Copyright Ó 2007 by the Genetics Society of America
DOI: 10.1534/genetics.107.077255
Mms22 Preserves Genomic Integrity During DNA Replication in
Schizosaccharomyces pombe
Claire L. Dovey and Paul Russell1
Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 90237
Manuscript received June 7, 2007
Accepted for publication June 29, 2007
ABSTRACT
The faithful replication of the genome, coupled with the accurate repair of DNA damage, is essential for
the maintenance of chromosomal integrity. The MMS22 gene of Saccharomyces cerevisiae plays an important
but poorly understood role in preservation of genome integrity. Here we describe a novel gene in
Schizosaccharomyces pombe that we propose is a highly diverged ortholog of MMS22. Fission yeast Mms22
functions in the recovery from replication-associated DNA damage. Loss of Mms22 results in the accumulation of spontaneous DNA damage in the S- and G2-phases of the cell cycle and elevated genomic instability.
There are severe synthetic interactions involving mms22 and most of the homologous recombination proteins but not the structure-specific endonuclease Mus81-Eme1, which is required for survival of broken
replication forks. Mms22 forms spontaneous nuclear foci and colocalizes with Rad22 in cells treated with
camptothecin, suggesting that it has a direct role in repair of broken replication forks. Moreover, genetic
interactions with components of the DNA replication fork suggest that Mms2 functions in the coordination
of DNA synthesis following damage. We propose that Mms22 functions directly at the replication fork to
maintain genomic integrity in a pathway involving Mus81-Eme1.
T
HE success of all living organisms depends on their
ability to distribute accurate copies of their genome
to daughter cells during each round of cell division.
This task must be accomplished against a backdrop of a
plethora of DNA lesions that can arise during normal
cellular metabolism or through the actions of exogenous genotoxins. To combat this, eukaryotic organisms
have evolved a variety of mechanisms that sense DNA
damage and facilitate its repair. These mechanisms include
specific DNA repair processes such as homologous recombination (HR), nonhomologous end joining, and
nucleotide excision repair, as well as checkpoint mechanisms that delay cell cycle progression and coordinate DNA
repair.
In the fission yeast Schizosaccharomyces pombe, one specific protein that has been implicated in DNA repair is
Brc1, which is a nonessential protein with six-BRCT
(BRCA1 C-terminal) domains. Brc1 is thought to function primarily in S-phase where it aids in the replication of
damaged DNA (Verkade et al. 1999; Sheedy et al. 2005).
Brc1 was initially identified as a high-copy suppressor of
smc6-74 (Verkade et al. 1999), which encodes a subunit of
the essential ‘‘structural maintenance of chromosomes’’
Smc5/6 complex that functions in chromosome organization and DNA repair (Lehmann et al. 1995; McDonald
et al. 2003; Harvey et al. 2004; Pebernard et al. 2004,
1
Corresponding author: Department of Molecular Biology, The Scripps
Research Institute, MB-3, 10550 North Torrey Pines Rd., La Jolla, CA
90237. E-mail: [email protected]
Genetics 177: 47–61 (September 2007)
2006). The Brc1-mediated rescue of smc6-74 is thought to
proceed via a novel Rhp18-dependent mechanism, utilizing multiple nucleases to facilitate the initial cleavage
of abnormal structures that arise due to compromised
Smc5/6 function and their subsequent processing by HR
(Sheedy et al. 2005; Lee et al. 2007). Loss of Brc1 function
results in sensitivity to a range of DNA-damaging drugs
that generate lesions specifically during S-phase or impede
DNA replication (Verkade et al. 1999; Sheedy et al. 2005).
Brc1 is related to the 6-BRCT repeat proteins Rtt107p
(Esc4p) in Saccharomyces cerevisiae and PTIP in humans,
both of which have been implicated in the response to
DNA damage ( Jowsey et al. 2004; Rouse 2004). Budding yeast Rtt107p promotes the resumption of DNA
synthesis after damage (Rouse 2004) and has been
postulated to act as a protein scaffold at stalled replication
forks where it recruits or modulates the function of other
proteins required for the restart of DNA replication
(Chin et al. 2006). In line with this, Rtt107p physically interacts with Slx4, a component of the Slx1-Slx4 structurespecific nuclease, and Slx4-dependent phosphorylation
of Rtt107p by Mec1 has been proposed to be critical for
replication restart following alkylation damage (Roberts
et al. 2006). Rtt107p has also been shown by yeast twohybrid analysis to interact with HR mediators and with
Tof1 (Chin et al. 2006), a subunit of the replicationpausing complex Tof1-Csm3, which associates with unperturbed and stalled replication forks and is required to
prevent uncoupling of Cdc45 and the MCM complex
from DNA synthesis during hydroxyurea (HU) arrest
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C. L. Dovey and P. Russell
(Katou et al. 2003). Similarly, Rtt107p was identified as an
interactor of the DNA repair protein Mms22p in a highthroughput study and also by yeast two-hybrid analysis
(Ho et al. 2002; Chin et al. 2006). MMS22 was initially
identified as a gene required for resistance to ionizing
radiation (IR) (Bennett et al. 2001); however, MMS22
mutants also display hypersensitivities to HU, methyl methanesulfonate (MMS), camptothecin (CPT), and etoposide, agents that result in DNA damage predominantly
in S-phase (Chang et al. 2002; Araki et al. 2003; Baldwin
et al. 2005).
MMS22 was also uncovered in a screen for mutants
that are lethal in combination with mcm10-1, a thermosensitive allele of the DNA replication factor MCM10
(Araki et al. 2003), suggesting that Mms22p functions to
resolve replication intermediates or to prevent damage
caused by blocked replication forks (Araki et al. 2003).
Mms22p is thought to function downstream in a pathway
involving the DNA repair protein Mms1p (Araki et al.
2003); however, the precise mechanism of repair employed by this pathway is unknown, and homologs of
Mms1p and Mms22p have not yet been identified in S.
pombe or in higher eukaryotes. In view of the importance
of this DNA repair complex in S. cerevisiae, we undertook
a detailed sequence analysis to attempt to identify a
fission yeast homolog of MMS22.
Here we report the identification and initial characterization of a novel fission yeast gene that is a putative
ortholog of budding yeast MMS22. Our studies indicate
that S. pombe mms221 has a central role in preserving
genome integrity during DNA replication and is vital for
viability following DNA replication-associated damage.
MATERIALS AND METHODS
Strains and genetic methods: Standard procedures and
media for S. pombe genetics were used as previously described
(Moreno et al. 1991). The entire open reading frame of mms221
was replaced with the hygromycin B (HphMx6) or kanamycin
(KanMx6) resistance markers as described (Bahler et al. 1998).
