Clamp Directs Localization of Mismatch Repair in Bacillus
subtilis
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Citation
Simmons, Lyle A., Bryan W. Davies, Alan D. Grossman, and
Graham C. Walker. “ Clamp Directs Localization of Mismatch
Repair in Bacillus subtilis.” Molecular Cell 29, no. 3 (February
2008): 291-301. Copyright © 2008 Elsevier Inc.
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http://dx.doi.org/10.1016/j.molcel.2007.10.036
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Elsevier
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Detailed Terms
Molecular Cell
Article
b Clamp Directs Localization
of Mismatch Repair in Bacillus subtilis
Lyle A. Simmons,1 Bryan W. Davies,1 Alan D. Grossman,1 and Graham C. Walker1,*
1Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
*Correspondence: gwalker@mit.edu
DOI 10.1016/j.molcel.2007.10.036
SUMMARY
MutS homologs function in several cellular pathways
including mismatch repair (MMR), the process by
which mismatches introduced during DNA replication are corrected. We demonstrate that the C terminus of Bacillus subtilis MutS is necessary for an
interaction with b clamp. This interaction is required
for MutS-GFP focus formation in response to mismatches. Reciprocally, we show that a mutant of
the b clamp causes elevated mutation frequencies
and is reduced for MutS-GFP focus formation.
MutS mutants defective for interaction with b clamp
failed to support the next step of MMR, MutL-GFP
focus formation. We conclude that the interaction
between MutS and b is the major molecular interaction facilitating focus formation and that b clamp
aids in the stabilization of MutS at a mismatch in
vivo. The striking ability of the MutS C terminus to
direct focus formation at replisomes by itself, suggests that it is mismatch recognition that licenses
MutS’s interaction with b clamp.
INTRODUCTION
Replicative DNA polymerases are responsible for duplicating entire
genomes with high fidelity (for review see Johnson and O’Donnell,
2005). Despite the high fidelity of this process (e.g., Kool, 2002),
replication errors still occur. Correction of these errors requires
the enlistment of mismatch repair (MMR) proteins to restore the
proper coding sequence (for review see Kunkel and Erie, 2005).
MMR is an important, highly conserved repair pathway that is
found in both prokaryotes and eukaryotes (Culligan et al., 2000).
In bacteria, deletion of MMR genes results in a several hundred-fold increase in mutation frequency (Cox et al., 1972). In humans, several primary tumors from cancers including hereditary
nonpolyposis colorectal carcinoma (HNPCC) and Turcot syndrome (Fishel et al., 1993; Hamilton et al., 1995) have MMR
defects, suggesting that increased mutation frequency could
contribute to the development of these cancers. The function
of MMR proteins is not limited to their clearly defined roles in
MMR. For example, in bacteria MMR proteins can also participate in an antirecombination mechanism preventing recombination between nonidentical DNA sequences and thereby serving
to preserve species barriers at a molecular level (Rayssiguier
et al., 1989). In eukaryotes, MMR proteins have been shown to
function in meiosis (Hunter and Borts, 1997; Williamson et al.,
1985) and are important for maintaining repetitive DNA
sequences (microsatellites) (Orth et al., 1994). Moreover, MMR
proteins contribute to several other cellular processes including
apoptosis (Hickman and Samson, 2004) and DNA damage
checkpoint activation (Yoshioka et al., 2006).
The central and most intensively studied role for MMR is in the
repair of mismatched nucleotides or insertion-deletion loops
introduced principally as polymerase errors. In both prokaryotes
and eukaryotes, MMR is initiated through the binding of a MutS
homolog to mismatched DNA (for review see Schofield and Hsieh,
2003). In the paradigmatic E. coli system, MutS recognizes a mismatch followed by the recruitment of MutL (Schofield et al., 2001).
MutL coordinates the actions of the nicking endonuclease MutH
(e.g., Hall and Matson, 1999) and the loading of UvrD helicase
(Hall et al., 1998) at the incised nick to result in the ATP-dependent
separation of the DNA strand encoding the mismatch (Oeda et al.,
1982). The mismatch-containing strand can then be degraded by
one of several exonucleases (Viswanathan et al., 2001). The
single-strand region is filled in by DNA Pol III and sealed by ligase
to complete the repair process (Lahue et al., 1989).
In eukaryotes, a heterodimer of MutS homologs (Saccharomyces cerevisiae MSH2-MSH6 [MutSa] [Prolla et al., 1994b] and
hMSH2-hMSH6 in humans [Acharya et al., 1996]) is responsible
for the recognition of most mismatches and small insertion or
deletion loops (Drummond et al., 1995). These proteins recruit
heterodimeric MutL homologs, (S. cerevisiae MLH1-PMS1
[MutLa], and human hMLH1-hPMS2 [Prolla et al., 1994a]), which
function analogously to E. coli MutL and appear important for
coordinating the actions of the remaining proteins in the pathway. The purified human proteins have been used to reconstitute
MMR in vitro (Dzantiev et al., 2004). These studies have revealed
that MMR excision from a 50 strand break requires MutSa, an
exonuclease (EXO1), and single-stranded binding protein
(RPA). MMR excision from a strand break 30 to the mismatch
requires the proteins mentioned above in addition to MutLa,
the clamp loader (RFC), and the replication processivity clamp
(PCNA). These experiments indicate that the protein assemblies
and mechanisms required for excision and repair are different
depending on the directionality of the strand break relative to
the mismatch. Theses studies also demonstrate the dependence on a processivity clamp for 30 directed repair.
