Visualization of Eukaryotic DNA
Mismatch Repair Reveals Distinct
Recognition and Repair Intermediates
Hans Hombauer,1,2,3,5Christopher S. Campbell,1,3,5Catherine E. Smith,1,2,3Arshad Desai,1,3
and Richard D. Kolodner1,2,3,4,*
1Ludwig Institute for Cancer Research
2Department of Medicine
3Department of Cellular and Molecular Medicine, Moores-UCSD Cancer Center
4Institute of Genomic Medicine
University of California School of Medicine, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0669, USA
5These authors contributed equally to this work
DNA mismatch repair (MMR) increases replication
fidelity by eliminating mispaired bases resulting
from replication errors. In Saccharomyces cerevi-
siae, mispairs are primarily detected by the Msh2-
Msh6 complex and corrected following recruitment
tional fluorescent versions of Msh2-Msh6 and Mlh1-
Pms1 in living cells. We found that the Msh2-Msh6
complex is an S phase component of replication
centers independent of mispaired bases; this local-
ized pool accounted for 10%–15% of MMR in wild-
type cells but was essential for MMR in the absence
of Exo1. Unexpectedly, Mlh1-Pms1 formed nuclear
foci that, although dependent on Msh2-Msh6 for
formation, rarely colocalized with Msh2-Msh6 repli-
cation-associated foci. Mlh1-Pms1 foci increased
when the number of mispaired bases was increased;
in contrast, Msh2-Msh6 foci were unaffected. These
machinery-coupled and -independent pathways for
mispair recognition by Msh2-Msh6, which direct
formation of superstoichiometric Mlh1-Pms1 foci
that represent sites of active MMR.
DNA mismatch repair (MMR) catalyzes a postreplication exci-
sion reaction that increases the fidelity of DNA replication by
eliminating mispaired bases resulting from replication errors
(Iyer et al., 2006; Kolodner, 1996; Kolodner and Marsischky,
1999). MMR defects cause increased mutation rates, and in
mammals this results in the development of different cancers
(Peltoma ¨ki, 2003). In addition, MMR acts on mispaired bases
in recombination intermediates andalso preventsrecombination
between divergent DNA sequences, preventing genome rear-
rangements (Datta et al., 1996; Matic et al., 1995; Putnam
etal.,2009).Mispaired bases arerecognizedbyMutS in bacteria
(Iyer et al., 2006) and by two partially redundant MutS-related
heterodimer complexes, Msh2-Msh6 or Msh2-Msh3, in eukary-
otes (Marsischky et al., 1996). Msh2-Msh6 is more abundant
than Msh2-Msh3 (Genschel et al., 1998; Ghaemmaghami
et al., 2003) and likely promotes most MMR in eukaryotes.
Msh2-Msh3 primarily corrects mispairs that are not efficiently
repaired by Msh2-Msh6 and acts when Msh2-Msh6 is absent
due to loss of Msh6 (Genschel et al., 1998; Marsischky et al.,
1996; Sia et al., 1997). After the mismatch recognition factors
bind a mispaired base, accessory factors including MutL in
bacteria and the Mlh1-Pms1 (S. cerevisiae Pms1 = human
Pms2) or Mlh1-Mlh3 complexes in eukaryotes are recruited,
targeting repair to the daughter DNA strand (Cannavo et al.,
2005; Flores-Rozas and Kolodner, 1998; Iyer et al., 2006; Kunkel
and Erie, 2005; Prolla et al., 1994).
Recent studies in S. cerevisiae using next-generation
sequencing to detect mutations in an MMR-defective mlh1
mutant indicate that the rate of accumulating mispair bases,
including both base:base and frameshift mispairs in repeat
sequences, is approximately 0.1 mispaired base per cell division
(Zanders et al., 2010). This rate is consistent with the rate of
accumulation of nucleotide changes in URA3 and CAN1 (Lang
and Murray, 2008) in wild-type S. cerevisiae multiplied by the
known increase in mutation rate at these genes in MMR-defec-
tive mutants. Thus, it appears that MMR must be able to recog-
nize 1 mispaired base per genome (?12,000,000 base pairs).
