol3-ts8, -ts15,
-ts28, and -ts29 were 6, 83, 42, 83, and 65%,
respectively (500 cells were counted). The pol3-ts15 mutant
had a smaller fraction of large-budded cells than the other mutants at
37°C and also displayed a less severe growth defect at this
temperature than the other mutants.
Each of these nonsense mutants elevated the rate of mutation at the
CAN1 locus between 5- and 14-fold. The mutation rates
(all
10
7/cell division; 95% confidence limits are shown in
parentheses)
for the wild type and
pol3-ts8,
-ts15,
-ts28, and
-ts29 were 2.4
(2.3 to 2.9), 20 (17 to 22), 12 (8 to 14), 20 (16 to 26), and
33 (30 to 55).
We examined the mutational spectra at
CAN1 for
the nonsense
mutants
pol3-ts8 and
pol3-ts15. As was observed
for
pol3-ts1 and
pol3-ts11, over one-half of the
mutations within
CAN1 generated in strains bearing
either of these two nonsense
mutations were deletions of 16 bp or
greater (Table
6) flanked
by imperfect
direct repeats (data not shown). One mutation identified
in the
pol3-ts8 strain had a deletion followed by a telomere
addition
that was similar to one of the observed mutations in the
pol3-ts11 strain.
Since
POL3 is an essential gene and several of the mutations
were located upstream of the catalytic domain of Pol3p, our recovery
of
viable cells with these mutations was very surprising. One
plausible
explanation for viability, the existence of a nonsense-suppressing
tRNA
gene in the genetic background, was unlikely, since such
suppressors in
yeasts are codon specific (
17) and we had identified
both
amber (UAG) and ochre (UAA)
pol3 mutants.
A different type of suppressor, which can elevate the readthrough
of all nonsense codons, is the prion-like
[
PSI+] factor (
31,
32,
59). The
[
PSI+] factor, encoded by the
SUP35
gene, is a misfolded form of the
translational termination factor
eRF-3. [
PSI+] can be cured from a yeast strain
by plating cells on enriched
medium containing low levels of GuHCl
(
59). To test for the
presence of a
[
PSI+]-like element in our host strain, we
examined the viability of
the strains containing the
POL3
nonsense mutations on medium containing
GuHCl. We found that all of the
temperature-sensitive
POL3 nonsense
mutants failed to grow
on enriched medium containing GuHCl while
an isogenic
POL3+ strain and all of the
POL3
missense mutants grew normally (data
not shown). Since loss of
viability under growth conditions that
cure yeast of
[
PSI+] was specific to the presence of
POL3 nonsense mutations, it
appears that
[
PSI+]-mediated nonsense suppression is
responsible for the viability
of strains containing these
POL3 mutations.
Reduced expression of POL3 elevates mutation
rates.
There are two explanations of the mutator phenotype
exhibited by the pol3 nonsense mutations. Since
[PSI+]-mediated suppression involves the
insertion of an amino acid in place of the termination codon, this
substitution may yield a mutator DNA polymerase. Alternatively, since
the efficiency of [PSI+]-mediated suppression
in yeast is reported to be very low (~1%) (30), the
mutator phenotype may reflect a very low level of fully functional
DNA polymerase. To distinguish between these alternatives, we
replaced the native promoter of POL3 with the galactose-inducible GAL1/10 promoter; in addition, we
inserted an epitope tag (HA) at the beginning of the gene in
order to monitor the level of the protein. From previous studies,
it has been shown that the level of gene expression from the
GAL1/10 promoter can be regulated by altering the
concentration of galactose (3). In most of our experiments,
cells were grown in a constant level of the noninducing carbon source
raffinose (3%) and the level of galactose was varied from 0.5 to 0%.
In a few experiments, we grew cells in 2% glucose. In
glucose-containing medium, the level of expression from the
GAL1/10 promoter is even lower than that from cells grown in
3% raffinose in the absence of glucose because glucose-grown cells
repress expression of the GAL1/10 promoter (45).
To monitor the level of DNA polymerase
, we performed Western blot
analysis (using antibodies directed against the HA epitope)
on protein
extracts of cells grown in various types of media.
As expected, the
level of DNA polymerase
was related to the
concentration of
galactose in the medium (Fig.
4). As a
control,
we monitored the level of DNA polymerase
in a strain in
which
the same HA tag was inserted into a
POL3 gene with its
native
promoter (Fig.
