MOLECULAR AND CELLULAR BIOLOGY, Nov. 2005, p. 9221–9231
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 25, No. 21
Novel PMS1 Alleles Preferentially Affect the Repair of Primer Strand
Loops during DNA Replication
Naz Erdeniz,1Sandra Dudley,1Regan Gealy,2Sue Jinks-Robertson,2and R. Michael Liskay1*
Molecular and Medical Genetics, Oregon Health and Science University, L103, 3181 SW Sam Jackson Park Road,
Portland, Oregon 97239,1and Department of Biology, Emory University, 1510 Clifton Rd., Atlanta, Georgia 303222
Received 14 March 2005/Returned for modification 11 April 2005/Accepted 21 July 2005
Null mutations in DNA mismatch repair (MMR) genes elevate both base substitutions and insertions/
deletions in simple sequence repeats. Data suggest that during replication of simple repeat sequences, poly-
merase slippage can generate single-strand loops on either the primer or template strand that are subsequently
processed by the MMR machinery to prevent insertions and deletions, respectively. In the budding yeast
Saccharomyces cerevisiae and mammalian cells, MMR appears to be more efficient at repairing mispairs
comprised of loops on the template strand compared to loops on the primer strand. We identified two novel
yeast pms1 alleles, pms1-G882E and pms1-H888R, which confer a strong defect in the repair of “primer strand”
loops, while maintaining efficient repair of “template strand” loops. Furthermore, these alleles appear to affect
equally the repair of 1-nucleotide primer strand loops during both leading- and lagging-strand replication.
Interestingly, both pms1 mutants are proficient in the repair of 1-nucleotide loop mispairs in heteroduplex
DNA generated during meiotic recombination. Our results suggest that the inherent inefficiency of primer
strand loop repair is not simply a mismatch recognition problem but also involves Pms1 and other proteins
that are presumed to function downstream of mismatch recognition, such as Mlh1. In addition, the findings
reinforce the current view that during mutation avoidance, MMR is associated with the replication apparatus.
DNA mismatch repair (MMR) contributes to genomic in-
tegrity by repairing mismatches generated during replication,
by chemical damage, and as “heteroduplex” intermediates dur-
ing recombination (7, 28, 31, 35, 44). In addition, the MMR
system in higher eukaryotes plays a role in response to DNA
damage (3, 6, 7, 62). Inherited MMR defects lead to a mutator
phenotype, which in humans and mice is associated with in-
creased cancer susceptibility (5, 7, 13, 16, 38, 50). The MMR
system of Escherichia coli has been reconstituted in vitro with
purified proteins, including the dedicated proteins MutS,
MutL, and MutH (44, 56). The MutS protein, a homodimer,
first binds the mispair, followed by recruitment of MutL, the
endonuclease MutH, the UvrD helicase, four exonucleases,
DNA polymerase, and ligase. Together with transient Dam-
mediated hemimethylation, these proteins impose strand spec-
ificity that leads to specific repair of the newly replicated strand
(10, 25, 26, 43, 44, 74).
In the budding yeast Saccharomyces cerevisiae, six MutS ho-
mologues (Msh proteins Msh1 to Msh6) and four MutL ho-
mologues (Mlh proteins, Mlh1 to Mlh3, and Pms1) function in
various MMR transactions (7, 28, 31, 35). Unlike E. coli, the
MutS and MutL activities of budding yeast and mammals are
each comprised of heterodimers. Mismatches in nuclear DNA
replication intermediates are recognized by the Msh2/Msh6
and Msh2/Msh3 heterodimers, which have partial functional
overlap (7, 35, 42). Msh2/Msh6 operates in the repair of base-
base mispairs and 1-nucleotide “insertion/deletion” loops (28,
32, 41), while Msh2/Msh3 functions in the repair of 1- to
4-nucleotide insertion/deletion loops (28, 32, 41). Similarly for
the MutL homologs, Mlh1 forms heterodimers with Pms1,
Mlh2, or Mlh3 (19, 28, 30, 49, 72). Genetic studies indicate that
the Mlh1/Pms1 heterodimer is the primary MutL activity in
MMR-mediated mutation avoidance, whereas the Mlh1/Mlh3
complex plays a minor role in Msh2/Msh3-dependent repair of
insertion/deletion loops (19, 24, 30, 49, 53, 54). Current data
suggest that initial recognition of the mismatch is by Msh2/
Msh6 or Msh2/Msh3, possibly aided by PCNA, which is sub-
sequently joined by Mlh1-Pms1 to form a higher order complex
on DNA. This complex is thought to be responsible for direct-
ing the downstream and less well characterized MMR events,
including strand discrimination, excision, and resynthesis (4, 7,
28, 35, 42).
Although the MutS and MutL proteins must interact during
MMR-mediated mutation avoidance, the nature of these and
other protein-protein interactions is not clear. Whereas
MLH1, PMS1, and MHS2 deletion mutations appear to result
in a null, or near null, MMR state for mutation avoidance,
specific pms1 or mlh1 mutant alleles might produce a novel
mutator phenotype, for example, by differentially impacting
interactions with Msh2/Msh3 or Msh2/Msh6. In turn, these
types of alleles might provide additional insights into the func-
tion of the MutL homologs, including interactions with other
MMR factors. Therefore, we have initiated genetic screens for
mutations of PMS1 or MLH1 that result in mutational spectra
different from the corresponding MMR-null strains. Here, we
report the isolation of two novel pms1 alleles, which in contrast
to a pms1? strain preferentially elevate ?1-bp frameshifts in
mononucleotide runs, with little or no effect on ?1-bp frame-
shifts. These pms1 alleles are in close proximity, affecting res-
idues near the end of the protein in a previously uncharacter-
ized but highly conserved amino acid motif. Further analyses
* Corresponding author. Mailing address: Molecular and Medical
Genetics, Oregon Health and Science University, L103, 3181 SW Sam
Jackson Park Rd., Portland, OR 97239-3098. Phone: (503) 494-3475.