Ectopic expression of GFP-Mms22 was from the pRep41-N-GFP
plasmid under control of the attenuated thiamine-repressible
nmt41 promoter. Strains used are listed in supplemental Table
S1 at http://www.genetics.org/supplemental/ (Nakamura et al.
2004).
Microscopy: Cells were photographed using a Nikon Eclipse
E800 microscope equipped with a Photometrics Quantix CCD
camera. Rad22-YFP-expressing strains were cultured in yeast
extract with supplements (YES) for at least 16 hr before foci
quantification and at least 250 nuclei were scored in three
independent experiments. Strains expressing pRep41-N-GFPmms221 were grown in EMM supplemented media containing
thiamine for 20 hr before imaging. For quantification of GFPMms22 foci following CPT treatment, cultures were grown for
20 hr and then split into two. To one culture, a final concentration of 30mm CPT in DMSO was added, while the other culture had 0.3% (v/v final) DMSO added as a control. Treatment
was for 3 hr at 30°. All microscopy was conducted on live,
midlog phase cells, except for cultures to be DAPI stained,
which were fixed in 70% ethanol for 10 min at room tem-
perature, washed, and pelleted before resuspension in 5 ml of
DAPI solution (500 mg/ml).
Minichromosome instability assay: Minichromosome loss
was measured as previously described (Allshire et al. 1995).
Briefly, 500–1000 cells from individual Ade1 colonies were
plated on adenine-limiting (12 mg/liter) plates, incubated at
30° for 3 days and then at 4° for 1 day to allow deepening of the
red color in Ade colonies. The number of chromosome loss
events per division was determined as the number of colonies
with a red sector equal to, or greater than, half the colony,
divided by the sum of white and sectored colonies.
Survival assays: For chronic exposures, midlog phase cultures were resuspended to 1 3 107 cells/ml and serially diluted
fivefold. Dilutions were spotted onto YES agar plates or YES
agar containing the indicated amounts of MMS, CPT, or HU
(selective minimal media was used for experiments involving
ectopic GFP-Mms22 expression). For acute exposures to IR
and UV-induced damage, 1000 cells were plated onto triplicate
YES agar plates and immediately irradiated with the indicated
dose. Alternatively, YES plates were spotted with serial dilutions of cells, as for chronic drug exposure, and irradiated with
the indicated dose. For survival of acute exposure to HU,
midlog phase cells were cultured in YES media containing 12
mm HU for 10 hr. At the indicated time points, samples were
taken, HU was washed out, and 1000 cells were plated onto
YES agar plates in triplicate. For all survival assays, recovery was
for 2–3 days at 30° unless otherwise stated.
RESULTS
Identification of S. pombe mms22 +: By focusing on an
N-terminal core homology region that is most highly
conserved between S. cerevisiae MMS22 and its budding
yeast homologs, we were able to use position-specific
iterated BLAST (Altschul et al. 1997; Schaffer et al.
2001) to identify a related gene in other divergent fungal
species, including S. pombe (represented in Figure 1A).
All of these genes encode very large proteins that range
in size from 1400 to 2700 amino acids. Fission yeast
SPAC6B12.02c1 encodes a protein of 1888 residues (Mw
217.4 kDa). Alignment of S. cerevisiae Mms22p and S.
pombe SPAC6B12.02c proteins with the EMBOSS pairwise alignment (needle) algorithm (EMBL-EBI) indicated that the two proteins share 18.4% identity and
32.3% similarity over their full-length sequences. On the
basis of our alignments, we predict that SPAC6B12.02c1 is
a putative homolog of MMS22, and the relatively low
identity may be explained by the fact that S. cerevisiae
and S. pombe are highly divergent yeasts. Therefore,
SPAC6B12.02c1 was named mms221.
Mms22 is important during a perturbed S-phase:
Deletion of mms221 produced viable haploid cells that
formed small colonies in the absence of any genotoxic
stress (Figure 1C). The mms22D colonies contained many
dead or elongated cells (Figure 2A) that are typical of
mutants defective in DNA replication or mutants that
are unable to repair DNA breaks that can arise spontaneously during DNA replication. In line with this, the
plating efficiency of mms22D mutants was, on average
40%, compared to 84% for wild type (data not shown).
Similarly, the average doubling time of mms22D mutants
Mms22 Functions in DNA Repair
49
Figure 1.—Identification of a putative homolog of MMS22 in S. pombe. (A) A schematic of putative MMS22 homologs in representative fungi showing areas of homology in the N and the C terminus and the corresponding amino acid alignments. The
N-terminal homology domain spans S. cerevisiae Mms22p residues 196–292, Coccidioides immitis CIMG_05124 residues 403–499,
Aspergillus nidulans AN6261.2 residues 522–614, and S. pombe SPAC6B12.02c (hereafter named Mms22) residues 246–337. The
C-terminal homology domain incorporates S. cerevisiae Mms22p residues 1055–1234, CIMG_05124 residues 1615–1815, AN6261.2 residues 1748–1949, and S. pombe Mms22 residues 1319–1517. White text on a solid background indicates amino acid identity and solid text
on a shaded background indicates conservative amino acid substitutions. (B) Survival curves of mms22D mutants exposed to increasing
doses of IR or UV. A total of 500–1000 cells were plated on YES agar in triplicate and immediately exposed to the indicated dose of IR or
UV irradiation. Colony numbers were counted following incubation at 30° for 2–3 days and the mean colony number for each dose
represented graphically (with untreated normalized to 100% survival). The sensitivity of a rad32D mutant was analyzed as positive control. (C) Phenotypes of mms22D mutants. Fivefold serial dilutions of cells were plated on YES agar exposed to the indicated DNA-damaging agent and incubated at 30° for 2–3 days.
was 181 min in rich media at 32°, compared to 118 min
for wild-type strains cultured under the same conditions
(data not shown). Quantitative analysis confirmed that
mms22D cultures contained an elevated number of uninucleate cells that are indicative of a checkpoint delay
in cell cycle progression (Figure 2A). In fission yeast,
Cds1 is the effector kinase of the DNA replication checkpoint that is activated when replication forks stall, whereas
Chk1 enforces the G2-M checkpoint that is activated when
DNA is damaged (Boddy and Russell 2001). Elimination of Chk1, but not of Cds1, reduced the elongated
phenotype of most mms22D cells (Figure 2A), indicating
that mms22D cells accumulate DNA structures that activate
the Chk1-dependent DNA damage checkpoint.