4, lane 8). Giving the level of DNA polymerase
in
this control strain a value/1, the approximate levels of DNA
polymerase
in cells grown in 3% raffinose plus 0.5% galactose
(lanes 1 and 2),
3% raffinose plus 0.05% galactose (lane 3), 3%
raffinose plus 0.1%
galactose (lane 4), 3% raffinose plus 0.005%
galactose (lane 5), and
3% raffinose without galactose (lane 6)
were 27, 8, 3, 0.07, and 0.04, respectively.
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FIG. 4.
Effect of galactose concentration on Pol3p levels in
GAL1-POL3 strain. Cell lysates were prepared and probed for
3×HA-Pol3p by Western blot analysis as described in Materials and
Methods. The location of 3×HA-Pol3p is indicated with a thick arrow,
and the position of a protein nonspecifically recognized by the HA
antibody (used as a loading control) is indicated with a thin arrow.
The positions of the molecular mass standards are shown on the right.
Lanes 1 to 6 contain lysates of RJK368-4
(GAL1-3×HA-POL3 pep4) grown in enriched medium
containing 3% raffinose and the following galactose concentrations:
lanes 1 and 2, 0.5%; lane 3, 0.05%; lane 4, 0.01%; lane 5, 0.005%;
lane 6, no galactose. Lane 7 contains a lysate of RJK366-5 (POL3
pep4) grown in 2% glucose; lane 8 contains a lysate of RJK394
(3×HA-POL3 pep4) grown in 2% glucose. The lysates in lanes
7 and 8 are from strains in which POL3 transcription is
driven from the native POL3 promoter. The amount of lysate
loaded was approximately 30 µg in lanes 2 to 8 and 6 µg in lane 1. The band pattern shown in lane 1 was generated from a much shorter film
exposure than those shown in lanes 2 to 8. The intensities of the Pol3p
bands in lanes 2 to 6 relative to the Pol3p band intensity in
lane 8 are 32, 11, 2.8, 0.084, and 0.060, respectively. Similar
relative intensities derived from a second independent set of lysates
were 6.1, 3.0, 3.0, 0.052, and 0.022. From a third set of
lysates, the relative intensities of the Pol3p band for the samples in
lanes 2 and 3 were 44 and 11, respectively. Averages among all the data
sets for each growth condition are presented in Results.
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|
The effects of reducing
POL3 expression on cell growth and
sensitivity to DNA damage were evaluated. When it was grown with
reduced levels of galactose (0.005%), we found the yeast strain
containing this
GAL1-POL3 construct exhibited a fairly high
level
of sensitivity to MMS but no apparent UV sensitivity (Fig.
5).
In addition, cell viability was
sensitive to the level of DNA
polymerase
in the absence of Rad52p.
As shown in Fig.
6, lowering
the
galactose concentration from 0.05 to 0.005% noticeably lowered
the
ability of a
GAL1-POL3 rad52 strain to sustain growth.
One
interpretation of this result is that a reduction in the level
of
DNA polymerase
leads to an increase in the level of DNA
double-strand
breaks.
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FIG. 5.
Effects of a reduction in the level of DNA polymerase
on DNA repair processes. Fivefold serial dilutions of strains MS71
(POL3; top row of each panel), RJK341 (GAL1-POL3;
second row), EAS102 (rad1; third row), and HM6
(rad52; bottom row) were spotted onto YPR plates
containing the indicated concentrations of galactose in the presence of
MMS or subjected to UV irradiation.
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FIG. 6.
A reduction in the level of Pol3p reduces cell viability
in the absence of Rad52p. Fivefold serial dilutions of strains RJK396
(POL3 RAD52; top row of each panel), RJK397 (POL3
rad52; second row), RJK398 (GAL1-POL3 RAD52; third
row), and RJK399 (GAL1-POL3 rad52; bottom row) were
spotted onto plates containing the indicated medium. All four strains
were derived from the same tetrad following sporulation and dissection
of diploid strain RJK372. The growth pattern shown here is identical to
that from two additional tetrads of the same genotypes.
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|
Surprisingly, we observed no apparent loss in viability or temperature
sensitivity when the growth medium contained low levels
of galactose in
a
RAD52 strain with the
GAL1-POL3 construct
(RJK398)
(Fig.
6 and data not shown). There was, however, a larger
percentage
of large-budded cells (40%) present during growth
with 0.005%
galactose compared to that seen during growth with
0.05% galactose
(10% large-budded cells). In the absence of galactose
(3% raffinose),
a condition in which there is a very low level of
DNA polymerase
, the growth rate is substantially reduced at both 30 and 37°C,
although there was still no apparent temperature
sensitivity under
these growth conditions. In addition, a much higher
percentage
of cells grown in the absence of galactose are large budded
(65%).