Fax: (503) 494-6886. E-mail: email@example.com.
suggest that these pms1 alleles do not differentially impact
MMR during leading- versus lagging-strand replication but,
rather, fail to efficiently repair single nucleotide loops arising
on the primer strand. The results presented here reinforce the
current view that MMR is associated with the replication ap-
MATERIALS AND METHODS
Media and growth conditions. All media were prepared as described (58)
except that synthetic medium contained increased leucine (60 mg/liter). Growth
and sporulation were at 30°C and at 18°C, respectively. Sporulation of diploid
cells and tetrad dissections were performed as previously described (15, 73).
Strain constructions. Yeast strains used for assaying CAN1 forward mutations
or his1-7, his7-2, and hom3-10 reversion are derivatives of a RAD5 CAN1
W303-1B strain (65) (Table 1). The hom3-10 allele was introduced at the HOM3
locus by a two-step recombination procedure using the plasmid pK8 linearized
with SpeI (41). Presence of the hom3-10 allele was verified by the inability of the
strain to grow on medium lacking threonine. The his1-7 (71) and his7-2 (57)
alleles were introduced by a cloning-free, PCR-based allele replacement ap-
proach (14). The sequences of primers are the following (with uppercase letter
sequences denoting the adaptamer sequences and lowercase letters denoting
HIS1 or HIS7 sequences): his1-7 Adap A (5?-AATTCCAGCTGACCACCATG
AAattgagagaaaaacgaaggg-3?) and his1-7 Adap B (5?-GATCCCCGGGAATTG
CCATGctgacaaatatgctacgaag-3?) for his1-7, and his7-2 Adap A (5?-AATTCCA
GCTGACCACCATGgcgtcgggctacaagcgc-3?) and his7-2 Adap B (5?-GATCCC
CGGGAATTGCCATGctgtgccaactgaacaggc-3?) for his7-2. Introduction of the
mutant allele was verified by the inability of the strain to grow on medium lacking
histidine and confirmed by sequencing analysis.
Strains containing 10C runs were constructed using a derivative of YPH45
(MATa ura3-52 ade2-101octrp1?1) in which the open reading frame of LYS2 was
replaced with a hygromycin resistance cassette (lys2?::hyg) by transformation
with a PCR fragment derived from hphMX4 (20). A wild-type LYS2 gene was
then inserted at the HIS4 locus on chromosome III by transformation with a
PCR fragment amplified from pDP6 (17) using primers with terminal homology
to the HIS4 locus. The primer pair Lys2FHis4F2 (5?-ACTTGGTTGAACAAT
gaggcatcgcacagttttagc-3?; base pairs complementary to HIS4 sequences are in
uppercase while those complementary to LYS2 are in lowercase) and
Lys2RHis4R (5?- GTTCGGTTTCCAAGTTAGAAATAATCTACTGGAAAT
CCTTTGGGATCAACCCAAGCTTACccgaaaagaagctaagtctt-3?) were used to
TABLE 1. Strains used in this study
MAT? ade2-1 CAN1 his3-11,15 leu2-3,112 trp1-1 ura3-1 RAD5
MAT? ade2-1 CAN1 his1-7 hom3-10 leu2-3,112 trp1-1 ura3-1 RAD5; HIS3 his1-7 derivative of NEY190
MATa ade2-1 CAN1 his7-2 hom3-10 leu2-3,112 trp1-1 ura3-1 RAD5; HIS3 his7-2 derivative of NEY190
NEY570 pms1::TRP1 msh2::LEU2
NEY570 pms1-G882E msh2::LEU2
NEY570 pms1-H888R msh2::LEU2
NEY570 pms1::TRP1 pol2-C1089Y
NEY570 pms1-G882E pol2-C1089Y
NEY570 pms1-H888R pol2-C1089Y
MATa ade2-101ochis4?::LYS2R-10C lys2?::hyg trp1? ura3-52
SJR1973 pms1::URA3 ogg1::TRP1
SJR1973 pms1-G882E ogg1::TRP1
SJR1973 pms1-H888R ogg1::TRP1
MATa ade2-101ochis4?::LYS2F-10C lys2?::hyg trp1? ura3-52
SJR1977 pms1::URA3 ogg1::TRP1
SJR1977 pms1-G882E ogg1::TRP1
SJR1977 pms1-H888R ogg1::TRP1
MAT? trp1 arg4 tyr7 ade6 ura3
MATa leu2 ade6 ura3 his4-713
9222ERDENIZ ET AL.MOL. CELL. BIOL.
generate a fragment for inserting LYS2 at HIS4 in the forward orientation
(his4?::LYS2F), with LYS2 transcribed in the same direction as HIS4. The
primer pair Lys2Fhis4R2 (5?-GTTCGGTTTCCAAGTTAGAAATAATCTACT
and Lys2RHis4F (5?-ACTTGGTTGAACAATTGAATGTACCAAAGGAGCG
TGTTGTTGTGGAAGAGAACGGTGTTTccgaaaagaagctaagtctt-3?) were used
to generate a fragment for inserting LYS2 at HIS4 in the reverse orientation
(his4?::LYS2R), with LYS2 transcription opposing that of HIS4. In strains with
the his4?::LYS2F allele, transcription and replication fork movement proceed in
the same direction; in strains with the his4?::LYS2R allele, the direction of
transcription opposes that of replication fork movement.
Mononucleotide run of 10C was inserted into the coding strand of the
his4?::LYS2 alleles using the delitto perfetto method (63). First, the CORE
cassette containing URA3 and Kan was amplified with the primer pair
TGGAAAGGACCCCTCgagctcgttttcgacactgg-3?; uppercase letters denote the
LYS2 sequence and lowercase letters denote CORE cassette sequence) and
GCCAAACGGAACAACTtccttaccattaagttgatc-3?), with the CORE cassette in-
serted after base pair position 406 relative to the LYS2 start codon. Following
transformation with the amplified fragment, Ura?, geneticin-resistant transfor-
mants were selected and screened for a Lys?phenotype. The CORE cassette
within LYS2 was then replaced with a 240-bp LYS2-10C fragment amplified from
SJR1354 (21). Lys?transformants were selected and screened for loss of CORE
sequences (Ura?, geneticin-sensitive phenotype). The presence of the 10C run
was confirmed by sequence analysis. Runs are named according to the nucleo-
tides on the coding strand; a LYS2-10C allele thus has a 10C run on the coding
strand and the complementary 10G run on the noncoding strand.