S. cerevisiae mms22D cells are hypersensitive to a variety
of DNA-damaging agents, including MMS, HU, CPT,
and topoisomerase II-mediated DNA damage, and are
moderately sensitive to UV and IR (Bennett et al. 2001;
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C. L. Dovey and P. Russell
Figure 2.—Deletion of Chk1 reduces the elongated phenotype of mms22 mutants. (A) Cells
were cultured to midlog phase in YES medium,
fixed in 70% ethanol for 10 min at room temperature, and DAPI stained to visualize the nuclei
and morphology of the cell. At least 250 cells
were scored in three independent experiments
and assigned to a particular cell cycle phase as
previously described (Noguchi et al. 2004). Mean
values were plotted with error bars representing
the standard deviation about the mean. (B)
Mms22 is not required for checkpoint activation
in response to HU. Cells were cultured to midlog
phase in YES medium and the culture was split
into two. One culture was pelleted, immediately
fixed in 70% ethanol for 10 min at room temperature, and DAPI stained, while the other culture
was incubated in the presence of 12 mm HU for
4 hr prior to fixation and DAPI staining. Representative images are displayed.
Chang et al. 2002; Araki et al. 2003; Baldwin et al.
2005); thus we analyzed whether fission yeast mms22D
cells were sensitive to DNA-damaging agents. IR generates a number of lesions, including single-strand and
double-strand breaks (DSBs), as well as base modification and damage, but its toxicity arises mainly from DSBs
(Ward 1988). Unlike HR mutants that are profoundly
sensitive to IR, we found that mms22D mutants were not
hypersensitive to IR (Figure 1B). Similarly, we found that
mms22D mutants were sensitive to UV only at high doses
(Figure 1B). These data indicate that Mms22 is not essential for repair of IR-induced DSBs or UV-induced
DNA lesions.
To address whether loss of Mms22 function results in
sensitivity to agents that impede DNA replication, we
assessed the growth of mms22D mutants following chronic
exposure to agents that stall replication forks. HU is a
ribonucleotide reductase inhibitor, which arrests replisome progression by depleting the cellular pool of
dNTPS, whereas MMS impedes replication fork progression by alkylating DNA template bases. Cells dis-
rupted for mms22 were sensitive to HU (Figures 1C and
3B) and hypersensitive to MMS (Figure 1C), suggesting
that the function of Mms22 is important in the recovery
from replication fork stalling.
We also examined whether Mms22 has a role in the
tolerance of S-phase-associated DNA breaks using the
topoisomerase-I (Top-I)-inhibitor CPT. CPT stabilizes
Top-I-DNA cleavage complexes, which can result in DSB
formation when the replication fork collapses on encountering Top-I-mediated nicks in the template DNA
(Pommier 2006). We observed that mms22D mutants
were hypersensitive to CPT, even at very low doses (Figure
1C), which, together with the sensitivity to HU and MMS,
indicates an important function for Mms22 in the recovery from replication fork damage and stalling.
Genetic interactions with checkpoint mutations: To
address whether Mms22 functions in the checkpoint
response to either DNA damage or replication stress, we
performed experiments to uncover genetic interactions
between mms22 and cds1 or chk1. We found that mms22D
chk1D cells were more sensitive to low doses (10–25
Mms22 Functions in DNA Repair
51
Figure 3.—Mms22 has checkpoint-independent
functions. (A) Phenotypes of mms22 mutants in
checkpoint kinase-deficient backgrounds. Fivefold serial dilutions of cells were plated on YES
agar exposed to the indicated DNA-damaging
agent and incubated at 30° for 2–3 days. (B) Phenotypes of mms22 mutants in checkpoint kinasedeficient backgrounds following acute exposure
to UV and HU. For UV exposure, 1000 cells were
plated onto triplicate YES agar plates and immediately irradiated with the indicated dose. For survival of acute exposure to HU, midlog phase cells
were cultured in YES media containing 12 mm
HU for 10 hr. At 0 hr, 1000 cells were plated onto
YES agar plates in triplicate and, at the indicated
time points, the same culture volume was taken,
HU was washed out, and the cells were plated in
triplicate. Survival was estimated relative to untreated cells. For all survival assays, recovery was
for 2–3 days at 30° unless otherwise stated.
J/m2) of UV than either mms22D or chk1D alone (Figure 3,
A and B), indicating that the modest UV survival defects
caused by the absence of Mms22 are enhanced in the
absence of a DNA damage checkpoint arrest. A similar
interaction was seen following IR and on chronic exposure to 2 mm HU (Figure 3A). Similarly, following
8–10 hr of acute exposure to 12 mm HU, the double
mms22D chk1D mutant showed reduced viability compared to mms22D or chk1D alone (Figure 3B). There was
no genetic interaction between mms22D and cds1D in
response to UV or IR (Figure 3, A and B), although the
double mms22D cds1D showed a growth defect and an
enhanced sensitivity to chronic low doses of HU (1–
1.25 mm) relative to mms22D or cds1D alone (Figure 3A),
as well as additive sensitivity following 1–4 hr of acute
HU exposure (Figure 3B). Moreover, loss of Mms22 was
detrimental to the growth of cells defective in both Cds1
and Chk1 in response to UV and IR (Figure 3, A and B).
Similarly, following chronic exposure to 1 mm HU, the
triple mutant was more sensitive than either mms22D or
the double cds1D chk1D strain (Figure 3A), suggesting that
Mms22 has a checkpoint-independent function at least
in the tolerance of these DNA-damaging agents. The hypersensitivity of mms22D to MMS and to CPT made accurate comparisons for sensitivity to these agents difficult.
Finally, we examined whether Mms22 is involved in
checkpoint activation in the response to HU. The majority of cells in mms22D, and both the mms22D cds1D
and the mms22D chk1D cultures, arrested division with
an elongated phenotype, whereas checkpoint failure in
response to HU in a cds1D chk1D background resulted
in a ‘‘cut’’ phenotype, which is diagnostic of checkpoint
failure and subsequent mitotic catastrophe (Figure 2B).
These data confirm that the intra-S-phase checkpoint
can be activated in response to HU independently of
Mms22. Together, these synergistic interactions of
mms22 with cds1 and chk1 suggest that Mms22 has
functions that are at least partially independent of
Cds1 and Chk1 and that Mms22 is not required
upstream for the activation of either checkpoint kinase
in response to HU.
Mms22 is required to maintain genomic stability: To
confirm that mms22 mutants display an elongated
phenotype due to the presence of DNA damage, we
analyzed the effect of mms22D on the formation of
Rad22 foci. Rad22 is the fission yeast homolog of the
single-stranded DNA (ssDNA)-binding protein Rad52,
which concentrates into bright visible foci at sites of
DSB repair (Du et al. 2003). Approximately 14% of asynchronous wild-type nuclei contained one or more
Rad22-YFP foci, compared to 53% in mms22D mutants
(Figure 4A), suggesting that loss of mms22 function
leads to elevated spontaneous DNA damage.