In B. subtilis, both MutS and MutL fused to green fluorescent
protein (GFP) localize as discrete foci in cells exposed to the
Molecular Cell 29, 291–301, February 15, 2008 ª2008 Elsevier Inc. 291
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b Clamp Recruits MutS
Figure 1. MutS Interacts with the b Clamp
through the C-Terminal 58 Amino Acids
The peptide array and far-western blot were
probed with b clamp bearing a single Myc tag
(b-Myc).
(A) Shown are 10-mer peptides of interest from the
MutS N terminus that failed to bind b or the Cterminal 58 amino acids that bound b-Myc. The
amino acid sequence is indicated, and the b clamp
binding motif or N-terminal motif is highlighted in
gray. Ponceau staining of the peptides is shown
in the left-most panel.
(B) b-Myc, MutS, MutS800, and His-DnaE probed
with b-Myc.
(C) Interaction between MutS or MutS800 with
b clamp covalently linked to a sensor chip was
completed using surface plasmon resonance as
measured with a BIAcore biosensor (Experimental
Procedures). Representative SPR traces for MutS
1.0 mM (blue) and 4.0 mM (red), and MutS800
2.0 mM (green) and 10.0 mM (purple) are shown.
mismatch-inducing agent 2-aminopurine (2-AP) (Smith et al.,
2001). A new challenge in understanding MMR is to determine
the protein interactions that allow for the coordinated assembly
of MMR foci in vivo. B. subtilis serves as a particularly useful
system for addressing this issue because B. subtilis is the only
bacterium in which MMR proteins have been visualized in vivo.
In addition, like eukaryotes and most bacteria, B. subtilis uses
a DNA methylase-independent mechanism for strand discrimination indicating that B. subtilis serves as a valuable model
system for understanding this process.
Experiments in several systems have shown that MutS homologs interact with replication processivity clamps (b clamp in
bacteria and PCNA in eukaryotes) (Gu et al., 1998; Kleczkowska
et al., 2001; Lau and Kolodner, 2003; Lee and Alani, 2006; Lopez
de Saro et al., 2006; Umar et al., 1996). Disruption of the interaction between MSH6 and PCNA in S. cerevisiae results in mild mutator phenotypes in vivo (for review see Schofield and Hsieh,
2003). The only modifications shown to result in strong mutator
phenotypes encompass large deletions of the N-terminal domain
that forms a flexible linker to PCNA (Shell et al., 2007). In E. coli,
internal deletion of a b clamp binding motif renders MutS refractory to b clamp binding in vitro, yet in vivo the corresponding mutS
allele confers a wild-type MMR phenotype (Lopez de Saro et al.,
2006). Collectively, MutS homologs from several organisms have
been shown to bind their resident processivity clamps; however,
the in vivo significance for this interaction is unclear.
We examined the mechanism that governs the focus formation
response of MutS to mismatches in B. subtilis. We studied an as-
sortment of MutS variants altered in their
C terminus and found that interaction between MutS and b clamp is required for
MutS-GFP focus formation. Furthermore,
we found that the C-terminal 58 amino
acids are not only necessary but also sufficient for focus formation and that these
foci exhibit the same subcellular distribution as the replisome. We also found that
MutS deleted for this C-terminal region is reduced for binding
to b clamp, although this purified mutant protein retains the ability
to bind a mismatch in vitro. Taken together, we conclude that interaction between MutS and b is the major molecular interaction
that facilitates focus formation and that b clamp aids in the stabilization of MutS at a mismatch in vivo.
RESULTS
The C Terminus of MutS Is Required for Interaction
with b Clamp
Several lines of evidence indicate that processivity clamps play
an important role in MMR (Kleczkowska et al., 2001; Lopez de
Saro et al., 2006; Umar et al., 1996). B. subtilis MutS contains
a putative b clamp binding motif (806QLSFF810). It has been
hypothesized that b clamp binding motifs modulate the interaction between a variety of proteins and the b clamp (Dalrymple
et al., 2001). In vitro studies of E. coli MutS have shown that
both its N- and C-terminal regions interact with b clamp (Lopez
de Saro et al., 2006). The N-terminal MutS motif is necessary
for MMR in E. coli, while the C-terminal MutS motif appears to
be dispensable (Lopez de Saro et al., 2006) in vivo.
As a first step toward determining whether b clamp contributes
to mismatch-dependent MutS-GFP focus formation in B. subtilis,
we used a peptide array approach to identify MutS sequences
capable of interacting with the b clamp. Unlike E. coli, we found
that peptides containing the conserved N-terminal motif
(9QQYL12) failed to bind b (Figure 1A). Instead, we found that
292 Molecular Cell 29, 291–301, February 15, 2008 ª2008 Elsevier Inc.
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b Clamp Recruits MutS
the predicted C-terminal b clamp binding motif of MutS
(806QLSFF810) does indeed bind b clamp (Figure 1A) and that
one other 10-mer peptide corresponding to a region within the
last 58 amino acids of MutS also bound b (Figure 1A).
We did find that several N-terminal MutS 10-mer peptides did
bind b (see Figure S1A available online). To determine whether
any of these regions were important for interaction with b in the
folded MutS protein, we performed a far-western blot with
purified intact MutS (amino acids 1–858) and a protein deleted
for the C-terminal 58 amino acids (referred to as MutS800). We
found that MutS bound a C-terminal Myc (single) tagged derivative of b clamp (b-Myc). The interaction was comparable to that
for DnaE (an essential polymerase) and b-Myc (Figure 1B). In
contrast, no interaction between MutS800 and b-Myc was
detected (Figure 1B).