Furthermore, most of the unbudded cells are enlarged and
irregular
in shape. The above-mentioned growth defects in the absence
of
galactose are further exacerbated when this strain is grown in
enriched medium containing only glucose (YPD). Cells incubated
in YPD
medium grew very slowly and were not further
evaluated.
When the strain bearing the
GAL1-POL3 construct was
examined for the
CAN1 mutation rate, we observed a gradual
increase in
the mutator phenotype as the level of Pol3p was reduced.
The rates
of
can1 mutations in the strain with the
GAL1/10-POL3 gene (all
10
7/cell division; 95%
confidence limits are in parentheses) when
grown in 3% raffinose plus
0.5% galactose, 3% raffinose plus 0.05%
galactose, 3%
raffinose plus 0.01% galactose, 3% raffinose plus
0.005%
galactose, and 3% raffinose without galactose were 1.5
(1.3 to
1.6), 1.7 (1.6 to 2.1), 5.6 (4.9 to 7.4), 15 (11 to 18),
and 56 (43 to
95), respectively. When the strain with the HA-tagged
POL3
gene and the native promoter was grown in 3% raffinose plus
0.05%
galactose, 3% raffinose without galactose, and YPD medium,
the
mutation rates (all 10
7/cell division; 95% confidence
limits are in parentheses) were
2.6 (2.2 to 3.6), 2.6 (2.1 to 3.6), and
4.4 (3.4 to 5.7), respectively.
Finally, when a strain with an untagged
version of the
POL3 gene
with its native promoter was grown
in medium containing 3% raffinose
plus 0.05% galactose, we observed a
rate of mutation of 1.4 (1.1
to 1.9) × 10
7/cell
division. We conclude from these studies that reducing the
level of
fully functional DNA polymerase
can substantially elevate
global
mutation rates, whereas overexpression of DNA polymerase
does not
result in a mutator
phenotype.
We also examined the spectra of mutations generated in cells with low
levels of DNA polymerase (Table
7).
Although cells
grown in medium with 3% raffinose plus 0.05% galactose
(no mutator
effect) had a spectrum of alterations similar to that
observed
in the wild-type strain, the same strain grown in medium with
3% raffinose and 0.005% galactose or no galactose had a significantly
elevated frequency of deletions between direct repeats. Thus,
the
mutator phenotype in strains with reduced levels of Pol3p
resembles
that observed for some of the
POL3 missense mutations
and
the
POL3 nonsense mutations.
| |
DISCUSSION |
The low level of mutations observed in wild-type cells is a
consequence of the serial operation of multiple levels of control of
the accuracy of DNA replication: base selectivity by DNA polymerase, exonucleolytic proofreading, and postreplicative DNA mismatch repair
(44). If frameshifts within simple repetitive DNA sequences and deletions between direct repeats reflect DNA polymerase slippage events, the low level of such alterations in wild-type cells is likely
to reflect the processivity of replicative DNA polymerases and the
efficiency of postreplicative DNA mismatch repair. One might expect,
therefore, mutator alleles of DNA polymerase that reduce base
selectivity, the efficiency of proofreading, or the processivity of
replication. In addition, a DNA polymerase mutation could
indirectly result in a mutator phenotype. For example, if the level of
an accurate replicative DNA polymerase was lowered, its role could be
partially taken over by an error-prone DNA polymerase.
One of the POL3 missense mutants, pol3-ts26,
elevated the rate of base substitution mutations with only a minor
effect on the rate of deletions. This mutant polymerase could have a
partial defect in base selectivity or proofreading. Strains with the
other two missense mutations (pol3-ts1 and
pol3-ts11), the nonsense pol3 mutations, or
reduced levels of DNA polymerase had elevated levels of deletions,
as well as base pair substitutions; these deletions, like those
observed in rfa1 strains (7), involved interactions between short dispersed repeats.
Mechanism of deletion formation.