As Mms22 is important for the survival of S-phasespecific damage, we hypothesized that the spontaneous
damage might arise during DNA replication. To explore
this, the cell cycle position of cells containing Rad22-YFP
foci was determined. We observed that Rad22-YFP foci
52
C. L. Dovey and P. Russell
Figure 4.—Spontaneous DNA damage occurs in the absence of Mms22. (A) Elevated Rad22-YFP foci arise in the Sand G2-phases of the cell cycle in an mms22D background.
Cells were cultured to midlog phase in YES medium and imaged live. The numbers of foci in at least 250 nuclei representing different phases of the cell cycle were scored in three
independent experiments, and mean values were plotted with
error bars representing the standard deviation of the mean.
(B) Elevated spontaneous minichromosome 16 loss associated with disruption of Mms22 function. Cells containing
the minichromosome are ade1 due to allelic complementation. Cells from individual Ade1 colonies were plated on
adenine-limiting plates and the percentage of chromosome
loss events per division was determined. The actual numbers
are displayed under the chart (at least half red/total colonies).
were specifically elevated in the S- and G2-phases in mms22D
cells (Figure 4A), suggesting the presence of damaged
DNA during these cell cycle phases in the absence of
Mms22 function.
To determine whether the DNA damage that we observed in mms22 strains translates into genomic instability,
we utilized strains that contained the three chromosomes
of haploid S. pombe, as well as a copy of minichromosome
16 (Ch16), to analyze spontaneous chromosomal instability (Prudden et al. 2003). Ch16 is a highly stable
530-kb linear minichromosome, containing a centric re-
gion of chromosome III (Niwa et al. 1986). Ch16 encodes
an ade6-M216 point mutation, which, when present in a
background with an ade6-M210 heteroallele on chromosome III, results in an ade1 phenotype through allelic
complementation (Figure 4B). The spontaneous loss of
Ch16 was monitored in wild-type and mms22D backgrounds by growth on media lacking adenine. In this
assay, mms22D mutants experienced an 35-fold increase
in spontaneous loss of the minichromosome per cell division relative to wild type (Figure 4B).
In summary, mms22D mutants display spontaneous
DNA damage as judged by the Chk1-dependent cell
cycle delay and elevated Rad22 foci, and together with
the increased spontaneous minichromosome 16 loss,
these data suggest that Mms22 function is essential to
maintain genomic stability in fission yeast.
Mms22 forms nuclear foci: A number of proteins
involved in DNA repair or checkpoint responses concentrate into foci in response to damage (Lisby et al.
2004), therefore we were interested to see if Mms22
localizes to punctate structures in S. pombe. Ectopic GFPMms22 was expressed under the control of the thiaminerepressible nmt promoter. In the presence of thiamine,
GFP-Mms22 formed spontaneous nuclear foci due to
leaky expression from the promoter (Figure 5, A and B).
Under repressed conditions, the pRep41-N-GFP-mms221
plasmid encodes a functional protein, as it rescues the
growth defect and MMS, CPT, and HU sensitivity of
mms22D cells (data not shown).
Fission yeast nuclei are composed of two hemispherical compartments, one encompassing the nucleolus
that excludes DAPI staining and is enriched with RNA
and the other that stains with DAPI and contains the
three chromosomes. Two chromatin protrusions containing the highly repetitive rDNA arrays extend into
the nucleolus (Uzawa and Yanagida 1992). Fission
yeast rDNA contains four closely spaced polar replication
barriers named RFB1–4, which are sites of programmed
fork pausing (Krings and Bastia 2004; SanchezGorostiaga et al. 2004). As Mms22 has an important
role in the recovery from drug-induced replication fork
stalling, we wondered whether the protein also functions
at sites of natural fork pausing, such as the rDNA repeats.
To address whether Mms22 foci form exclusively at the
sites of the rDNA repeats, we transformed GFP-Mms22 into
cells expressing the red fluorescent protein (RFP)-tagged
small-nucleolar-RNA-associated protein Gar1 from its
endogenous locus. While GFP-Mms22 foci were frequently proximal to the Gar1-RFP signal, they were also
observed at non-nucleolar sites (data not shown), suggesting that Mms22 does not function exclusively at the
nucleolus.
We wanted to determine if GFP-Mms22 concentrates
into foci that increase in number following DNA damage.
GFP-Mms22 foci number levels varied between experiments due to the expression from the ectopic plasmid;
however, in multiple independent experiments, the
Mms22 Functions in DNA Repair
Figure 5.—Mms22 forms nuclear foci that increase in response to DNA damage. (A) Cells expressing ectopic GFPMms22 were cultured for 20 hr to midlog phase in selective
medium containing thiamine. The culture was split into
three, one of which was analyzed immediately as the starting
culture, and the other two of which were treated with CPT or
DMSO as stated in the materials and methods section. For
each culture, foci were scored in at least 250 live nuclei in
three independent experiments. A representative data set
from one experiment is shown due to variable GFP-Mms22 expression between individual experiments. (B) Mms22 foci
represent sites of DSBs following CPT treatment. The majority
of spontaneous GFP-Mms22 and Rad22-RFP foci do not colocalize, whereas an increased overlap in signal is observed following DNA damage. Representative images are shown. (C)
Quantification of the percentage of GFP-Mms22 foci with
an overlapping Rad22-RFP focus before and after CPT treatment. For each culture, foci were scored in three independent experiments and mean values were plotted with error
bars representing the standard deviation of the mean.
number of nuclei containing Mms22 foci increased twoto threefold following CPT-induced DNA damage (a
representative data set is shown in Figure 5A), suggesting
that Mms22 foci may represent sites of DNA damage. To
address microscopically whether GFP-Mms22 foci represent sites of DSBs, we generated a strain expressing
53
endogenous Rad22-RFP and ectopic GFP-Mms22 to
assess whether the two proteins colocalize within common foci. In an asynchronous culture, the majority of
Rad22-RFP and GFP-Mms22 foci did not colocalize, with
only 5% of GFP-Mms22 foci having an overlapping
Rad22-RFP focus (Figure 5, B and C). This suggests that
spontaneous GFP-Mms22 foci largely do not localize to
sites of HR-mediated repair, or alternatively, that Rad22RFP and GFP-Mms22 may localize to the same sites but in
a sequential manner. However, following CPT treatment,
an increased association was observed, with 45% of GFPMms22 foci having a colocalizing Rad22-RFP signal
(Figure 5, B and C), implying that at least within the
limits of our microscopy system, a proportion of GFPMms22 foci represent sites of DSBs after CPT treatment.