PMS1 and OGG1 disruptions were constructed in relevant haploid strains by
a PCR-based gene disruption method (2) utilizing TRP1 and URA3 for PMS1
and TRP1 for OGG1 as selective markers and the following primer pairs (up-
percase letters indicate sequences complementary to PMS1 or OGG1 sequences
and lowercase letters denote selective marker sequences): PMS1-L1 (5?-CGAA
gtactgag-3?) and PMS1-L2 (5?-ATAATGTATTTGTTAATTATATAATGAAT
GAATATCAAAGCTAGAtgtgcggtatttcacacc-3?; OGG1-L1 (5?-TTTGAAGCG
3?) and OGG1-L2 (5?-TTCGGTCGCGTGCTTTTATCGTGGTATTTACTAT
GACTTTTTAAGtgtgcggtatttcacacc-3?). Each disruption was confirmed by PCR.
The alleles pms1-G882E and pms1-H888R were each integrated into the ge-
nome by the cloning-free, PCR-based allele integration approach described
above (14). Primers used to amplify either pms1-G882E or pms1-H888R are the
following (with uppercase letters denoting the adaptamer sequence and lower-
case letters for PMS1 sequence): PMS1 C-term Adap A (5?-aattccagctgaccacca
tgGAAGACGGTGGGTTACGAAG-3?) and PMS1 C-term Adap B (5?-gatcccc
gggaattgccatgCAAGCATCTTCAATGCACGAG-3?). The fusion fragments
were cotransformed into relevant strains and Ura?transformants were selected.
Subsequent retention of a single copy of the mutant pms1 allele after 5-fluoroo-
rotic acid selection was verified by PCR and sequencing using primers PMS1
C-term Adap A and PMS1 C-term Adap B.
pol2-C1089Y was introduced by a two-step allele replacement method (55).
AgeI linearized p173-rsa (33) was transformed into cells, followed by selection on
medium lacking uracil. Ura?transformants were purified and grown in YPD
medium (containing yeast extract, peptone, and glucose) and streaked to 5-fluo-
roorotic acid plates to select for Ura?colonies, which were screened for the
pol2-C1089Y mutation by PCR and diagnostic digestion with RsaI.
Plasmid constructions. Plasmid pRS414-PMS1 was constructed by inserting a
4-kb chromosomal BglII/SalI fragment that contains the 2,715-bp PMS1 open
reading frame (ORF) into BglII/SalI-digested pRS416 (URA3-CEN vector ).
Plasmids used in two-hybrid assays were constructed by inserting coding se-
quences of MLH1 and PMS1 into pBTM116 and pGAD424, which contain the
LexA DNA-binding domain and Gal4 activation domain, respectively (49). The
pms1-G882E and pms1-H888R alleles were introduced into the pGAD424-PMS1
plasmid by gap repair (40, 48). Three independent plasmids from each transfor-
mation were sequenced to verify the pms1-G882E and pms1-H888R mutations.
Construction of a randomly mutagenized PMS1 library. PCR mutagenesis of
the PMS1 ORF was carried out in two separate reactions (75). The primer pair
PMS1-MluI (5?-GCACAGATTAATACCGATTC-3?) and PMS1-BsaBI (5?-GC
GTAGAGTATTCCACTGGC-3?) and the pair PMS1-ClaIF (5?-CGCAGAGA
TTGAGCCAGTTG-3?) and PMS1-ClaIRev (5?-GACGATTGAAGGAGACG
CTAG-3?) were used to mutagenize the first approximately one-third (fragment
I) and the last approximately one-third (fragment II) of the PMS1 ORF, respec-
tively. The PCR mixture contained the following components: 10 mM Tris-HCl,
1.5 mM MgCl2, 50 mM KCl (pH 8.3), 0.25 mM MnCl2, a 200 ?M concentration
of each deoxynucleoside triphosphate, a 1 ?M concentration of each primer, 5 U
of Taq DNA polymerase, and 10 ng of plasmid pRS414-PMS1. The PCR con-
ditions were 5 min at 94°C and 40 cycles of 15 s at 94°C, 15 s at 50°C, and 1 min
at 72°C, followed by 10 min at 72°C.
The PCR-mutagenized fragments I and II were gel purified and cotransformed
individually into yeast strain NEY186 with pRS416-PMS1 linearized with MluI/
BsaBI or ClaI, respectively. Transformants were selected on synthetic complete
medium lacking uracil. In vivo homologous gap repair recombination between
the PCR fragments and the vector DNA produced a library of mutagenized pms1
alleles on a CEN plasmid. Control transformations with only the gapped vector
indicated ?95% efficiency in gap repair. A total of 2,000 transformants of each
mutagenized fragment were patched onto medium lacking uracil. After 2 days,
the patches were replica plated onto synthetic complete medium lacking uracil
and arginine but containing canavanine and onto medium lacking uracil and
threonine to score mutation at CAN1 and hom3-10, respectively. Plasmid DNAs
from two transformants that consistently exhibited a mutator phenotype on
canavanine, but not in the hom3-10 reversion assay, were used to retransform
Rate measurements and statistical analyses. The method of the median was
used to calculate the mutation rate (37). Data from at least 20 independent
cultures (typically four cultures from each of five independent isolates) were used
for each rate determination. In all cases, different isolates behaved the same as
evidenced by side-by-side comparisons. Briefly, purified colonies were grown in
liquid yeast extract-peptone-dextrose medium to saturation. Appropriate dilu-
tions were plated onto complete synthetic medium to determine the viability of
the cells. Medium lacking histidine was used to select His?prototrophs; medium
lacking threonine was used to select revertants of hom3-10; medium lacking
arginine but containing canavanine was used to select can1 mutants; and medium
containing alpha amino-adipic acid (8) was used to select Lys?mutants. Colonies
were counted 3 to 5 days after selection plating. Statistical analyses were per-
formed using Prism 3.0 software (GraphPad Software Inc.). The efficiencies of
mismatch correction (repair efficiencies) were determined by comparing the
rates of instability at the LYS2-10C locus in pms1? strains to the rates observed
in wild-type, pms1-G882E, and pms1-H888R strains (59). If we denote the rate of
instability as Rwt, Rpms1?, Rpms1-G882E, and Rpms1-H888Rin wild-type, pms1?,
pms1-G882E, and pms1-H888R strains, respectively, then the repair efficiency is
calculated by substracting the mutation rate after repair (Rwt, Rpms1-G882E, or
Rpms1-H888R) from mutation rate before repair (Rpms1?) and then dividing this
difference by the mutation rate before repair (Rpms1?).