By covalently coupling b-Myc to a flow cell surface, we were
able to use the more sensitive technique of surface plasmon
resonance (SPR) to show that a very minimal interaction with
b can be detected when MutS800 is present at high concentrations (Figure 1C). In these experiments, binding is indicated by
a trace with resonance units above zero. For each trace shown,
we subtracted the injection signal for each protein over a blank
surface. We found that when we injected MutS with increasing
concentrations (Figure 1C) we observed an increase in binding
and an increase in retention of MutS on the b-Myc-coupled
surface (Koff) indicative of protein-protein interaction over a range
of 1.0–4.0 mM. In contrast, when MutS800 was injected we
observed no interaction with b-Myc above the blank control
surface at 2.0 mM (Figure 1C, green trace). When we increased
the concentration of MutS800 to 10.0 mM we observed a binding
signal less then MutS at 4.0 mM (Figure 1C). We attempted this
analysis with MutS850; however, this protein showed significant
binding to the blank surface, precluding further use of this
technique to evaluate MutS850 interaction with b clamp (data
not shown).
With these data, we conclude that MutS binds b clamp and
MutS800 is significantly reduced for binding b clamp. We also
conclude that the weak binding observed by SPR analysis
between MutS800 and b clamp is likely mediated by the multiple
N-terminal peptides that we identified in our peptide array analysis (Figure S1A). We further conclude that the C-terminal 58
amino acids represent the crucial site for interaction with b clamp
in vitro. It seems likely that after MutS interacts with a mismatch it
could change conformation in such a way that the other interaction sites that we identified in the peptide array could become
important for forming a productive MMR complex between
MutS and b clamp. Intriguingly, our observations reveal that
the functional interaction between MutS and b clamp is different
for E. coli and B. subtilis. In E. coli, the N-terminal site of interaction between MutS and b is critical for MMR activity in vivo
(Lopez de Saro et al., 2006). In B. subtilis, the MutS C terminus
is critical for interaction between MutS and b clamp and for
MMR activity in vivo (see below). It should be noted that we
considered a role for MutL in contributing to MutS-GFP localization, but we did not find support for this possibility. We describe
in the Supplemental Data that in a DmutL strain background
MutS-GFP foci are unaffected (Supplemental Results and
Table S1).
The C Terminus of MutS Is Required for MMR and Focus
Formation In Vivo, but Not Mismatch Binding In Vitro
We show that at least two nonoverlapping 10-mer peptides in the
C-terminal 58 amino acids have the capability to bind b clamp.
We also found that MutS800 is significantly reduced for binding
to b, suggesting that the entire C-terminal region might be involved in interaction with b and thus might influence MMR and
focus formation. Therefore, we constructed a set of mutS-gfp alleles that successively removed segments of this 58 amino acid
C-terminal region from MutS within the MutS-GFP fusion context
(Figure 2A). All of these truncated mutS alleles were present at
the normal mutS gene location on the chromosome and provide
the only source of MutS protein in the cell. Because integration of
these alleles disrupts mutL expression, mutL was ectopically
expressed from the amyE locus (Experimental Procedures).
All of the C-terminal mutS deletion mutants exhibited profound
deficiencies for MMR as assayed by mutation frequency
(Figure 2B). Strikingly, truncation of only the last 8 amino acids
of MutS (MutS850-GFP) resulted in nearly a complete loss of
MMR activity in vivo (Figure 2B). Furthermore, all of the MutS
deletion proteins accumulated to levels similar to those
observed for wild-type protein in vivo (Figure 2B). To ensure
this result was not influenced by the GFP component of MutSGFP, we also replaced the native mutS gene with each corresponding mutS truncation allele lacking the GFP fusion. We
found that all of these mutS C-terminal deletion alleles resulted
in profound MMR deficiencies (Figure S2).
All of the MutS-GFP derivatives with truncation of the MutS
C terminus exhibited major defects in focus formation when cells
were treated with 2-AP to introduce replication errors (Figures
3C and 3F). These results suggest that interaction between
MutS and b clamp through the MutS C terminus might contribute
to stabilizing MutS at mismatches in vivo. Another explanation
could be that the MutS C-terminal truncation mutants are unable
to bind mismatches. To discriminate between these possibilities,
we tested MutS800 and MutS850 for mismatch binding in vitro.
We found that mismatch binding by purified MutS800 and MutS
was nearly indistinguishable (Figure 3D). Because MutS850 only
truncated the last eight amino acids, we examined this protein
for binding to b clamp by far-western blotting. We found that
MutS850 was reduced for binding b but was not completely
defective, and this protein also bound a mismatch in vitro (Figures S1B and S1C). Taken together, the only biochemical
difference that we have identified between MutS and MutS800
(or MutS850) is interaction with b clamp.
In E. coli MutS, removal of the C-terminal region (53 amino
acids) causes defects in tetramer formation and mismatch binding (Bjornson et al., 2003), and the truncated protein is defective
in vivo when expressed from the native locus (Calmann et al.,
2005). Whether or not tetramer formation by E. coli MutS provides an in vivo function is unclear (Mendillo et al., 2007; Miguel
et al., 2007). Our finding that MutS800 and MutS850 bind a mismatch in vitro suggests that our deletion mutants do not suffer
from the same biochemical defects as the E. coli protein. These
data support the model that removal of C-terminal residues from
MutS disrupts interaction between MutS and b clamp, which
uncovers a defect in the cellular response to mismatches in vivo,
but not mismatch binding in vitro. We conclude that, in vivo,
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b Clamp Recruits MutS
Figure 2. The MutS C Terminus Is Required
for MMR Activity In Vivo
(A) Diagram of B. subtilis MutS domain structure
and segments of the protein absent from the truncated mutants. The domains were determined by
alignment with Thermus aquaticus MutS, and the
domain nomenclature follows that of the crystal
structure (Obmolova et al., 2000). Briefly, domains
I and IV are involved in mismatch binding, domain
III is the central core of the protein, domain II
resembles an RNase H-like fold, and domain V is
an ABC ATPase.