Genetic interactions with HR mutants: On the basis
of the observation that Mms22 and Rad22 form largely
distinct spontaneous foci, we speculated that Mms22
may act in an alternative DNA repair pathway to HR.
Consequently, the elevated spontaneous Rad22 foci
observed in mms22D cells may represent DNA damage
that in the absence of Mms22 require HR for repair. To
address this, we crossed mms22D strains with mutants
defective for HR and analyzed the progeny by tetrad
dissection. The mms22D strains that were also defective
in rad22 (S.c. RAD52), rhp51 (S.c. RAD51), or rhp54 (S.c.
RAD54) showed severe synthetic phenotypes. Double
mutants did not arise with the expected frequency (this
effect was most noted for the mms22D rad22D doubles),
and when colonies did form, the double mutants showed
a severe growth defect (Figure 6A). Similarly, double
mms22D rhp57D (S.c. RAD57) mutants, while not as severe,
showed additive growth defects and DNA damage sensitivities (Figure 6B). Taken together with the elevated
numbers of Rad22 foci in mms22D mutants, these data
suggest that, in the absence of Mms22, cells experience an
elevated occurrence of spontaneous-replication-associated
DNA damage that is repaired by HR.
Genetic interactions between mms22 and DNA repair
mutants: As Mms22 seems to function in a checkpointindependent non-HR mechanism, we sought to identify
other members of the pathway using genetic epistasis
analysis. To this end, we performed genetic crosses to
disrupt mms22D in strain backgrounds defective in a
number of different DNA repair processes, including
post-replicative repair (PRR) and replication restart.
Synergistic genetic interactions between mms22 and
srs2 or rhp18 suggested that Mms22 does not function
exclusively with these proteins in the PRR pathway (data
not shown). Therefore, we addressed the participation
of Mms22 in fork recapture and replication restart by
disruption of the gene in backgrounds defective in the
DNA-processing enzymes Mus81 or Rqh1. Mus81, in a
complex with its partner Eme1, comprises a structurespecific endonuclease that cleaves cruciform DNA structures such as D-loops or Holliday junctions (HJs) that
arise during homologous recombination (Boddy et al.
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C. L. Dovey and P. Russell
Figure 6.—Mms22 functions in a non-HR DNA repair
pathway (A) Tetrad dissection of genetic crosses of mms22D
and HR mutants. Representative spores from three asci are
shown for each cross. (B) Synthetic additivity of mms22 and
rhp57 mutations. Fivefold serial dilutions of cells were exposed to the indicated DNA-damaging agent and incubated
at 30° for 2–3 days.
2001; Kaliraman et al. 2001; Gaillard et al. 2003; Osman
et al. 2003; Whitby et al. 2003; Cromie et al. 2006; Gaskell
et al. 2007). In our genetic analysis, mus81D mutants were
as slow growing and sensitive to DNA damage and replication stress as mms22D mus81D cells, suggesting that
mus81 is epistatic to mms22 with respect to growth and
damage tolerance (Figure 7A).
Another class of highly conserved enzymes important
for the maintenance of genomic stability is the RecQ
helicase family of proteins (Bachrati and Hickson
2003). In S. pombe, the RecQ helicase Rqh1 has been
shown to be required for processing of aberrant chromosome structures arising from DNA replication (Win
et al. 2005). Mechanistically, RecQ-like DNA helicases
can act directly to dissolve HR intermediate structures
such as D-loops (Van Brabant et al. 2000), and in vitro
they are capable of dissolving a DNA substrate containing two HJs into a single noncrossover product in a
process termed double-junction dissolution (Wu and
Hickson 2003). On the contrary to the situation with
mus81, double mms22D rqh1D mutants displayed slower
growth and elevated sensitivities to UV, IR, MMS, CPT,
and HU compared to either mms22D or rqh1D alone,
indicating that Mms22’s function is important for growth
in the absence of rqh1 and vice versa (Figure 7A). Together
with the knowledge that mus81D rqh1D cells are inviable
(Boddy et al. 2000), these data suggest that Mms22 functions with Mus81 in the repair of DNA replication fork
abnormalities, and in the absence of this pathway cells
require the function of Rqh1.
Finally, we examined the genetic relationship between
mms22 and brc1. On the basis of putative interaction
between budding yeast Mms22p and Rtt107p (Ho et al.
2002; Chin et al. 2006), we hypothesized that if mms221 is
the ortholog of MMS22, then mms22 and brc1 should
show genetic epistasis if the two proteins function exclusively together. We observed that brc1D mutants grow
with wild-type kinetics, but are sensitive to the same
spectrum of DNA-damaging agents as mms22D, albeit
at higher doses, and are not hypersensitive to IR or UV
(Verkade et al. 1999; Sheedy et al. 2005; our own observations). Cells disrupted for both mms22 and brc1
showed a moderate synergistic growth defect compared
to mms22D alone (Figure 7B), suggesting that the two
proteins do not function exclusively together. Yet, taking
this growth defect into account, the mms22D brc1D mutant was just as sensitive as mms22 cells to DNA damage
and replication stress (Figure 7B). Together, these data
suggest that Brc1 and Mms22 have some independent
functions that affect cell viability; however, their functions in tolerating DNA damage during S-phase may
involve a common pathway. However, it is of importance
to note that brc1 mutants are still viable at these low doses
of CPT (0.3 mm) and MMS (0.002%) and do not show
sensitivity until exposure to higher doses (5 mm CPT and
0.01% MMS; our unpublished observations), which may
result in additive sensitivity of the double mutant being
undetected at these low doses.
Mms22 interacts genetically with components of the
replication fork: Together, our data suggest a role for
Mms22 in the recovery from DNA damage that occurs
during the process of DNA replication. As no detectable
functional domains are apparent in Mms22, it seems
unlikely that the protein is involved in the direct processing of abnormal replication structures. Rather, taking
into account the large size of the Mms22 protein, we
speculate that it might instead function as a protein
interaction platform important for the restart of DNA
replication after forks have paused or stalled. To address
this genetically, we constructed double mutants of mms22D
with either swi1D or swi3D. Swi1 and Swi3 form the DNA
replication fork protection complex (FPC), which plays
important roles in the stabilization of stalled replication
forks and activation of the DNA replication checkpoint
(Noguchi et al. 2004). Swi1 and Swi3 are required for
fork pausing at specific sites in S. pombe (Krings and
Bastia 2004), and their function is conserved in their S.
cerevisiae counterparts Tof1p and Csm3p (Calzada et al.