Two-hybrid analysis. Protein-protein interactions were assayed by the two-
hybrid method as previously described (49).
Sequence analysis of mutants. Independent isolates for sequence analysis
were obtained by streaking individual colonies onto appropriate selective me-
dium. The relevant gene regions were amplified from the yeast genome by
“colony PCR” (39). PCR products were sequenced using the following primers:
for CAN1, CAN1 For (5?-CTTAACTCCTGTAAAAAC-3?), CAN1 Seq1 (5?-C
ATTGGCCGCACCAAATGC-3?), CAN1 Seq2 (5?-TTCATCCCTGTTACATC
C-3?), CAN1 Seq3 (5?-CCAAATGCAGCAGTAACG-3?), and CAN1 Rev (5?-G
AAATGGCGTGGGAATGT-3?); for LYS2, LYS2 5? (5?-GCTACATATTCGT
TACAGC-3?) and LYS2 3? (5?-GGTCCGCAACAATGGTTACTC-3?); and for
his1-7, HIS1 For (5?-CTCCTATTAACGGTTTGAATC-3?) and HIS1 Rev (5?-
To understand better the role of the Mlh1/Pms1 (MutL?)
heterodimer in MMR, we set out to identify alleles of PMS1
that differentially impact MMR-mediated mutation avoidance.
Thus, we mutagenized the most conserved regions of the yeast
PMS1 ORF and screened for alleles that resulted in a mutator
phenotype different from that seen in a pms1? strain. A library
of randomly mutagenized PMS1 genes in a CEN vector was
screened initially in a pms1? strain using two mutation assays:
reversion of hom3-10 and forward mutation to canavanine
resistance at CAN1, which report ?1-bp frameshifts in a 7A/7T
run and multiple types of mutations, respectively (Fig. 1a and
b). Previous work demonstrated that in a pms1? strain, rever-
sion of hom3-10 was increased approximately 1,000-fold while
VOL. 25, 2005PMS1 ALLELES AND PRIMER STRAND LOOP REPAIR9223
forward mutation at the CAN1 locus was increased 30-fold,
with more than 60% of the can1 mutations being ?1-bp frame-
shifts in short mononucleotide runs (67). In a pms1? strain,
elevated mutation levels at hom3-10 and CAN1 can be de-
tected by an increased number of papillae on the appropriate
selective media (Fig. 2a). Whereas most mutator strains in-
creased papillation in both assays, two exceptional mutants
consistently conferred a strong increase in the number of can1
FIG. 1. Schematic of four mutator assays. (a) hom3-10 reversion assay measures ?1-bp frameshifts in a stretch of 7A/7T bp. (b) CAN1 forward
mutation assay detects multiple types of mutations. (c) his1-798 (his1-7) reversion assay measures intragenic missense suppressor mutations near
the 3? end of HIS1. (d) his7-2 reversion assay reports ?1-bp frameshifts in a run of 7A/7T bp.
FIG. 2. (a) Papillation phenotypes of the wild-type, pms1?, pms1-G882E, and pms1-H888R strains. The relative mutator effects at hom3-10,
his7-2, and CAN1 alleles in wild-type, pms1?, pms1-G882E, and pms1-H888R strains were detected initially by monitoring reversion at hom3-10
and his7-2 and the forward mutation of CAN1 alleles individually by replica plating patches of cells onto appropriate selective media. (b) Pms1
domains and location of altered Pms1 residues. The ATPase domain (residues 54 to 144) and the Mlh1-interacting domain (residues 692 to 904)
are indicated by dotted and black boxes, respectively. Also shown are the residues corresponding to the COOH termini of yeast and human
homologs. The mutated residues, G882 and H888, are shaded gray. The 13-amino-acid domain, identical between yeast and human homologs, is
depicted below the alignment of yeast and human homolog sequences. Numbers correspond to the amino acid position in the protein.
9224 ERDENIZ ET AL.MOL. CELL. BIOL.
papillae but no detectable increase in reversion at hom3-10
(Fig. 2a). We did not detect any candidates with the opposite
phenotype. To address strain-specific effects, we tested the two
pms1 alleles on CEN plasmids in two additional strain back-
grounds, GCY35 (45) and AMY125 (59), and observed similar
results (data not shown). DNA sequencing analysis of these
two unusual pms1 alleles revealed in each strain the presence
of a single mutation resulting in a change of glycine to gluta-
mate at residue 882 (pms1-G882E) or histidine to arginine at
residue 888 (pms1-H888R) (Fig. 2b).
Pms1-G882E and Pms1-H888R interact efficiently with
Mlh1 and are expressed at wild-type levels. Because residues
G882 and H888 both lie in the C-terminal region of Pms1,
which is essential for interaction with Mlh1 (49), we tested the
ability of the mutant proteins to interact with Mlh1 using a
two-hybrid assay. As shown in Table 2, both mutant forms of
Pms1 interacted efficiently with Mlh1. In addition, we deter-
mined the expression levels of Pms1-G882E and Pms1-H888R
at the endogenous PMS1 locus. No significant differences be-
tween the levels of FLAG-tagged Pms1-G882E, Pms1-H888R,
or wild type Pms1 were observed using Western blot analysis
(data not shown). Together, comparison of Pms1 protein levels
and the two-hybrid assay suggest that the pms1-G882E and
pms1-H888R alleles have little if any effect on protein stability
or interaction with Mlh1.
