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Molecular and Cellular Biology, December 2000, p. 8758-8766, Vol. 20, No. 23
Departments of Molecular Biology and Cell
Biology, The Scripps Research Institute, La Jolla, California
920371; Department of Microbiology,
University of Washington, Seattle, Washington
981952; and Division of Biological
Sciences, Sections of Microbiology and Molecular & Cellular
Biology, University of California, Davis, Davis, California
956163
Received 30 June 2000/Returned for modification 1 September
2000/Accepted 7 September 2000
Cds1, a serine/threonine kinase, enforces the S-M checkpoint in the
fission yeast Schizosaccharomyces pombe. Cds1 is required for survival of replicational stress caused by agents that stall replication forks, but how Cds1 performs these functions is largely unknown. Here we report that the forkhead-associated-1 (FHA1) protein-docking domain of Cds1 interacts with Mus81, an evolutionarily conserved damage tolerance protein. Mus81 has an endonuclease homology
domain found in the XPF nucleotide excision repair protein. Inactivation of mus81 reveals a unique spectrum of
phenotypes. Mus81 enables survival of deoxynucleotide triphosphate
starvation, UV radiation, and DNA polymerase impairment. Mus81 is
essential in the absence of Bloom's syndrome Rqh1 helicase and is
required for productive meiosis. Genetic epistasis studies suggest that Mus81 works with recombination enzymes to properly replicate damaged DNA. Inactivation of Mus81 triggers a checkpoint-dependent delay of
mitosis. We propose that Mus81 is involved in the recruitment of Cds1
to aberrant DNA structures where Cds1 modulates the activity of damage
tolerance enzymes.
Genome integrity is vulnerable
during DNA replication. The act of replication can convert a relatively
benign single-strand DNA break to a cytotoxic double-strand break. A
cyclobutane dimer, formed by UV irradiation, is sufficient to block the
progression of DNA polymerases and to cause replication fork collapse
(17). To cope with these problems, eukaryotic organisms have
developed mechanisms for replicating DNA through and around damage in
ways that cause minimal genome instability. Notable among the proteins involved in these processes are lesion bypass polymerases that can
replicate through cyclobutane dimers (45). Recombination and double-strand break repair enzymes are also important,
participating in the direct repair of DNA structure abnormalities that
arise during DNA replication or permitting continued replication around sites of damage (17). These systems, and perhaps others that remain undiscovered, collectively form a genome defense system that
allows tolerance of damage during DNA replication.
In the fission yeast Schizosaccharomyces pombe, the
checkpoint kinase Cds1 is thought to regulate DNA damage tolerance
systems (26, 28, 31). Cds1 is the presumptive homolog of
Rad53 in the budding yeast Saccharomyces cerevisiae and Cds1
(also called Chk2) in humans (7, 9, 16, 27). One of its
functions is to delay the onset of mitosis when genome replication
encounters difficulties (8, 26, 47). Cds1 enforces this S-M
checkpoint by regulating the Cdc25 and Mik1 mitotic control proteins
(2, 8, 18, 47). In addition, Cds1 regulates the way in which DNA is replicated under conditions that cause replicational
stress. For example, Cds1 is required to slow the replication
of DNA that has been damaged by UV irradiation (26,
34). A similar function has been ascribed to Rad53 in
budding yeast (32). Slowing of DNA replication partly
involves the suppression of late-firing replication origins
(35, 37). Activation of Cds1 or Rad53 has been linked
to changes in the phosphorylation states of several different proteins
(4, 11, 20, 21, 24, 33, 36, 38, 44), some of which are
involved in DNA replication. Notably, human Cds1 has been
implicated in the phosphorylation of BRCA1, a protein involved in DNA
repair (24). BRCA1 does not exist in yeast; hence,
evolutionarily conserved targets of Cds1 remain to be discovered.
Indeed, the actual roles of Cds1 in DNA damage tolerance and of the
proteins that it interacts with remain obscure.
Rad53 contains two forkhead-associated (FHA) domains (41).