2005). Strikingly, we observed that deletion of either
swi1 or swi3 leads to a substantial rescue of the slowgrowth phenotype of mms22 and also to a partial rescue
Mms22 Functions in DNA Repair
55
Figure 7.—Genetic relationship between Mms22 and other DNA repair proteins. Fivefold serial dilutions of cells were exposed
to the indicated DNA-damaging agent and incubated at 30° for 2–3 days.
of the sensitivity of mms22 mutants to MMS (Figure 8A).
A slight rescue was also observed following exposure to
low doses of HU and CPT, but this was not evident at
doses above 2 mm and 0.3 mm, respectively.
Another component of the traveling and paused replication fork is the checkpoint mediator Mrc1; however,
unlike Swi1 and Swi3, Mrc1 is dispensable for fork
pausing at a protein–DNA barrier (Calzada et al. 2005).
The partial rescue of mms22D by swi1D or swi3D that we
observed was specifically attributable to defective pausing, as deletion of Mrc1 did not improve the growth or
sensitivity of mms22 mutants (Figure 8A). This suggests
that Mms22 may function in the recovery of paused
replication forks after they have encountered a block or
obstacle.
If Mms22 has a role in the restart of replication after
fork pausing or stalling, then it might follow that mms22
interacts genetically with components of the DNA replication fork. Indeed, budding yeast MMS22 was previously
isolated in a screen for mutations that are synthetically
lethal with the mcm10-1 allele (Araki et al. 2003). Mcm10p
is an essential eukaryotic DNA replication factor, which has
been shown to be required for recruitment of Cdc45p
to replication origins (Sawyer et al. 2004) and for the
stabilization and targeting of DNA polymerase a (Pola) to
chromatin (Fien et al. 2004; Ricke and Bielinsky 2004).
To establish whether mms221 displays interactions analogous to MMS22, we disrupted mms221 in a DNA replication-defective background using a hypomorphic mutation
in cdc23, the fission yeast homolog of MCM10.
The cdc23-M36 allele was isolated as an S-phasedefective temperature-sensitive mutant, which arrested
with a cdc phenotype at the restrictive temperature of
36°, but is viable at the intermediate temperature of 30°
(Nasmyth and Nurse 1981). At the permissive temperature of 25°, the double cdc23-M36 mms22D mutant was
viable, with a slight growth defect and elevated sensitivity to damage (Figure 8B). Moreover, the restrictive
temperature of cdc23-M36 was reduced to 30° in an
mms22D background (Figure 8B), with the cells displaying an aberrant and elongated terminal morphology.
Therefore, similarly to the situation for mms22 and mcm10
in budding yeast, deletion of mms221 shows a synthetic
additive interaction with cdc23 when grown at the semipermissive temperature.
As mms22 displays a synthetic interaction with mcm10,
we generated mms22D strains carrying defective DNA
polymerase alleles to address whether Mms22 also has
roles in coordinating elongation of DNA synthesis.
Pola–primase is the only enzyme capable of initiating
de novo DNA synthesis (Burgers 1998) and is required
for the initiation of DNA synthesis on the leading and
lagging strand (Waga and Stillman 1998). We generated mms22D strains carrying the pol1-1 mutation in the
catalytic subunit of Pola. pol1-1 thermosensitive mutants
are defective in DNA synthesis and arrest with a cdc
phenotype in late S-phase at the restrictive temperature
(D’Urso et al. 1995; Murakami and Okayama 1995). In
line with the interaction between mcm10 and mms22, at
25° the double mutant was viable with a slight synergistic
growth defect and sensitivity to DNA damage (Figure
8B). However, at 30°, which permits growth of the pol1-1
hypomorph, the mms22D pol1-1 mutant did not form
colonies (Figure 8B). We found the same interaction
with another thermosensitive mutant of the catalytic
subunit of Pola, swi7-H4, confirming that the additivity
is not specific to the pol1-1 allele (data not shown).
To further explore the link between mms22 and components of the replication fork, we examined genetic
interactions with the replicative polymerases d and e.
Like Pola, Pold and Pole are both multi-subunit, essential
enzymes, but the precise roles of the two polymerases in
DNA elongation are controversial (reviewed in Garg and
Burgers 2005). We utilized thermosensitive alleles of the
catalytic subunits of Pole (cdc20-M10) and Pold (cdc6-23)
to address interactions with both polymerases.
56
C. L. Dovey and P. Russell
Figure 8.—Genetic interactions between Mms22 and components of the
replication fork. Fivefold serial dilutions
of cells were exposed to the indicated
DNA-damaging agent. Plates were incubated at the indicated temperatures for
2–4 days. All DNA-damaging treatments
were conducted at 25°.
Curiously, the cdc6-23 allele of Pold improved the
growth of mms22D at 25°, which was also evident in response to DNA-damaging treatments (Figure 9A). Conversely, at the semipermissive temperature of 30°, the
mms22D cdc6-23 displayed a synergistic growth defect
compared to parental strains (Figure 9A). The same
trend was observed with cdc27-K3 (data not shown), an
allele of a Pold subunit that is required for full polymerase
processivity (Zuo et al. 2000). This opposing effect was not
observed when introducing the Pole cdc20-M10 allele into
the mms22 background. No additivity was observed at 30°
(Figure 9B) and growth was not drastically improved at
25° (Figure 9B). While we cannot rule out a requirement
for Mms22 function in processing DNA damage that arises
due to the DNA polymerase mutations, the genetic interactions suggest that the function of Mms22 in replication
restart may be mediated at the replication fork.
DISCUSSION
In this study, we present the identification of the novel
gene mms221 that is important for the recovery from
S-phase-associated DNA damage in S. pombe. A lack of
Mms22 protein results in elevated DNA damage specifically in the S- and G2-phases of the cell cycle. While we
assume that this indicates that damage occurs during
these stages, we cannot rule out the possibility of the
lesions actually arising during another phase, such as
mitosis. Moreover, loss of Mms22 function leads to elevated genomic instability as judged by increased minichromosome loss. We demonstrate through a series of
genetic analyses that Mms22 likely functions in a pathway important for the processing of abnormal structures
that arise in S-phase involving the proposed HJ resolvase
Mus81-Eme1 and that loss of Mms22 function requires
an intact HR pathway for full viability. On the basis of
genetic interactions with thermosensitive alleles of essential DNA replication factors, we propose that Mms22
carries out its function in replication restart after stalling directly at the replication fork.