Further analysis of the pms1-G882E and pms1-H888R mu-
tator phenotype. To confirm the differential effects of pms1-
G882E and pms1-H888R in the hom3-10 and CAN1 mutator
assays, the plasmid-encoded alleles were introduced at the
endogenous PMS1 locus in wild-type strain NEY190. As shown
in Table 3, both pms1-G882E and pms1-H888R strains dis-
played significantly elevated mutation rates at CAN1, although
both alleles caused less of a mutator phenotype than seen in a
pms1? strain (3-fold and 5-fold increases, respectively, versus a
17-fold increase in pms1?). In contrast, whereas pms1? re-
sulted in a 1,100-fold increase in hom3-10 reversion, the pms1-
G882E and pms1-H888R strains displayed only 3- to 4-fold
increases in hom3-10 reversion relative to wild type (Table 3).
To gain additional insight into the MMR defects, we tested
the pms1-G882E and pms1-H888R alleles using his1-798
(his1-7) reversion, which reports intragenic missense suppres-
sor mutations near the 3? end of HIS1 (Fig. 1) (71). First, the
his1-7 allele was integrated and characterized in wild-type and
pms1? backgrounds. pms1? strains displayed a ninefold ele-
vated his1-7 reversion rate compared to the wild type (Table
3). As predicted (71), all 20 revertants from the pms1? strain
were sequenced and found to be intragenic missense suppres-
sors at HIS1 locus (data not shown). The pms1-G882E strain
did not display a significant increase in the base substitution
rate. However, the pms1-H888R strain showed a small but
significant threefold increase over the wild type in base substi-
tutions (Table 3).
Next, we tested the pms1 alleles using the his7-2 reversion
assay, which reports ?1-bp frameshifts in a run of 7A/7T bp
(Fig. 1). With the his7-2 assay, the pms1? strain displayed a
172-fold increase in ?1 frameshifts compared to a wild-type
strain (Table 3). Strikingly, in contrast to their behavior with
hom3-10 reporter, both pms1-G882E and pms1-H888R were
similar to pms1? with the his7-2 reporter. The reversion rate
for his7-2 was elevated 56- and 153-fold in the pms1-G882E
and pms1-H888R strains, respectively (Table 3).
Based on rate analyses with four mutator reporters (CAN1,
hom3-10, his1-7, and his7-2), the pms1-G882E and pms1-
H888R alleles both appeared to preferentially elevate ?1-bp
frameshifts in mononucleotide runs. To confirm this interpre-
tation, 20 to 30 can1 mutants from each strain were sequenced.
Strikingly, in pms1-G882E and pms1-H888R strains, approxi-
mately 40% of the can1 mutations were ?1-bp frameshifts,
whereas in wild-type and pms1? strains, this percentage was
only 5% (Table 4).
Taken together, the most notable finding from the mutation
analyses is that pms1-G882E and pms1-H888R alleles increase
?1-bp frameshifts in mononucleotide repeats to an extent
comparable to that associated with a pms1? allele, while hav-
ing little or no effect on ?1-bp frameshifts and no or little
effect on base substitutions. The preferential increase in the
?1-bp frameshifts is in contrast to pms1? or other MMR-null
strains, which generally show a greater increase in ?1-bp
frameshifts in mononucleotide reporters (21). Our findings
suggest that the pms1-G882E and pms1-H888R alleles are pref-
erentially defective in repairing single-strand loops arising on
the primer strand during DNA replication (Fig. 3).
pms1-G882E and pms1-H888R mutator effects are similar
during leading- and lagging-strand synthesis. The mutation
data presented above suggest that the pms1-G882E and pms1-
TABLE 2. Mlh1/Pms1 two-hybrid interactions
pBT-MLH1 ? pGAD-PMS1
pBT-MLH1 ? pGAD-pms1-G882E
pBT-MLH1 ? pGAD-pms1-H888R
TABLE 3. Mutation rates at CAN1, his1-7, hom3-10 and his7-2
1 2.6 (1.1–4)
aValues in parentheses are 95% confidence intervals.
bIncreases are calculated relative to PMS1.
VOL. 25, 2005 PMS1 ALLELES AND PRIMER STRAND LOOP REPAIR 9225
H888R alleles differentially affect the repair of ?1-bp versus
repair of mispairs due to 1-nucleotide loops on the primer strand
than 1-nucleotide loops on the template strand. However, be-
cause these data were derived using different genes at different
positions in the yeast genome, the mutation pattern might instead
reflect sequence effects (e.g., more efficient repair of extra A than
of extra T) and/or differences in the repair of errors generated
during leading- versus lagging-strand synthesis.
A more direct comparison of the repair efficiencies of ?1
versus ?1 frameshift intermediates requires the use of a single
mutational target for both types of events. Therefore, we con-
structed strains with an in-frame 10C run on the coding strand
of the LYS2 gene (LYS2-10C). As shown previously, a 10N run
is sufficiently long to insure that the majority of lys2 forward
mutations occur within the run (21, 29, 66). Whether a given
sequence resides on the leading- or lagging-strand template
during replication was controlled by positioning the LYS2-10C
allele at the HIS4 locus on chromosome III, which is replicated
from ARS306 ?90% of the time (46, 76). We define the for-
ward (LYS2F) orientation as that in which the transcriptional
machinery and replication fork move in the same direction; in
the reverse (LYS2R) orientation, they converge (Fig. 4). To
change the location of a given run on the leading- versus
lagging-strand template, we inverted the entire LYS2-10C gene.
As illustrated in Fig. 4, the 10C run is on the lagging-strand
template in strains containing the LYS2F-10C allele but on the
leading-strand template in strains containing LYS2R-10C.