Deletions could result from
DNA polymerase slippage (Fig. 2B) or single-strand annealing (SSA)
(Fig. 2C). Our conclusion that many of the deletions result from
DNA polymerase slippage is based primarily on a comparison of the sizes
of the deletions generated in pol3 mutant strains and in
strains with both pol3 and msh3 mutations. In
single-mutant pol3-ts1 and pol3-ts11 strains, all deletion mutations involving the CAN1 locus were
single-base-pair deletions in homopolymeric regions or deletions of at
least 16 bp. In contrast, in a strain with both the pol3-ts1
and msh3 mutations, over 70% of the deletions were more
than 1 bp but less than 16 bp (Table 3). Msh3p is involved in the
repair of DNA loops ranging in size from 1 to 14 bp (48,
60). We suggest, therefore, that the pol3-ts1 and
pol3-ts11 mutations result in increased levels of DNA
polymerase slippage and increased rates of formation of DNA
loops. Since DNA loops smaller than 16 bp are efficiently corrected in
single-mutant pol3 strains, we observe an increase in
mutation frequency only in deletions of 16 bp or larger. In the
double-mutant pol3 msh3 strains, deletions of both classes are observed, as expected by the DNA polymerase slippage model. This
model also predicts the synergistic effect of pol3-ts1 and msh3 on mutation rates at the CAN1 locus and the
synergistic destabilization of the poly(GT) tract.
An alternative model for the generation of deletions is
by SSA (Fig.
2C). Although SSA requires
MSH3 (as
well as
MSH2,
RAD1,
and
RAD10), if the
distance between interacting repeats is greater
than 60 bp
(
39), deletions smaller than 60 bp could be generated
by
this mechanism. However, since this mechanism does not explain
the lack
of deletions of less than 16 bp in the
pol3-ts1 strain
and
the prevalence of these deletions in the
pol3-ts1 msh3
strain,
we prefer the DNA polymerase slippage model. Several additional
points concerning this conclusion should be mentioned. First,
our
conclusion is rigorous only for deletions of 16 bp or less,
although it
seems reasonable that somewhat larger deletions are
also generated by
DNA polymerase slippage. Second, it is likely
that very large deletions
(greater than 1 kb) represent SSA events.
Chen et al. (
7)
found that a mutant allele of
RFA1 elevated
frequencies of
large deletions (mostly larger than 6 kb) in the
CAN1
locus; they reported that deletion formation was dependent
on
Rad10p, as expected for SSA events. It should be pointed out
that,
although Rad1p and Rad10p have roles in the meiotic repair
of large DNA
loops (
21),
rad1 strains do not appear to be
deficient
in the mitotic repair of large DNA loops (E. Sia and T. Petes,
unpublished
data).
Rad52p is required for most types of homologous recombination in yeast,
including SSA (
40). In strains with mutations in
POL3 and
RAD52, very few deletions are observed.
This observation
can be interpreted in two ways. One possibility is
that deletions
are generated by SSA; as described above, this
possibility is
unlikely for deletions of less than 16 bp. An
alternative possibility
is that Rad52p promotes DNA polymerase slippage
between short
dispersed repeats. Following DNA strand dissociation,
Rad52p could
help the nascent strand seek out regions of homology in
single-stranded
regions of the replication fork. Using a different
assay for mutations,
Tran et al. (
55) observed Rad52p
dependence of deletion formation
and suggested a nonrecombinational
role for Rad52p in this
process.
We observed a substantial increase in the rate of base substitutions in
a
rad52 strain and a synergistic effect between
pol3-ts1 and
rad52. It has previously been shown
that loss of Rad52p function
also increases the rate of base
substitutions at the
SUP4 locus
and that this increase is
dependent on the presence of the error-prone
DNA polymerase
(
42). Since DNA polymerase
normally functions
in
translesion DNA synthesis (
11), the elevated mutation rates
observed in the
rad52 strain may be a consequence of
error-prone
repair synthesis at regions of spontaneous DNA damage. The
synergistic
effect of
pol3-ts1 and
rad52 on
the rate of base substitutions
may reflect an increase in DNA damage
associated with the
pol3-ts1 mutation.
One interpretation of the phenotypes associated with the
pol3-ts1 and
-ts11 mutations is that the mutant
polymerases are less
processive. Reduced processivity associated with
phage T7 DNA
polymerase lacking the processivity factor thioredoxin and
a mutant
of the Klenow fragment DNA polymerase has been correlated with
increased microsatellite instability (
26,
36). For the yeast
DNA polymerase mutants, reductions in processivity may be a consequence
of defective interactions with the processivity factor PCNA. Regions
within the N-terminal region of Pol3p appear to interact with
PCNA, and
the processivity of DNA polymerase
depends on this
interaction
(
5,
61). Alternatively, increases in frameshift
mutagenesis
could reflect defective interactions within the polymerase
active site,
as observed for a mutant human DNA polymerase
(
38).