The role of Mms22 following DNA damage: As an
epistatic relationship between two genes is classically
interpreted as an involvement of the gene products in a
common pathway, the synthetic interactions between
mms22 and many homologous recombination genes in
the absence of exogenous DNA damage suggests that
Mms22 is unlikely to participate in such a pathway, at
Mms22 Functions in DNA Repair
57
Figure 9.—Genetic interactions between Mms22 and DNA polymerases d and e. Fivefold serial dilutions of cells were exposed to
the indicated DNA damaging agent. Plates were incubated at the indicated temperatures for 2–4 days. All DNA-damaging treatments were conducted at 25°.
least not exclusively. In agreement with this, the DNA
damage sensitivity conferred by the mms22 deletion is
synergistic with that resulting from deletion of rhp57
(Figure 6B). By these criteria, it would appear that
mms221 is required for a pathway of spontaneous and
MMS-, UV-, and CPT-induced damage recovery that is
not exclusively mediated through Rhp51-dependent
recombination.
The elevated numbers of S- and G2-phase-associated
Rad22 foci, taken together with the requirement for HR
in the absence of Mms22 function, suggests that mms22
mutants experience an elevated occurrence of spontaneous DNA damage that needs HR for repair. Deletion
of Chk1 did not negatively impact on the growth of the
mms22 strain (Figure 3A), suggesting that the Chk1dependent cell cycle delay caused by mms22D is not due
to elevated DSBs in the mms22 background. Furthermore, mus81D cells are hypersensitive to CPT and yet
mus81D has no genetic interaction with mms22D, suggesting that loss of Mms22 function does not lead to
broken forks. Instead, the increased Rad22 foci may be
indicative of exposed ssDNA regions arising during Sphase due to uncoupling of leading- and lagging-strand
synthesis at stalled or paused replication forks (Branzei
and Foiani 2007). It will be of interest to determine the
exact structure(s) that accumulate in the absence of
Mms22, which may help to clarify the role in plays in the
prevention of genomic instability. Genetic data suggest
that mms22 and mus81 probably function together in the
repair of abnormal DNA structures. The two mutants
are epistatic during unperturbed growth and also in
terms of HU-, CPT-, and MMS-induced damage sensitivities and also share common genetic interactions with
other repair factors. Rqh1 is essential for viability in
strains that lack either Mus81-Eme1 or Slx1-Slx4 (Boddy
et al. 2001; Coulon et al. 2004) and in strains that lack the
Swi1-Swi3 FPC complex (Noguchi et al. 2003; Coulon
et al. 2004). While we did not observe a synthetic lethal
interaction between rqh1 and mms22, the double mutant
did have a severe synergistic growth defect. Also, we
have observed that deletion of Slx1 does not negatively
impact on the growth of mms22D strains or on their
survival following damage (C. L. Dovey and P. Russell,
unpublished data). The knowledge that rqh1 rhp51
double mutants are viable (Murray et al. 1997) supports
our hypothesis that Mms22 acts in a pathway involving
the structure-specific endonuclease Mus81-Eme1 (and
possibly Slx1-Slx4), which is distinct from Rqh1 and
Rhp51.
The Rhp51 mediators Swi5/Sfr1 and Rhp55/57 are
not exclusively redundant and may process DSBs differently (Akamatsu et al. 2007). Mus81-Eme1 has been
suggested to function in the Swi5-dependent pathway of
HR repair, and depending on the DNA substrate, Swi5
can generate either Mus81-dependent crossovers or
Mus81-independent noncrossovers (Hope et al. 2007).
It will interesting to determine the specific substrates that
require Mms22 for repair and how these are channeled
into alternative repair pathways in its absence. The phenotypes of mms22D are similar, but not as severe as
those of mus81D; therefore, we propose that Mus81 has
Mms22-independent functions. In line with this, while
Mus81 is required for the production of viable spores in
meiosis (Boddy et al. 2001), there is no such requirement for Mms22 (data not shown).
The function of Mms22 at the replication fork: The
Swi1-Swi3 FPC promotes the stabilization of replication
forks and on fork stalling is required for effective Cds1
activation (Noguchi et al. 2004). Fork pausing at the
MAT locus imprinting site and replication termination
at RTS1 are dependent on Swi1 and Swi3 (Dalgaard
and Klar 2000). Like the FPC, Mrc1 similarly travels
with replication forks (Katou et al. 2003; Osborn and
Elledge 2003) and mediates the activation of Cds1 on
stalling (Tanaka and Russell 2004). Mrc1 phosphorylation also contributes to the stability of stalled replication forks (Katou et al. 2003; Osborn and Elledge
2003); however, in a system for studying transient pausing
58
C. L. Dovey and P. Russell
of DNA replication forks at a protein–DNA barrier in
budding yeast, fork pausing depended on Tof1p and
Csm3p, but not on Mrc1p (Calzada et al. 2005). Similarly, fork stalling at the rDNA RFB actively requires
Tof1p, but not Mrc1p (Tourriere et al. 2005). In this
study, we observed that deletion of the Swi1-Swi3 FPC, but
not of Mrc1, partially rescued the phenotypes of a mms22
mutant. This suggests that, in the absence of Mms22,
restart of replication after fork pausing is defective, and
consequently by eliminating Swi1-Swi3-mediated pausing,
Mms22 becomes less important for viability in unperturbed conditions and following DNA damage by agents
that physically stall the progression of the replication fork.
If Mms22 functions in replication restart directly at
the replisome, one would predict mms22 to interact
genetically with other components required for the resumption of DNA synthesis. Indeed, mms22 shows synthetic additivity with cdc23 (mcm10) and pola at the
semipermissive temperature of 30°. While double mutants
of mms22 and cdc23 or pola are viable at 25°, the phenotypes
of mms22D are enhanced by the pola thermosensitive
mutations. However, we observed different genetic interactions with alleles of the replicative polymerases Pold and
Pole. At 25°, defective pold actually improved the growth
and damage tolerance of mms22 strains (Figure 9B),
whereas at 30° the double mms22 pol d grew less well than
mms22 or pol d alone. These interactions were not observed
with pole. These data may be explained if Mms22 functions
in response to DNA structures that are generated specifically by the Pold-associated replisome. In cdc6-23 or cdc27K3 strains, Pole may be able to substitute for defective Pold,
alleviating the cellular need for Mms22 to process aberrant
replication structures. However, the absence of Mms22
under conditions of dysfunctional Pold at 30° results in
the cellular burden exceeding the threshold for viability,
leading to a synthetic interaction. Interestingly, it has been
demonstrated that mutants of DNA Pola and Pold, but not
of Pole, accumulate HJs in the rDNA repeats (Zou and
Rothstein 1997), which is consistent with our proposed
role for Mms22 in the processing of aberrant DNA structures that arise at replication forks. As Mms22 foci increase
in response to damage, it is likely that Mms22 functions
directly on chromatin, most likely at broken or stalled
replication forks. It will be important to determine the
mechanisms by which this recruitment occurs and its
functional consequences.