For each LYS2-10C allele, we determined the forward mu-
tation rate and mutational spectrum in PMS1, pms1?, pms1-
G882E, and pms1-H888R backgrounds (Table 5). In the wild
type, the forward mutation rates for the LYS2F-10C and
LYS2R-10C alleles were similar, and an approximately 10:1
bias for ?1 events was observed, suggesting less efficient repair
of ?1 than of ?1 frameshift intermediates. In spite of this
apparent bias, however, it should be noted that the MMR
efficiency for both ?1 and ?1 intermediates exceeded 95%.
The forward mutation rates were elevated approximately 100-
fold in the pms1? strains, with ?1 events outnumbering ?1
events approximately 2:1. These data from PMS1 and pms1?
backgrounds agree well with observations reported previously
for strains containing the same LYS2-10C alleles at the LYS2
locus in a different strain background (21). Although the for-
ward LYS2 mutation rates in the pms1-G882E and pms1-
FIG. 3. DNA polymerase slippage model for instability in mononucleotide runs. Following a transient dissociation of the primer and template
strand during DNA replication, the strands can reanneal in misaligned configuration, resulting either in a displaced single-strand loop on the
primer (upper) or the template (lower) strand. If the resulting mismatches are not corrected before the next round of replication, the mispaired
loops will give rise to unit size insertions or deletions, depending on whether the unpaired loop was in the primer or the template, respectively.
TABLE 4. Spectra of can1 mutations
CAN1 mutation rate
No. of base substitutions/
no. of mutants sequenced
No. of frameshifts/no. of mutants
sequenced (fold increase)b
aValues in parentheses are 95% confidence intervals.
bIncreases are calculated relative to PMS1.
9226ERDENIZ ET AL.MOL. CELL. BIOL.
H888R strains were comparable to the pms1? strain rates, the
distributions of ?1 versus ?1 events within the 10C/10G runs
were not. There was a very strong bias for ?1 events (39:1),
which is the reverse of that seen in the pms1? strains, but very
similar to the bias in the PMS1 strains. The C/G runs of the
LYS2-10C alleles thus behave similarly to the A/T runs of the
hom3-10 and his7-2 alleles and support the conclusion that the
primary MMR defect conferred by the pms1-G882E and pms1-
H888R alleles is in the repair of primer strand loop mispairs
Although the above data demonstrate that the inefficiency in
primer strand loop repair conferred by the pms1-G882E and
pms1-H888R alleles is not sequence specific, the analysis did
not address the possibility of differential effects during leading-
versus lagging-strand synthesis. Addressing the leading/lagging
issue requires knowledge of which strand the mutation origi-
nated on, which can be deduced using a strain deficient in
OGG1, a glycosylase that specifically initiates repair of 8-oxo-
7,8-dihydroguanine (GO) lesions (70), which can base pair
efficiently with adenine as well as cytosine. In an ogg1 mutant,
the resulting increase in GC to TA transversions reflects spe-
cifically G-A rather than C-T mispairings, thus assigning the
strand on which the original mispair occurred (51). We rea-
soned that if the presence of a GO lesion in the template can
stimulate DNA polymerase slippage, we should observe ele-
vated frameshifts within the 10C/10G runs of the LYS-10C
alleles in ogg1 mutants. Using the same reasoning as applied to
the transversions of GC to TA in ogg1 mutants, we could assign
a strandedness to the underlying slippage events because the
initiating GO lesions would always be on the template strand.
FIG. 4. Schematic representation of replication forks emerging from ARS306. The location of ARS306 is depicted as a gray box. The coding
(nontranscribed) LYS2 strand is illustrated as a thick black arrow, whereas the bi-directional arrow above ARS306 indicates the directionality of
replication from ARS306. The forward (LYS2F) orientation is defined as that in which the transcriptional machinery and replication fork move in
the same direction; in the reverse (LYS2R) orientation, they converge. The 10G run is on the leading-strand template whereas the 10C run is on
the lagging-strand template in strains containing the LYS2F-10C allele. With the LYS2R-10C, the 10G run is on the lagging-strand template, and
the 10C run is on the leading-strand template.
TABLE 5. Forward mutation in LYS2-10C strains
10G lagging-strand template (LYS2R-10C) 10G leading-strand template (LYS2F-10C)
No. of frameshiftsc
No. of frameshiftsc
aValues in parentheses are 95% confidence intervals.
bIncreases are calculated relative to PMS1.
cNumber/total number of mutants sequenced.
VOL. 25, 2005 PMS1 ALLELES AND PRIMER STRAND LOOP REPAIR 9227
An ogg1? allele was introduced into the PMS1, pms1?,
pms1-G882E, and pms1-H888R strains containing the LYS2F-
10C allele or the LYS2R-10C allele, in which GO lesions
should be present within the leading- or lagging-strand tem-
plate run, respectively (see Fig. 4). In each ogg1 mutant, the
rate of frameshifts in the 10G/10C run was increased at least
fivefold relative to the rate in the corresponding OGG1 parent
strain (Table 5). In either the PMS1 ogg1 or the pms1? ogg1
strain, the rate of slippage within the 10G/10C run was same
regardless of whether the G run was on the leading- or the
lagging-strand template. Therefore, at least in the case of the
LYS2-10C alleles used here, there appears to be neither a
strand-related difference in the rate of polymerase slippage
within G/C runs nor a strand-specific bias in the efficiency of
MMR. In addition, the very strong bias for the accumulation of
?1 frameshifts was evident in the pms1-G882E ogg1 and the
pms1-H888R ogg1 strains, regardless of whether the 10G run
was on the leading- or lagging-strand template. We estimate
that the repair efficiencies of template strand loops in these
strains was ?90%, while that of primer strand loops was less
than 1%. These data confirm that the novel pms1 alleles re-
ported here are specifically defective in the repair of primer
strand loops generated during leading- and lagging-strand
pms1-G882E and pms1-H888R impact MMR and synergize
with the ?1-bp frameshift mutator allele, pol2-C1089Y. Previ-
ously, a mutation in DNA polymerase ε, pol2-C1089Y, was
reported to elevate preferentially ?1-bp frameshift mutations
within mononucleotide runs in yeast (33). As expected for a
DNA polymerase defect, pol2-C1089Y synergized with msh2?