(B) Corresponding mutation frequencies associated with strains lacking the indicated C-terminal
amino acids of MutS. Relative stability of the resultant proteins was determined by immunoblot analysis and is presented above the histogram.
(C) Mutation frequency resulting from alanine
scanning mutagenesis of the MutS b clamp binding motif. An immunoblot of the in vivo stability of
the resulting proteins is also shown. The alanine
substitutions in the MutS b clamp binding motif
are indicated in bold type. Error bars represent
the standard deviation from at least triplicate
experiments.
interaction between MutS and b clamp helps stabilize MutS at
mismatches and stimulate focus formation.
The Putative b Clamp Binding Motif Is Important
for MMR and MutS Focus Formation In Vivo
To examine the importance of the putative b clamp binding
motif to MMR, we performed comprehensive alanine scanning
mutagenesis of this penta-amino acid motif (Figure 2C). We
found that substitution of the second to last amino acid position (806QLSFF810) in this motif with an alanine indicated that
it was important for MMR activity in vivo. We also found that
substituting the first two positions (806QLSFF810) or the last
two positions (806QLSFF810) with alanines conferred an increase in mutation frequency similar to replacing the entire
motif with alanines. Even so, replacement of the entire motif
with alanines (MutS5A) resulted in only an 8-fold increase in
mutation frequency (Figure 2C) compared with 50-fold for
the DmutS strain when both are normalized to the mutS-gfp
strain.
These results suggest that, although this motif contributes to
MutS function in vivo, considerable MMR can occur in its
absence. We asked whether a correlation exists between the
mild mutator phenotype observed for MutS5A and a decrease
in focus formation. We challenged our MutS5A-GFP strain with
2-AP and observed an 2-fold (MutS5A-GFP 21%, n = 1102;
MutS-GFP 42%, n = 1309) reduction in focus formation (Figures
3B and 3F) indicating that MutS5A is less proficient for focus
formation than MutS-GFP in response to
replication errors. We also determined
the subcellular position of MutS5A-GFP
relative to MutS-GFP. We found that the
position of MutS5A-GFP and MutS-GFP
were nearly identical, indicating that the
frequency of MutS5A-GFP focus formation is altered, but not
the subcellular position at which foci form (Figure 3G).
Immunoblot analysis of these MutS-GFP b clamp binding
motif mutants revealed that the levels of these proteins were
indistinguishable from wild-type MutS fused to GFP in vivo
(Figure 2C). Finally, as an additional control we constructed
fusions of MutS, MutS5A, and MutS800 to a monomeric version of GFP and found that the localization properties of these
proteins were almost identical to the more commonly used
GFPMUT2 employed in the above analyses (Table S2). We
conclude that the b clamp binding motif of B. subtilis MutS
contributes to both MutS focus formation and MMR; however,
the system can function moderately well in the absence of the
motif.
MutS800 and MutS5A Are Reduced for MutL
Recruitment
Previous experiments examining MutS-GFP and MutL-GFP
localization in B. subtilis demonstrated that 2-AP treatment failed
to stimulate MutL-GFP focus formation in strains lacking the
mutS gene (Smith et al., 2001). Taken together with biochemical
studies from E. coli demonstrating that MutL directly binds to
MutS (Wu and Marinus, 1999), these observations indicate that
MutS-GFP foci must form prior to MutL-GFP foci in vivo. We
therefore asked whether our C-terminal MutS variants impaired
for their own focus formation and MMR were also impaired for
supporting MutL-GFP focus formation.
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b Clamp Recruits MutS
Figure 3. The C Terminus of MutS Is Required for Focus Formation
All samples shown are representative. Cells were
treated with 600 mg/ml 2-AP to induce mismatches. Shown are (A) MutS-GFP foci, (B)
MutS5A-GFP, and (C) MutS800-GFP. (D) MutS,
MutS800, and () control (no protein) were incubated with homoduplex DNA (T/A) or mismatched
heteroduplex DNA (T/G) as described (Biswas and
Hsieh, 1996). MutS bound to DNA was retained on
the nitrocellulose membrane and exposed to film.
(E) Percent of cells with foci untreated. (F) Percent
of cells with foci 45 min following addition of 2-AP.
(G) Comparison of the cellular position of MutSGFP single foci (black bar) and MutS5A-GFP foci
(white bar) following 2-AP addition. The number
of cells scored for untreated and 2-AP treated in
(D) and (E) are as follows: MutS-GFP (n = 837,
n = 1309); MutS5A-GFP (n = 1044, n = 1102);
MutS800-GFP (n = 396, n = 958); MutS810-GFP
(n = 567, n = 410); MutS830-GFP (n = 382,
n = 944); MutS840-GFP (n = 595, n = 1022),
MutS850-GFP (n = 771, n = 801). The cell membrane is stained with FM4-64 and is colored in
red, while MutS-GFP foci are in green. The white
bar indicates 2 mm. White dots are adjacent to
MutS-GFP foci.