Identification of Mms22 as a homolog of budding
yeast MMS22: We identified mms221 as a putative homolog of MMS22 on the basis of its regions of sequence
similarity to budding yeast Mms22p (Figure 1A). Similarly to HR mutants, budding yeast mms22 strains
are hypersensitive to topoisomerase II-generated DNA
damage (Baldwin et al. 2005); however, Mms22p has
been placed in a novel pathway for repair, distinct from
HR due to synergistic genetic interactions with rad51,
rad52, and rad54 (Araki et al. 2003; Baldwin et al. 2005).
We report that mms22 mutants show synthetic growth
defects with rad22, rhp51, rhp54, and also with rhp57,
suggesting that fission yeast Mms22 also probably acts
within a pathway distinctive to single-strand invasion.
However, the frequency of etoposide-induced HR in
budding yeast mms22 cells is reduced, arguing against
the two pathways being simply an alternative to each
other and instead suggesting that, although separate,
the pathways must be linked (Baldwin et al. 2005).
S. pombe Mms22 may function analogously to convert
aberrant DNA structures into a form that can be subsequently resolved by another repair pathway.
Budding yeast Mms22p interacts with the cullin
Rtt101p and also with Rtt107p (Ho et al. 2002). Rtt107p
functions in the restart of replication after damage and
has been proposed to modulate the function of proteins
that are required for DNA synthesis resumption (Chin
et al. 2006). Similarly, Rtt101p promotes fork progression through DNA lesions or naturally occurring protein–
DNA pause sites (Luke et al. 2006). On the basis of these
and other studies, the proteins have been placed along
with Mms1p, another protein required for the repair
of replication-dependent damage, in a common DNA
repair epistasis group with Mms1p functioning upstream
of Mms22p in a sequential pathway (Araki et al. 2003).
Despite this, previous genetic data suggested that
Mms1p and Mms22p might also have distinct functions.
Disruption of the HR genes RAD51 or RAD52 in a mms1
background was reported to not result in a synergistic
growth defect (Hryciw et al. 2002; Araki et al. 2003),
unlike disruption of MMS22 in a rad51 or rad52 mutant
background (Araki et al. 2003), which suggested that
Mms22p may have non-HR repair functions that are
independent of Mms1p despite its location within the
same epistasis group. However, recent large-scale studies
have uncovered synthetic interactions between HR
genes and both mms1 and mms22 (Pan et al. 2006;
Collins et al. 2007). Despite these conflicting studies,
the potential for genetic variation among individual
members of an epistasis group may explain why in S.
pombe we found that double mms22 brc1 mutants showed
a modestly enhanced growth defect compared to mms22
alone (Figure 7B).
Thus, Mms22 and Brc1 may have independent functions, or alternatively the functions of the individual
members of the S. cerevisiae MMS22 complex may not be
completely conserved in S. pombe, resulting in differing
genetic relationships. In support of this, S. cerevisiae
Rtt107p function has been shown to be partially independent of HR in response to MMS (Chin et al. 2006),
whereas fission yeast brc1 rhp51 double mutants display
sensitivity similar to the single mutants, suggesting that
Brc1 acts in the same pathway as Rhp51 for repair of
MMS-induced damage (Sheedy et al. 2005; our unpublished data). Therefore, while we have no definitive
evidence that Mms22 is the true functional homolog of
Mms22p, our initial genetic characterization does not
dispute this. Further, ongoing investigations into the
Mms22 Functions in DNA Repair
function of Mms22 should shed more light on the role
of this new protein in DNA repair and on whether a
functional Mms22p-like complex exists in fission yeast.
Taking all of our data into consideration, we propose
the following model for Mms22 function during the
coordination of leading- and lagging-strand replication
at sites of stalled replication or in lagging-strand synthesis
following restart. In response to a fork-blocking lesion
on the DNA template, the Swi1-Swi3 FPC complex mediates Pold-associated replisome stalling. Mms22, possibly in a complex with Brc1, associates with the stalled
replisome and may act either to facilitate processing by
repair factors such as Mus81-Eme1 or, alternatively, to
block the action of other repair pathways such as HR. In
the absence of Mms22, damage-induced toxic structures
are generated at the replication fork, leading to an
increased requirement for processing by HR and the
helicase Rqh1. Under these conditions, replication
restart depends on the optimal function of the replisome, as defects in cdc23 and Pol a are deleterious in the
absence of Mms22. The phenotypes of mms22 mutants
can be partially rescued by deletion of the Swi1-Swi3
FPC, which alleviates the need for Mms22-mediated
repair by destabilizing the fork and permitting direct
processing by other repair pathways such as HR.
Finally, we note that an independent study of S. pombe
Mms22 appeared while this article was in preparation
(Yokoyama et al. 2007). In agreement with the findings
reported here, Yokoyamaet al. (2007) found that Mms22,
which they termed Mus7, is involved in the repair of
replication-associated DNA damage in a pathway likely
involving Mus81. The authors found that the rate of
spontaneous Rhp51-dependent gene conversion was reduced in mus7D cells, despite the accumulation of Rad22YFP foci, suggesting that Mus7 functions downstream of
Rad22 in the Rhp51-dependent conversion-type recombination pathway. Finally, Yokoyama et al. (2007) noted
that fission yeast mus7 cells share many phenotypes with
budding yeast mms1 mutants and they therefore proposed that Mus7 and Mms1p might be functional
homologs whose sequences have diverged beyond recognition (Yokoyama et al. 2007). The phenotypes of
budding yeast Mms1p and Mms22p mutants are very
similar and are consistent with the phenotype that
arises on loss of fission yeast Mus7/Mms22. However,
our discovery of Mms22 through it sequence similarity
specifically to Mms22p suggests that it is more likely that
Mus7/Mms22 is an ortholog of Mms22p.
The authors thank Charly Chahwan for the initial identification of
S. pombe mms221 and for helpful suggestions throughout the course of this
study. We thank John Prudden and Tim Humphrey ( JP970), Victoria
Martin (VM166, VM257), and Yoshiki Yamada (YY218) for strains, as well
as Oliver Limbo for strains OL4087 and OL4088 and the pRep41-N-GFPmms221 plasmid. Members of the Russell lab and the Cell Cycle Groups at
The Scripps Research Institute are thanked, particularly Yoshiki Yamada,
Li-Lin Du, Jessica Williams, and M. Nick Boddy for helpful comments and
critical reading of the manuscript. This research was funded by National
Institutes of Health grant GM59447 awarded to P.R.
59
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Communicating editor: E. Alani