FHA1 is located near the amino terminus and is conserved in human Cds1
and fission yeast Cds1, while FHA2 is in a carboxyl-terminal extension
that is unique to Rad53. FHA2 interacts with Rad9, a protein that is
essential for the G2-M DNA damage checkpoint in budding
yeast (41). We report here the initial result of a search for proteins that interact with the FHA1 domain of fission yeast Cds1.
This search has uncovered a link between Cds1 and Mus81, a novel DNA
damage tolerance protein that is highly conserved among eukaryotes.
Yeast two-hybrid and UV assays.
The Cds1 FHA domain
(residues 1 to 155) was amplified by PCR using primers AL56
(5'-CATATGGCCATGGCCATGGAAGAACCAGAAGAAGC-3') and AL57
(5'-CGGGATCCTTAATCTCGTGAATGTTAAC-3'). The PCR product was
digested with NcoI and BamHI and ligated into
NcoI-BamHI-digested pAS2 to create pAL78.
S. cerevisiae strain HF7c was transformed with pAL78 to
generate strain AL319. The expression of the fusion protein
gal4(1-147)HA-Cds1FHA was monitored in the strain AL319 by immunoblot
analysis using antibodies against the hemagglutinin (HA) epitope. A
library (a gift from S. Elledge, Baylor College) of S. pombe
cDNAs expressed 3' of the Gal4 activation domain in pACT was
transformed into strain AL319. HIS3/3-AT selection gave more than
106 transformants that were subsequently screened for high
lacZ expression. Mus81-myc was generated as described
previously (3). Mus81-myc cells are indistinguishable from
wild-type cells, including the response to UV and hydroxyurea (HU)
treatment (our unpublished data). All UV sensitivity assays (see
Fig. 1, 3, and 5) were performed two or more times, and representative
results are shown.
Protein and immunofluorescence techniques.
For all
procedures cells were lysed in buffer A (50 mM Tris [pH 8], 150 mM
NaCl, 2 mM EDTA, 10% glycerol, 0.2% Nonidet P-40, 5 µg of
leupeptin, pepstatin, and aprotinin per ml, and 1 mM
phenylmethylsulfonyl fluoride). Protein concentrations were normalized
using optical density readings at a wavelength of 280 nm and resolved
by electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide gels
(acrylamide/bisacrylamide ratio, 200:1). Proteins were transferred to
Immobilon membranes for Western blotting. Blots were probed with
antibodies to the HA or myc epitopes following blocking of
membranes in 5% milk in Tris-buffered saline and 0.3% Tween 20. The
Cds1 kinase assay was performed as previously described (8).
Phosphatase treatments were carried out with lambda phosphatase
according to the guidelines in the New England Biolabs catalogue. The
DNA stain DAPI (4',6'-diamidino-2-phenylindole) was used to visualize
nuclei. Cells were photographed using a Nikon Eclipse E800 microscope
equipped with a Photometrics Quantix charge-coupled device camera.
Images were acquired with IPlab Spectrum software (Signal Analytics Corporation).
Coimmunoprecipitations.
Cells were lysed in buffer A, and
protein concentrations were normalized using optical density
readings at a wavelength of 280 nm. A mixture of protein
A-Sepharose and polyclonal anti-myc antiserum (Babco) was added to the
lysates, followed by incubation at 4°C for 1.5 h. Complexes were
collected by centrifugation and washed three times with lysis buffer
before they were resuspended in sodium dodecyl sulfate-polyacrylamide
gel electrophoresis loading buffer. Immunoblotting was performed as
described above.
Strains.