To this end, we utilized strains in which the mutS800 and
mutS5A alleles were located at their native chromosomal
locus. The mutL-gfp allele was integrated at an ectopic locus
(amyE) and placed under control of an IPTG-inducible promoter. We used this strategy because the integration of the
mutS alleles disrupts expression of the mutL gene directly
downstream (Smith et al., 2001). MutL-GFP forms foci in
response to 2-AP treatment from an ectopic locus at the
same frequency as MutL-GFP expressed from the native
locus (Smith et al., 2001). The mutS800 allele supported
only a small frequency of MutL-GFP focus formation (3%,
n = 2866), a level nearly identical to the DmutS allele (2%,
n = 1042), indicating that mutS800 is completely defective
for recruiting MutL-GFP to mismatches (Figures 4C, 4D, and
4F). The mutS5A allele displays an intermediate ability in
forming its own focus (2-fold [n = 1102] reduction) and
a similar, although more severe, reduction for supporting
MutL-GFP foci (a 4-fold [n = 690] reduction) (Figures 4B
and 4F). The MutL-GFP foci that formed in a mutS5A genetic
background were the same, qualitatively, as those that
formed in a mutS+ background. We conclude that localization
of MutL-GFP to mismatches requires an MMR-proficient
mutS allele.
The MutS C Terminus Is Necessary
and Sufficient to Localize YFP to
b Clamp In Vivo
Biochemical studies have revealed that
E. coli MutS homodimers form clamplike structures that can slide away from
a mismatch along the DNA (Acharya
et al., 2003). This led to the demonstration that a single mismatch could stimulate the loading of multiple MutS homodimers (Acharya et al.,
2003). This mechanism provided a facile explanation for the
MutS focus formation we had observed (Smith et al., 2001). Nevertheless, we asked whether the C terminus of MutS is not only
necessary but also sufficient for MutS focus formation.
We used a strain expressing yellow fluorescent protein (YFP)
from an ectopic locus using an IPTG-inducible promoter. As
expected, YFP alone was diffuse in the cell and failed to show
any specific localization pattern (Figure 5A). However, when we
linked YFP to the C-terminal 58 amino acids of MutS (MutSCYFP), we found that this protein localized as foci in virtually
every cell in a wild-type background in the absence of 2-AP
addition. A similar result was obtained in a strain deleted for
both mutS and mutL (Figure 5B). Thus focus formation by the
MutSC-YFP protein does not require interaction with intact
MutS or MutL. The foci formed in the mutSL+ strain are qualitatively more intense, suggesting that intact MutS may increase
the number of MutSC-YFP in each focus even though intact
MutS is not absolutely required (Figure 5).
MutSC-YFP formed foci in 82% of cells (n = 326) in a DmutSL
strain, indicating that in vivo the C terminus of MutS is sufficient
to localize as a focus. This result was very intriguing because it
suggests that DNA binding by MutS is not required for focus
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b Clamp Recruits MutS
Figure 4. MutS Mutant Proteins Are Impaired for Supporting MutL-GFP Focus
Formation
Cells were grown in S750 minimal medium and
treated with 600 mg/ml 2-AP to induce mismatches
45 min prior to visualization. Cell images shown
are representative of several experiments. Shown
are (A) MutL-GFP in a wild-type mutS background,
(B) MutL-GFP in a mutS5A (b clamp binding motif
replaced with alanines) background, (C) MutLGFP in a mutS800 background, and (D) MutLGFP in a DmutS background. (E) Percent of cells
with MutL-GFP foci untreated. (F) Percent of cells
with MutL-GFP foci 45 min following the addition
of 2-AP. (G) Comparison of the subcellular position
of MutL-GFP foci in a mutS (black bars) and
mutS5A (white bars) genetic background (n =
150). Number of cells scored for the graphs in (E)
and (F) with the untreated number followed by
the 2-AP-treated number are as follows: MutLGFP (n = 1396, n = 891) in a wild-type mutS
background; (n = 1028, n = 1042) in a DmutS background; (n = 2543, n = 2866) in a mutS800
background; and (n = 1142, n = 884) in a mutS5A
background.
formation, but instead interaction with b clamp is sufficient for focus formation. It has been shown in both B. subtilis and E. coli
that b clamp fused to GFP formed discrete foci, suggesting
that b clamp is concentrated to sites of replication in vivo (Kongsuwan et al., 2002; Meile et al., 2006). We performed a similar
analysis, except that b-GFP was expressed from its native promoter at the native chromosomal locus, and this fusion protein
also formed discrete foci in vivo (Figure 5E). The simplest explanation is that MutSC-YFP binds to the b clamp, and it is this interaction that allows for visualization of foci in vivo.
The Subcellular Position of MutSC and b Clamp
Is Consistent with the Replisome
We asked whether both b-GFP and MutSC-YFP formed foci at
cellular positions consistent with the replisome (defined as replicative DNA polymerase and associated proteins at the replication fork). Indeed, both b-GFP and MutSC-YFP localization for
single-focus cells was indistinguishable from the subcellular position of DnaX-GFP (replisome) (Figure 5F). An alternative explanation for the MutSC-YFP focus is that the C terminus is highly
multimerizing, which allows for us to
visualize this protein fragment as a focus.
Because MutSC-YFP shows the same
localization pattern as the replisome
(DnaX-GFP), we find this explanation unlikely. Our observation that the frequency
of MutSC-YFP foci in cells untreated with
a mismatch-inducing agent was so high
(82%) relative to the frequency of focus
formation by MutS-GFP (R5%) was of
interest. These data suggest that, in the
absence of a mismatch, MutS conformation is such that interaction with b clamp
is weak or nonexistent. In contrast, in the presence of a mismatch, MutS undergoes a conformational change licensing the
MutS C terminus to interact with b clamp.