for ?1-bp frameshifts in mononucleotide runs (33). To address
whether the pms1-G882E and pms1-H888R strains indeed re-
flect defects in MMR rather than some aspect of replication
per se, we constructed double mutant strains containing pms1
alleles together with either msh2? or pol2-C1089Y. As shown
in Table 6, pms1?, pms1-G882E, and pms1-H888R mutations
displayed epistatic interactions with msh2?, most relevantly for
the his7-2 ?1-bp frameshift assay. In contrast, all three pms1
alleles showed synergistic interactions with pol2-C1089Y in
both the CAN1 forward and his7-2 reversion assays. Further-
more, pol2-C1089Y did not synergize with pms1-G882E or
pms1-H888R using the ?1-bp frameshift specific reporter,
hom3-10 (data not shown). These results strongly suggest that
both pms1-G882E and pms1-H888R alleles specifically impact
mismatch repair of ?1-nucleotide primer-strand loops.
pms1-G882E and pms1-H888R efficiently repair 1-nucleotide
loop mispairs during meiotic recombination. In addition to a
mitotic mutation avoidance role, MMR proteins also function
during meiotic recombination. During recombination, se-
quence nonidentities between recombining alleles can result in
mismatches in heteroduplex DNA intermediates (47, 52). Such
mismatches are normally subject to mismatch correction,
which can lead to gene conversion. However, failure of MMR
will result in two nonidentical daughter cells when persisting
heteroduplex DNA is replicated, which is termed postmeiotic
segregation (PMS). Hence in wild-type cells, efficient hetero-
duplex repair will result in relatively high levels of gene con-
version and low levels of PMS. In contrast, MMR-deficient
strains will display increased levels of PMS at the expense of
gene conversion events.
To determine the effect of pms1-G882E and pms1-H888R on
the correction of 1-nucleotide loop mispairs in meiotic hetero-
duplex DNA, we used the haploid backgrounds AS4 and PD24.
The resultant diploids are identical in sequence for the HIS4
locus, except for being heterozygous for his4-713, a 1-bp inser-
tion near the C terminus of HIS4. Furthermore, these diploids
show high levels of non-Mendelian (aberrant) segregation ini-
tiated by double-strand breaks in the HIS4 promoter region
(11, 15). As shown in Table 7, the overall percentage of aber-
rant segregants, i.e., 6:2 plus 5:3, was similar in all diploid
strains tested (20 to 30%). In wild type, pms1-G882E and
pms1-H888R diploids, high levels of gene conversion, i.e., 6:2,
and a low levels of PMS, i.e., 5:3, were observed, indicating
efficient MMR of the 1-nucleotide loop mispair at his4-713
(Table 7). Given that the two HIS4 chromosomes used in the
diploids strains experience double-strand breaks with identical
frequencies (J. L. Arqueso and T. D. Petes, personal commu-
nication), the observed equal ratios of 6:2 versus 2:6 tetrads in
the PMS1, pms1-G882E, and pms1-H888R strains suggest that
there is no bias in the repair of the 1-nucleotide loop hetero-
duplex. As expected for MMR deficiency, pms1? diploids dis-
played significantly increased levels of PMS and reduced levels
TABLE 6. Genetic interactions between pms1 alleles and msh2?, pol2-C1089Y
PMS1 MSH2 POL2
aValues in parentheses are 95% confidence intervals.
bIncreases are calculated relative to PMS1.
9228ERDENIZ ET AL.MOL. CELL. BIOL.
of gene conversion. (Table 7, 45% gene conversion and 55%
PMS). Whereas differences between PMS levels in the pms1?
mutant and the wild type (P ? 0.0001), pms1-G882E (P ?
0.0001), and pms1-H888R (P ? 0.0003) are highly significant,
the differences in PMS levels between wild type and pms1-
G882E and pms1-H888R were not significant (data not shown).
Thus, these results indicate that neither pms1-G882E nor
pms1-H888R significantly alters the repair efficiency of 1-nu-
cleotide loop mismatches in heteroduplex DNA during meiotic
To understand better the function of MutL? during MMR-
mediated mutation avoidance, we screened for alleles of PMS1
that exhibit novel effects on mutational spectra. We identified
two alleles, pms1-G882E and pms1-H888R, that greatly ele-
vated the rate of ?1-bp frameshifts in mononucleotide runs,
while having relatively little effect on the rates of ?1-bp frame-
shifts or base substitutions. The repair bias initially was ob-
served in the hom3-10 and his7-2 frameshift reversion assays,
which report ?1-bp and ?1-bp frameshifts in A/T runs, re-
spectively, as well as in forward mutation spectra at CAN1
(Tables 3 and 4). The generality of the strikingly inefficient
repair of ?1 frameshift intermediates was confirmed using a
novel assay (Fig. 4), which determined mutational rates and
spectra for C/G mononucleotide runs during leading- versus
lagging- strand replication (Table 5). Using this assay, we
showed that the pms1-G882E and pms1-H888R alleles were
predominantly defective in the repair of primer strand loops
generated during both leading- and lagging-strand DNA rep-
lication. Finally, the observed efficient repair of 1-nucleotide
loops in meiotic heteroduplex DNA intermediates in pms1-
G882E and pms1-H888R mutants (Table 6) suggests that the
repair bias for primer strand versus template strand loops is
related to mismatch repair that occurs in the context of DNA
replication and not meiotic recombination. Interestingly, the
mutations in the pms1-G882E and pms1-H888R alleles are in
close proximity, affecting the COOH-terminus of Pms1 in a
previously uncharacterized but highly conserved sequence mo-
Frameshift intermediates in repeat tracts are generated dur-
ing DNA synthesis when the template and primer strands dis-
sociate transiently and then reanneal in a misaligned configu-
ration, resulting in single-strand loops of one or more repeats.