Remarkably, in vitro, mismatch recognition proteins exhibit
only modestly higher affinity for mispaired DNA than for DNA
containing only base pairs ranging from 3- to 20-fold (Alani,
1996; Iaccarino et al., 1998; Jiricny et al., 1988; Marsischky
and Kolodner, 1999) to recently reported 60- to 400-fold affinity
differences depending on the specific mispair (Huang and
Crothers, 2008). Mispair binding licenses an ATP-binding-
induced conversion of MutS, Msh2-Msh6, and Msh2-Msh3 to
1040 Cell 147, 1040–1053, November 23, 2011 ª2011 Elsevier Inc.
ATP induces direct dissociation of these proteins from DNA
(Acharya et al., 2003; Gradia et al., 1999; Mendillo et al., 2005).
In addition, the ATP-binding-dependent formation of ternary
complexes between MutS and MutL (or their eukaryotic homo-
logs) requires binding of the mispair recognition proteins to
a mispaired base (Acharya et al., 2003; Blackwell et al., 2001;
Mendillo et al., 2005). These mechanistic features amplify the
specificity of mispair recognition. Regardless, the specificity of
mispair binding in vitro is unlikely to account for the specificity
of MMR in vivo.
Onehypothesis forhowmismatch recognition occursin vivois
that MMR is coupled to DNA replication, which would localize
MMR proteins to where mispaired bases are formed. Two lines
of evidence suggest this. First, MMR in vitro requires single-
strand breaks in the DNA (Iyer et al., 2006; Kunkel and Erie,
2005), suggesting that MMR might be targeted to strand breaks
in the nascent DNA strands during DNA replication. Second,
Msh2-Msh6 and Msh2-Msh3 (Clark et al., 2000; Flores-Rozas
et al., 2000) as well as Mlh1-Pms1 (Dzantiev et al., 2004; Lee
and Alani, 2006) complexes interact with the proliferating cell
nuclear antigen (PCNA). Because PCNA is part of the replication
machinery, these interactions could link MMR to DNA replica-
tion, binding of MMR proteins to PCNA could target MMR to
regions of newly synthesized DNA (Shibahara and Stillman,
1999). However, the interaction between PCNA and Msh6
and Msh3 is not absolutely required for MMR (Flores-Rozas
et al., 2000; Shell et al., 2007). Furthermore, PCNA is required
at many steps during MMR including the activation of the human
Pms2 (Pms1 in S. cerevisiae) endonuclease (Pluciennik et al.,
2010) and the resynthesis step at the end of MMR (Gu et al.,
1998; Umar et al., 1996). Therefore a PCNA requirement does
not necessarily reflect coupling of MMR to DNA replication.
Here, we have used functional fluorescent-tagged MMR and
replication proteins to study MMR in situ. Our results indicate
that the Msh2-Msh6 mismatch recognition complex is an S
phase component of replication centers independent of the
presence of a mispaired base. Unexpectedly, Mlh1-Pms1
formed nuclear foci that, although dependent on Msh2-Msh6
for their formation, rarely colocalized with the Msh2-Msh6
replication-associated foci. The presence of mispaired bases
or defects downstream of mispair recognition increased the
formation of Mlh1-Pms1 foci but not Msh2-Msh6 foci. These
findings suggest the presence of replication machinery-coupled
and -independent pathways for mispair recognition by Msh2-
Msh6, which in turn direct formation of superstoichiometric
Mlh1-Pms1 foci that represent sites of active MMR.