A Mutant b Clamp Is Reduced for Supporting MutS-GFP
Focus Formation and MMR In Vivo
The data presented above are consistent with a MutSb clamp
interaction providing an important step in MMR. In support of
this hypothesis, we have determined that a previously isolated
allele (dnaN5) encoding a mutant form of b confers a mutator phenotype (Figure 6), and this allele confers a reduction in MutS-GFP
focus formation (see below).
The dnaN5 (referred to as bG73R) allele was isolated because it
confers a temperature-sensitive replication phenotype at 49 C
(Karamata and Gross, 1970). Sequencing of the dnaN5 allele
revealed that it encodes a G73R missense mutation (Ogasawara
et al., 1986). We found that strains bearing bG73R had elevated
mutation frequencies (Figures 6A and 6B). The bG73R conferred
an 10-fold mutator at 30 C and 25-fold at 37 C (Figures 6A
and 6B). An epistasis analysis demonstrated that the mutation
296 Molecular Cell 29, 291–301, February 15, 2008 ª2008 Elsevier Inc.
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b Clamp Recruits MutS
sult. MutS-GFP foci were reduced 3-fold (13%, n = 679) at 37 C. In
contrast, the percent of cells with MutS-GFP foci in a wild-type
background was unchanged at 37 C (36%, n = 699) (Figure 6F).
We performed an immunoblot analysis of wild-type b and
bG73R using anti-serum against b and found that these proteins
accumulated to indistinguishable levels in vivo at 30 C and 37 C
(Figure 6E). The amino acid substitution in B. subtilis bG73R is
located in a surface-exposed region on the outer rim of the protein
(Figures 6C and 6D). This position in b might be important for MutS
binding to b. In support of this, the G73R mutation in B. subtilis b is
located in a position of b corresponding to a position in E. coli b that
binds d0 , a component of clamp loader, and the little finger domain
of DinB (Pol IV) and UmuC (Pol V) (Bunting et al., 2003; Jeruzalmi
et al., 2001). We conclude that the mutator phenotype of bG73R
results from decreased focus formation of MutS-GFP in vivo.
DISCUSSION
Figure 5. The C Terminus of MutS Is Both Necessary and Sufficient
for Focus Formation
YFP was expressed from the amyE locus and visualized by fluorescence
microscopy. Shown are (A) YFP and membrane; (B) MutSC-YFP in a mutSL
strain and membrane; (C) MutSC-YFP and membrane in a wild-type strain;
(D) MutSC-YFP and membrane in a mutL strain. The white bar indicates
2 mm. The percent of cells with foci 45 min after addition of IPTG are shown
in each panel, and the numbers of cells scored are indicated in each panel.
(E) Representative b clamp-GFP foci are shown during normal growth in
S750 minimal medium. (F) Subcellular position of MutSC-YFP (white bars),
b clamp-GFP (gray bars), and DnaX-GFP (black bars) are presented as the
percent of cells versus the distance from the nearest pole.
frequency conferred by bG73R was not additive or synergistic with
a DmutSL allele (Figures 6A and 6B), indicating that the mutator
phenotype of bG73R results from loss of MutSL-dependent
MMR. To further confirm that the bG73R mutator was MMR dependent and DNA damage independent, we examined phage induction in single cells by visualizing a YFP fusion to a PBSX-regulated
gene (Britton et al., 2007). We found that bG73R did not display any
detectable PBSX induction (Figure S3) above our wild-type control
strain.
The MutSL-dependent mutator phenotype caused by the
bG73R mutation stimulated us to examine whether MutS-GFP focus formation was decreased in this background. Indeed, we found
that MutS-GFP was reduced for focus formation in cells treated
with the mismatch-inducing agent at 30 C (24%, n = 1248) relative
to MutS-GFP foci in a wild-type background (36%, n = 799). We
found that increasing the temperature to 37 C exacerbated this re-
In this study, we found that the C terminus of MutS is required for
MMR and localization as a focus in response to replication
errors. We have shown that this region of MutS is also required
for interaction with b clamp. Furthermore, the C terminus of
MutS is also sufficient for focus formation, even in the absence
of mismatches. We interpret these results to mean that interaction between MutS and b is the major molecular interaction
that facilitates focus formation and that b clamp aids in the
stabilization of MutS at mismatches in vivo. This conclusion is
supported by experiments demonstrating that a mutant form of
MutS impaired for focus formation is a mutator in vivo, and this
protein still binds a mismatch in vitro. These data strongly argue
that, in a living cell, mismatch binding and focus formation are
more complicated than MutS simply recognizing and binding
to the error and recruiting several more MutS dimers to the site
of the mismatch. These data also reinforce the idea that focus
formation is physiologically important for MMR activity in vivo
as we find a distinct correlation between mutS alleles that are
defective for MMR activity and focus formation.
In a reciprocal approach, we show that a mutant form of the
b clamp is impaired for MMR and MutS-GFP focus formation.
This observation strengthens our argument that b clamp is
critical for MMR and MutS-GFP focus formation. We also show
that a DmutSL allele is epistatic to our b clamp mutant, indicating
these proteins function in the same pathway.