If left unrepaired, 1-nucleotide loop mispairs will give rise, in
the next round of replication, to 1-bp insertions or deletions,
depending on whether the extra nucleotides are in the primer
or the template strand, respectively (Fig. 3). While DNA poly-
merase generates comparable numbers of misaligned nucleo-
tides on the template and primer strands when replicating
simple sequence repeats, the MMR system appears to be in-
herently more efficient at repairing template strand loops than
primer strand loops (21, 64, 68). One explanation for the more
efficient repair of template strand loops in wild-type strains is
that the MutS complexes differentially recognize primer strand
versus template strand loops. The results reported here with
pms1-G882E and pms1-H888R alleles suggest, however, that
this inherent inefficiency of MMR is not a MutS recognition
issue but, rather, reflects a processing difference dependent on
Pms1 and, likely, Mlh1.
Although mutations in PMS1 most likely affect the repair
and not the generation of mutational intermediates, we asked
first whether the pms1-G882E or pms1-H888R mutations im-
pact MMR, per se, and then whether they might in some
manner alter the polymerization fidelity through mononucle-
otide repeats. To address these questions, we performed ep-
istasis analysis between pms1 mutations and msh2?, as well as
the DNA polymerase ε allele, pol2-C19089Y, which preferen-
tially elevates ?1-bp frameshifts in monoucleotide runs. We
found that the pms1?, pms1-G882E, and pms1-H888R alleles
all displayed epistatic interactions with msh2?. As observed
previously (33), when combined with a msh2? allele, pol2-
C1089Y synergized with pms1? for ? 1-bp frameshifts in
mononucleotide runs (Table 6). In contrast to the epistatic
relationship with msh2?, pms1-G884E and pms1-H888R both
synergized with the pol2-C1089Y for ?1-bp frameshifts in the
his7-2 assay (Table 6). These results strongly suggest that
pms1-G882E and pms1-H888R indeed impact mismatch repair
of 1-nucleotide primer strand loops rather than affecting some
aspect of the actual replication process.
To gain further understanding into the defects of pms1-
G882E and pms1-H888R, we investigated the frameshifts gen-
erated in C/G runs during leading- versus lagging-strand syn-
thesis. In yeast, previous studies exploiting the OGG1
deficiency to assign the strand on which mutations arise sug-
gested that mismatch repair of base/base mispairs was more
efficient during lagging-strand than leading-strand replication
(51). We addressed this issue for frameshifts in mononucle-
otide runs by a similar approach and incorporated ogg1? into
strains containing the G/C run LYS2 reporter located near an
origin of replication (Fig. 4). In these strains, the G run was
present on either the leading- or lagging-strand template dur-
ing replication. ogg1? increased frameshifts in the runs and
therefore allowed the marking of the template that contained
the GO lesion. In addition, ogg1? displayed synergistic inter-
actions with pms1?, pms1-G882E, and pms1-H888R (Table 5),
TABLE 7. Meiotic mismatch repair of a 1-nucleotide loop heteroduplex at HIS4a
Total no. of
No. of tetrads
No. of other
VOL. 25, 2005PMS1 ALLELES AND PRIMER STRAND LOOP REPAIR9229
suggesting increased polymerase slippage due to unrepaired
GO lesions. Importantly, whereas most mutations in the
pms1? ogg1? strains were ?1 frameshifts, more than 95% of
the frameshifts in the pms1-G882E ogg1? and pms1-H888R
ogg1? were ?1 frameshifts. Taken together, these data indi-
cate that both pms1 alleles primarily affect repair of primer
strand loops during both leading- and lagging-strand synthesis.
In turn, these findings are consistent with the current view for
association between MMR and replication machinery (28, 31,
Further insight into the defect of the pms1-G884E and pms1-
H888R mutants emerged from examining the effects of these
alleles on heteroduplex correction during meiotic recombina-
tion. Relative to a pms1? strain, both mutant strains efficiently
repaired meiotic heteroduplexes containing 1-nucleotide loops
(Table 7). This implies that there is a difference between the
processing of replication- and recombination-associated mis-
pairs in these mutants. Although there are many factors that
may contribute to this difference, we suggest that the recogni-
tion of loop mispairs during meiotic recombination is likely to
be independent of the DNA synthesis machinery, whereas
MMR-mediated mutation avoidance is linked to replication (4,
9, 18, 22, 34, 36, 51, 69).
The pms1-G882E and pms1-H888 mutations that cause the
preferential elevation of ?1-bp frameshifts in mononucleotide
runs map in the C-terminal 200 amino acids of Pms1 to a
13-amino-acid motif that is highly conserved in eukaryotic
Pms1 homologs. Because the C-terminal region of Pms1 is
required for interaction with Mlh1 (49), the novel mutator
phenotypes might simply reflect abnormal MutL? heterodimer
formation. However, the pms1-G882E and pms1-H888 muta-
tions had no detectable effect on Pms1 stability or interaction
with Mlh1 as determined with the two-hybrid assay. The C-
terminal domain of MutL has been shown to be important for
homodimerization (1, 12), interactions with MutH and UvrD
(25, 26), and DNA binding (27). Furthermore, based on the
recently solved crystal structure of the COOH-terminus of
MutL (23), the residues affected by the pms1 alleles studied
here appear to be in an exposed region of the protein and,
therefore, may be important for interaction with other proteins
and/or DNA during MMR. Further studies of these pms1 al-
leles and other mutations with specific mutator effects should
shed additional light on eukaryotic MMR, possibly including
the mechanism of strand discrimination.
We thank Gray Crouse, Rodney Rothstein, Marcel Wehrli, Jennifer
Johnson, and Ashleigh Miller, for critical reading of the manuscript.
This work was supported by National Institutes of Health (NIH)
grant 5 R0l GM45413 to R.M.L. and NIH grant GM038464 to S.J.R.;
N.E. was supported by NIH fellowship F32 GM20342, and R.G. was
partially supported by the Graduate Division of Biological and Bio-
medical Sciences at Emory University.
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