Strains used in this study were as follows: PR109,
wild type; NB2554, mus81::kanMx6; PS2345,
rhp51::ura4+; PS2403, uve1::LEU2
rad13::ura4+; PS2402, uve1::LEU2;
NR1587, rad13::ura4+; NB2573,
cds1::ura4+ uve1::LEU2
rad13::ura4+; NB2574, cds1-fha* (a
mutant allele; see
below):2HA6His:ura4+:leu1+
uve1::LEU2 rad13::ura4+; NB2117,
cds1::ura4+; NR1826,
rad3::ura4+; NB2575,
chk1::ura4+; BF2115,
cds1::ura4+ chk1::ura4+;
NB2566, mus81::kan chk1::ura4+; NB2555,
mus81::kan rhp51::ura4+; NB2556,
mus81::kan rad3::ura4+; NB2557,
mus81::kan rad13::ura4+; NB2558,
mus81::kan rad13::ura4+ uve1::LEU2;
NB2559, mus81::kan uve1::LEU2; AL2228,
pol1-1; AL2229, cdc6-23; NB2560,
mus81::kan pol1-1; NB2561, mus81::kan
cdc6-23; NB2562,
cds1-fha*:2HA6His:ura4+:leu1+;
NB2576, cds1:2HA6His:ura4+ mus81:13myc:kan;
NB2564, cds1-kd:2HA6His:ura4+
mus81:13myc:kan; NB2577,
cds1-fha*:2HA6His:ura4+:leu1+
mus81:13myc:kan; NB2565, cds1::ura4+
mus81:13myc:kan; NB2578, mus81:HA
nmt1-GST:cds11-190; NB2579, mus81:HA
nmt1-GST:cds1-fha*1-190; NB2580,
cds1:2HA6His:ura4+ nmt1-GST:mus81-F;
NB2581, cds1:2HA6His:ura4+ nmt1-GST:mus81-N;
NB2582, cds1:2HA6His:ura4+
nmt1-GST:mus81-C; NB2583, cds1-kd:2HA6His:ura4+
nmt1-GST:mus81-F; NB2584,
cds1-kd:2HA6His:ura4+ nmt1-GST:mus81-N;
NB2585, cds1-kd:2HA6His:ura4+ nmt1-GST:mus81-C; NB2586, nmt1-GST:mus81-F; NB2587,
nmt1-GST:mus81-N; NB2588, nmt1-GST:mus81-C;
NB2589, mus81:13myc:kan; NB2590, mus81:13myc:kan cdc25-22.
Cds1 is important for tolerance of unrepaired UV lesions.
Replication of UV-damaged DNA is facilitated by lesion bypass
polymerases and recombination enzymes. These enzymes do not excise UV
photoproducts; hence, they function in what are termed damage tolerance
mechanisms (17, 45). As defined in bacterial studies, damage
tolerance proteins enhance the survival rates of mutant strains that
cannot excise UV photoproducts. The survival of these mutants depends
on the successful replication of the UV-damaged DNA, leading to a
gradual dilution of the damage through cell division. It has been
hypothesized that Cds1 is important for damage tolerance
(31). To test this hypothesis, we determined whether Cds1
contributed to the survival of a mutant strain defective in nucleotide
excision repair (NER) and UV damage excision repair (UVER). NER and
UVER account for all detectable UV damage repair in fission yeast
(46). NER was eliminated by the inactivation of
rad13+, which encodes the homolog of
S. cerevisiae Rad2p and human XPG. UVER was
inactivated by a mutation of uve1+, which
encodes the UV damage endonuclease. The cds1 mutation substantially enhanced the UV sensitivity of rad13 uve1
cells (Fig. 1A). These data support the
proposition that Cds1 is important for UV damage tolerance. This
finding correlates with the requirement for Cds1 in establishing an
intra-S checkpoint (26, 34).
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Damage Tolerance Protein Mus81 Associates with the
FHA1 Domain of Checkpoint Kinase Cds1
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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FIG. 1.
Identification of Mus81. (A) Cds1 is important for DNA
damage tolerance. The uve1 rad13 and cds1 uve1
rad13 strains were assayed for UV survival (all results shown for
UV sensitivity assays are representative examples of two or more
experiments). (B) The cds1-fha* allele impairs DNA damage
tolerance. uve1 rad13 and cds1-fha* uve1 rad13
cells were assayed for UV survival. (C) Schematic representation of
Mus81 from S. pombe (SpMus81) and S. cerevisiae
(ScMus81) and human XPF (HuXPF) (572, 632, and 905 amino acids,
respectively). Percent identities between putative endonuclease (endo)
and helix-hairpin-helix (H) domains are shown. The nonconserved
helicase domain of XPF is shown (helic). Light shading depicts regions
sharing more than 27% identity. (D) Alignment of endonuclease domains
of Mus81 homologs and XPF family members. Sp, S. pombe; Sc,
S. cerevisiae; At, Arabidopsis thaliana; Ce,
Caenorhabditis elegans; Dm, Drosophila
melanogaster, Hu, human. Shading highlights homologous residues,
and the boxes show identities (PSI-BLAST results; alignment with
ClustalW). (E) Alignment of the helix-hairpin-helix domains of Mus81
homologs and XPF family members.