A
final alternative is that the
pol3-ts1 and
-ts11
mutations result
in lower levels of DNA polymerase, producing a mutator
phenotype
by the mechanisms described below for strains with low levels
of DNA polymerase
.
Nonsense mutations of POL3.
We recovered viable
POL3 nonsense mutations because of
[PSI]-mediated readthrough of the nonsense codons. Since
this suppression is inefficient (30), the mutator phenotype
of the POL3 nonsense mutations (increased frequencies of
deletions with a smaller effect on base pair substitutions) presumably
reflects the same mechanisms (described below) observed in strains with
low levels of DNA polymerase . The temperature-sensitive phenotype
of the nonsense mutations is likely to be a consequence of the
temperature-sensitive efficiency of [PSI]-dependent
nonsense suppression (18). One interesting consequence of
this mechanism is the fact that, in strains with the nonsense
POL3 mutations, the [PSI] factor (usually
dispensable) is essential.
Mutator phenotype generated by depleted levels of DNA polymerase
.
When the level of DNA polymerase was reduced, we
observed elevated rates of deletions (presumably reflecting
elevated rates of DNA polymerase slippage) and base pair substitutions.
Since slippage could occur during the transition between
discontinuous synthesis (associated with dissociation of DNA
polymerase from the template) and continuous synthesis (associated
with attachment of DNA polymerase to the template), a reduction
in the level of wild-type DNA polymerase molecules could result in
increased slippage. Similarly, delayed DNA synthesis on the lagging
strand could lead to increased single-stranded regions in the
replication fork and the opportunity for the occurrence of slippage
events that involve dispersed small repeats, resulting in deletions.
Although there is a strong inverse correlation between polymerase
levels and mutation rates in the
GAL1-POL3 strain, there
is
one apparent discrepancy. Growth of this strain in YPR medium
containing 0.01% galactose results in a threefold increase in
the
level of DNA polymerase
over that observed in a strain in
which
POL3 is normally expressed under the control of its
native
promoter. However, the mutation rate of the
GAL1-POL3
strain under
these growth conditions is twice that of the normal
observed rate.
One possible explanation is that
GAL1-POL3
expression is constitutive
while
POL3 expression under its
native promoter occurs primarily
during S
phase.
Although the most obvious mutator phenotype of reducing the level of
DNA polymerase
was an effect on the rate of deletion
formation, base pair substitutions were also elevated. If reducing
the
level of DNA polymerase
increases the size of single-stranded
regions at the replication fork, there may be more spontaneous
DNA
damage. Such damage could have two consequences: (i) reduction
in
viability in strains with a
rad52 mutation (as observed) and
(ii) increased levels of mutations resulting from repair of damaged
DNA
by an error-prone DNA polymerase, such as DNA polymerase
.
It is
somewhat surprising that gross overproduction of DNA polymerase
had
no mutator phenotype, since dominant-negative effects caused
by
overproducing one subunit of a multienzyme complex are quite
common in
yeast (
41).
Our observations may be relevant to understanding certain human
diseases associated with genomic instability. One type of
genomic
instability (microsatellite instability) in patients with
hereditary
nonpolyposis colorectal cancer is associated with mutations
in genes
involved in DNA mismatch repair (
9). We suggest that
mutator
variants of DNA polymerase
or epigenetic reduction in
the level of
DNA polymerase
could be another source of genome
destabilization.
Mutations of the human DNA polymerase
gene
have been detected in
cancer cell lines, although the functional
significance of these
mutations has not yet been established (
10).
In order for
mutations in DNA polymerase genes to promote tumor
formation, they
would need to have a strong mutator phenotype
without a strong growth
defect, possibly a difficult balance to
achieve.
| |
ACKNOWLEDGMENTS |
We thank the members of the Petes lab for helpful discussions and
suggestions throughout the course of this project, A. McKenzie III for
advice on the construction of the GAL1-POL3 strain, P. Burgers for supplying pBL304, and R. Rybczynski for advice on the use
of the scanning densitometer. We also thank Y. Chernoff for helpful
discussions regarding [PSI].
This work was supported by NIH grants GM52319 (T.D.P.) and GM17879
(R.J.K.).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biology and Curriculum in Genetics and Molecular Biology, University of
North Carolina, Chapel Hill, NC 27599-3280. Phone: (919) 962-1445. Fax:
(919) 962-8472. E-mail: tompetes{at}emailunc.edu.
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Abstract
Introduction