Our findings that the C terminus of B. subtilis MutS is critical for
interaction with b and MMR activity in vivo are in contrast to
observations in E. coli. In E. coli, the C-terminal b clamp binding
motif of MutS is important for interaction with b clamp in vitro,
although the N-terminal interaction site is required for MMR
activity in vivo (Lopez de Saro et al., 2006). The differences in
b clamp binding observed between these two systems could
reflect the use of methylation-directed repair by E. coli. Taken
together, we conclude that b clamp helps recruit MutS into a
focus at mismatches in B. subtilis.
b Clamp Recruits MutS-GFP into a Focus in Response
to Mismatches
Because we deleted the entire domain structure of MutS responsible for DNA binding and still observed foci, we propose that the
Molecular Cell 29, 291–301, February 15, 2008 ª2008 Elsevier Inc. 297
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b Clamp Recruits MutS
Figure 6. A Mutant Form of the b Clamp
Is Reduced for Supporting MutS-GFP Formation
(A) Relative mutation frequency of the bG73R
strain compared with MMR-deficient strains at
30 C, and (B) 37 C. The error bars represent the
standard deviation from at least triplicate experiments. (C) Homology model of B. subtilis b clamp
with the G73R substitution shown (green). Residues colored in red and yellow are conserved
with residues in E. coli b that comprise the hydrophobic pocket (red) and conserved residues
important for interaction between E. coli clamp
loader component d0 and b (yellow). (D) Enlargement of the region containing the G73R mutation
(green). (E) Immunoblot of b and bG73R protein
levels in whole-cell lysates at 30 C and 37 C. Protein load was normalized to cell number. b is in
lanes 1 and 3, and bG73R is in lanes 2 and 4. (F)
Quantitation of MutS-GFP focus formation in
wild-type and bG73R backgrounds at 30 C and
37 C.
MutS C terminus has an intrinsic ability to bind b and localize as
a focus. We propose that, in the absence of a mismatch, intact
MutS assumes a conformation that negatively controls the interaction between MutS and b clamp. Once MutS binds a mismatch,
MutS assumes a conformation that licenses interaction with
b clamp through the MutS C terminus. We propose that this conformational change takes place in MutS and not b clamp because
our MutS C-terminal fragment appears to bind b, indicating that
b clamp is constitutively able to interact with the MutS C terminus.
Several models have been proposed suggesting that MutS
recognizes and then translocates or diffuses away from the
mismatch (Allen et al., 1997; Gradia et al., 1999). Placing these
observations into context with ours, we propose that formation
of the MutSb clamp complex is a required step to stimulate
the repetitive loading of MutS dimers at a mismatch in a living
cell. Another interpretation could be that MutS is bound to
b clamp during replication and it dissociates from b once a mismatch is detected, resulting in the recruitment of more MutS
dimers and focus formation. We find this explanation unlikely.
We show that b clamp forms discrete foci during normal growth.
If MutS constitutively associates with b during replication, we
should be able to visualize MutS-GFP foci in the absence of
mismatches by virtue of the interaction with b clamp. To the contrary, we only observe MutS foci after inducing mismatches or by
expression of the MutS C terminus and thus bypassing the
requirement for a mismatch to visualize foci.
Processivity Clamps May
Contribute to Strand
Discrimination in Organisms
Lacking dam-Directed Methylation
All MMR systems must recognize the
mismatch and identify the strand bearing
the mismatch. Early studies of the methylation-independent MMR system (hex)
of S. pneumoniae led to the suggestion
that single-strand breaks in the nascent
strand were used to direct the repair system (Lacks et al.,
1982). Examination of MMR using purified components from
the human system has demonstrated both 30 and 50 repair
from a single-strand break (Dzantiev et al., 2004; Zhang et al.,
2005). Considered together with ours, these results indicate
that interaction between MutS and b clamp might help localize
MutS to the site of replication, bringing MutS into close proximity
with the 30 terminus or the 50 end of an Okazaki fragment at a replication fork, allowing for the system to direct mismatch removal
from these sites.
Recently, it was shown that human MutL homolog hPMS2
contains an endonuclease activity that is required for MMR in
vitro from a 30 strand break (Kadyrov et al., 2006). The endonuclease active site is conspicuously conserved among many
organisms that lack methyl-directed MMR, including B. subtilis
MutL (Kadyrov et al., 2006). Moreover, in human, S. cerevisiae,
E. coli, and now B. subtilis, MutS homologs and some MutL
homologs have been shown to interact with their cognate processivity clamps (PCNA or b) (Kleczkowska et al., 2001; Lee and
Alani, 2006; Lopez de Saro et al., 2006; Shell et al., 2007).
Together with our localization results, these data collectively
support the model that MMR proteins assemble at mismatches
located at sites of replication, stimulating the formation
of MutS and MutL repair complexes through the direction of
b clamp. We propose a model where MutS is stabilized by
b clamp at mismatches in vivo located at or near the site of
298 Molecular Cell 29, 291–301, February 15, 2008 ª2008 Elsevier Inc.
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b Clamp Recruits MutS
DNA replication. MutS then recruits MutL where MutL could
incise a nick to help direct removal and repair of the mismatch.
EXPERIMENTAL PROCEDURES
tibodies used to detect b clamp were raised against b and are from Dr. C. Lee
(Massachusetts Institute of Technology). The primary was used in a 1:5000
dilution, and the goat anti-rabbit HRP-conjugated secondary antibody was
used in a 1:200 dilution.
Growth Conditions, Medium, and Mutagenesis Experiments
For microscopy all strains were grown at 30 C in S750 minimal medium.
The S750 medium was supplemented with 1% glucose, 0.1% glutamate,
40 mg/ml tryptophan, and 40 mg/ml phenylalanine or 0.5% casamino acids
for Figure 5. Antibiotic concentrations were the same as previously described
(Smith et al., 2001). Mutagenesis experiments were accomplished as
described (Smith et al., 2001) except that for the data in Figure 2, 600 mg/ml
2-AP was added for 1 hr prior to harvesting and plating of the cells.