Msh6 Forms Foci that Colocalize
with Replication Factories
DNA replication in eukaryotic cells takes place at discrete glob-
ular foci or clusters within the S phase nucleus, sometimes
referred to as replication factories (Hoza ´k et al., 1993; Kitamura
et al., 2006; Newport and Yan, 1996). To test for an association
between MMR and DNA replication in live cells, we used decon-
tagged DNA replication and MMR proteins (Tables S1 and S2
available online; the replication proteins are essential and hence
functional) expressed at normal levels (Figure S3A) from their
native chromosomal loci in S. cerevisiae. Consistent with
previous studies (Kitamura et al., 2006), Pol2-4GFP (catalytic
subunit of DNA polymerase ε) formed multiple foci within the
nucleus(Figure1A). Thesefociwere frequently observedinsmall
or medium budded cells (S phase cells) and were essentially
absent in unbudded cells (G1 cells) and cells with large buds
(R3 mm) (G2/M cells) (Figures 1A and 1B). Msh6-mCherry also
formed nuclear foci (Figure 1A) that were more abundant in
S phase cells (2.6 ± 1.4 [mean ± standard deviation (SD)],
n = 103, foci per S phase cell) (Figures 1B and S1A). These
Msh6-mCherry foci almost always colocalized with Pol2-4GFP
foci (Figures 1A and 1B), suggesting that a portion of Msh2-
Msh6 is present in replication factories. Importantly, a rad52D
mutation that eliminates mitotic recombination, replication-
dependent recombination intermediates and toxic recombina-
tion intermediates thought to arise from damaged replication
forks (Fabre et al., 2002; Zou and Rothstein, 1997), and an
msh3D mutation that eliminates targeting of Msh2 to recombina-
tion intermediates (Evans et al., 2000) did not significantly affect
the frequency of Msh6 foci (Figure 2B).
We next tested colocalization of Msh6 with other replisome
components including the following: Pol30 (PCNA), Pol3 (cata-
lytic subunit of DNA polymerase d), Pol1 (catalytic subunit of
DNA polymerase a), Rfa1 (large subunit of replication protein
A, RPA), and Mcm2 and Mcm4 (subunits of the MCM helicase
complex Mcm2-7). Colocalization of Msh6 foci with Pol30,
Pol3, and Pol1 foci was similar to that seen with Pol2 foci (Fig-
ure 1C). Rfa1 formed many foci, and Msh6 foci frequently colo-
calized with these, although a high percentage of Rfa1 foci did
notcolocalize with Msh6foci. Wecould notdetect colocalization
of Msh6 with the MCM subunits Mcm2-4GFP, Mcm4-4GFP (Fig-
ure 1C), or Mcm7-4GFP (Figure S1B); Mcm7-mCherry also did
not colocalize with Pol2-4GFP (Figure S1C). The lack of colocal-
ization of MCM subunits with replication fork components in
microscopy studies has been reported and remains a matter of
discussion (Dimitrova et al., 1999; Laskey and Madine, 2003).
Msh2-Msh6 Foci Depend on Interaction with PCNA
We next tested whether colocalization of Msh6 with the replica-
tion machinery is mediated by an interaction with PCNA, via the
PIP (PCNA interacting protein) box located at the N terminus of
Msh6 (Clark et al., 2000; Flores-Rozas et al., 2000). An msh6-
F33AF34A-GFP mutant, which disrupts the PIP box and the
interaction with PCNA in vitro, displayed a severe reduction in
the percentage of nuclei with Msh6-GFP foci in unsynchronized
(Figures 2A and 2B) and S phase cells (Figures S2A and S2C).
Similar results were obtained with the msh6-2-51D mutation
that deletes the Msh6 PIP box and, like the msh6-F33AF34A
mutation, causes only modest MMR defects (10%–15% reduc-
tion in MMR) (Shell et al., 2007). These results are consistent
with prior observations in human cells transfected with
a construct expressing a nonfunctional Msh6 lacking the first
77 N-terminal amino acids (msh6-D77) that does not interact
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