Surface Plasmon Resonance
A BIAcore 3000 biosensor was used for our analysis of MutS or MutS800 binding to b clamp-Myc. Standard research-grade CM5 sensor chips were used for
the immobilization of 10,200 resonance units of b clamp. Immobilization of
b was accomplished using the amine coupling kit (BIAcore) following the manufacturer’s recommendations. b clamp was immobilized by using 10 mM
sodium acetate buffer (pH 4.0). MutS and MutS800 were diluted in binding
buffer prior to injection of the blank or b clamp-coupled surfaces.
Construction of B. subtilis Strains Expressing Mutant Forms of mutS
All of the mutS alleles were constructed using plasmid pBTS240 that has been
previously described (Smith et al., 2001). To verify that the intended mutS missense mutations were properly integrated, we PCR amplified the 30 end of the
full-length mutS gene fused to gfp from the indicated strains and sequenced
the PCR product (data not shown). The mutS alleles encoding the C-terminal
deletion mutants and the mutS5A allele were cloned into plasmid pJL74
(LeDeaux and Grossman, 1995) using restriction sites EcoRI and XhoI. These
alleles were integrated into the chromosome by transformation at the normal
mutS gene location placing expression under the native mutS promoters.
Supplemental Data
Supplemental Data include Supplemental Results, Supplemental Experimental Procedures, Supplemental References, three tables, and three figures and
can be found with this article online at http://www.molecule.org/cgi/content/
full/29/3/291/DC1/.
Cell Treatment and Live Cell Microscopy
Microscopy and cell treatment for microscopy were done essentially as
described (Smith et al., 2001). Briefly, cells were grown in S750 minimal medium at 30 C. Where indicated, cultures were split and one was treated with
2-AP for 1 hr in mid-log phase (OD600 0.2–0.4), while the control culture was
untreated. Aliquots (300 ml) of cells were extracted from each culture and
treated with the vital membrane stain FM4-64 (Molecular Probes) and DAPI
to visualize the DNA. Images were captured with a Nikon E800 microscope
and OpenLab software (Improvision). Images were adjusted, colored, and
merged using OpenLab software.
Protein Purification, Far-Western Blotting, Peptide Array Analysis,
and Immunoblotting
MutS, MutS800, MutS850, and b-Myc were cloned into pET11T and overexpressed (primer sequences available upon request) in E. coli BL21DE3 cells
using standard procedures. Cell pellets containing b-Myc, MutS, MutS800,
and MutS850 were resuspended in buffer A (0.02 M K2HP04 [pH 7.4],
50 mM KCl, 10% glycerol, 0.1 M EDTA, and 0.1 M DTT) + 150 mM KCl, 20
mM SpCl3, 1 mM AEBSF, and 0.3 mg/ml lysozyme. For each protein, cells
were ruptured by freeze/thaw lysis after incubation on ice for 20 min. The lysate
was clarified by centrifugation prior to dilution with buffer A and application to
a monoQ column for purification. An elution gradient of 0.1–1 M KCl was used
with each protein eluting near 700 mM KCl. Purity of proteins was determined
to be greater than 90% as judged by SDS-PAGE. Purified His6-DnaE was from
Dr. Neil Brown (University of Massachusetts, Worcester).
Far-western blotting was done with slight modification from a procedure
already described (Ludlam et al., 2001). Briefly, equal amounts of the indicated
proteins were immobilized onto a nitrocellulose membrane (Protran,
Whatman) by spotting. Following protein immobilization, each membrane
was blocked in phosphate-buffered saline (PBS) + 2% nonfat milk for 1 hr.
After three washes for 10 min with TS/Tween, 2.6 mg/ml b-Myc was added
to binding buffer (40 mM HEPES-KOH [pH 7.6], 10 mM MgCl2, 4 mM DTT, 1
mM ATP, 0.1 mg/ml BSA, and 0.1% glutaraldehyde) for 1 hr. Each membrane
was washed 3X in TS/Tween for 20 min. The membranes were then incubated
overnight at 4 C with anti-Myc monoclonal antibodies (1:1000) in TS/Tween
with 2% nonfat milk. Membranes were washed 3X for 30 min with TS/Tween
followed by incubation with goat anti-mouse HRP-conjugated (Pierce)
antibodies (1:500) for 1 hr. Following three washes in TS/Tween for 30 min,
membranes were developed with chemiluminescence (Super-Signal Altra,
Pierce). Peptide arrays were performed as described (Beuning et al., 2006),
and immunoblots were done as described (Simmons et al., 2007). Primary an-
ACKNOWLEDGMENTS
We thank Dr. Brad Smith, Dr. Adam Breier, Mary Ellen Wiltrout, and members
of the Walker and Grossman labs for careful reading of this manuscript. We
thank Dr. Alexi Goranov for help with construction of the dnaN-GFP allele.
We would also like to thank Dr. Caroline Koehrer for help with the SPR analysis
and Drs. Uttam RajBhandary and H. Gobind Khorana for use of their BIAcore
3000 instrument. G.C.W. is an American Cancer Society Research Professor
and is supported by NCI grant CA21615-27 and the Massachusetts Institute
of Technology Center for Environmental Health Sciences. This work was
also supported by NIH grant GM41934 to A.D.G. and a postdoctoral fellowship
from the NCI to L.A.S. A graduate scholarship from the National Sciences and
Engineering Research Council of Canada supported B.W.D.
Received: August 7, 2007
Revised: September 28, 2007
Accepted: October 31, 2007
Published: February 14, 2008
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