FHA1 domain is required for UV damage tolerance function of Cds1. Cds1 contains an FHA1 domain hypothesized to mediate protein-protein interactions (15, 25, 41). To explore if the FHA1 domain is required for UV damage tolerance, cds1+ was replaced with cds1-fha*, an allele encoding mutations (S79A and H82A) at two highly conserved residues in the FHA domain. The cds1-fha* mutation substantially increased the UV sensitivity of rad13 uve1 cells (Fig. 1B). These data showed that the FHA1 domain is important for DNA damage tolerance.
FHA1 domain interacts with Mus81, a conserved DNA damage survival protein. A yeast two-hybrid screen was performed with the region formed by amino acids 1 to 155 of Cds1, which contains the FHA1 domain. Three distinct but overlapping clones of a novel gene were identified. These clones failed to interact with a 1-to-155 construct that expressed mutant FHA (FHA*). Database searches revealed substantial identity to S. cerevisiae MUS81 (Fig. 1C). Budding yeast Mus81 interacts with the recombinational repair protein Rad54 in the two-hybrid system and leads to UV and methylmethanesulfonate sensitivity when inactivated (22). The yeast genes have uncharacterized sequence homologs in plants, nematodes, and fruit flies (Fig. 1D), as well as in humans (J. Vialard, personal communication).
Mus81 has two motifs found in the XPF family of NER endonucleases (1). Mutations of the human XPF gene ERRC1 cause a form of xeroderma pigmentosum (39). XPF homologs contain a highly conserved sequence, ERKX3D, which is proposed to form part of the endonuclease catalytic site (1). Mus81 contains this motif but lacks the amino-terminal helicase signature found in other members of the XPF family (Fig. 1D). Mus81 contains a predicted helix-hairpin-helix signature (amino acids 493 to 526) found in XPF and other proteins involved in DNA metabolism (Fig. 1E). A second helix-hairpin-helix signature has also been identified in the N terminus of Mus81 homologs (22). This domain is thought to allow nonspecific DNA binding via the phosphate backbone (14).Coprecipitation of Cds1 and Mus81.
Immunoprecipitation studies
established that Cds1 associates with Mus81 in vivo. Strains that
expressed epitope-tagged Mus81:myc and Cds1:HA from genomic
loci were used for these studies. Wild-type and kinase-dead
Cds1:HA coprecipitated with Mus81:myc, whereas mutant
Cds1-FHA*:HA did not (Fig. 2A).
Cds1-FHA*:HA localization, stability, abundance, and
solubility appeared to be equivalent to those of wild-type Cds1
(M. N. Boddy and P. Russell, unpublished data). Activation of
Cds1:HA by HU treatment, which stalls replication forks by blocking
deoxynucleotide triphosphate synthesis, did not significantly alter its
interaction with Mus81:myc (Fig. 2A). For these studies we estimated
that approximately 2 to 5% of the Cds1 was associated with Mus81
(M. N. Boddy and P. Russell, unpublished data). The association
between Mus81 and Cds1 was confirmed in studies that expressed
glutathione S-transferase (GST) fused to Mus81 or the Cds1
FHA domain in fission yeast. Mus81:HA coprecipitated with GST fused
to the 1-to-190 region of Cds1 (GST-FHA1-190) but not
with mutant GST-FHA*1-190 (Fig. 2B). A combination of
two-hybrid and coimmunoprecipitation studies defined 141 amino acids in
the N terminus of Mus81 (amino acids 175 to 314) capable of mediating
the interaction with the Cds1 FHA domain (M. N. Boddy and P. Russell, unpublished data).
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) served as a control. The bottom panel (total) is
an immunoblot of Cds1:HA present in samples prior to
immunoprecipitation. Note that lower-mobility forms of Mus81:myc were
detected only in wild-type cells. (B) The 1-to-190 region of the Cds1
wild type (WT) or the FHA mutant (FHA*) was expressed from the nmt1
promoter as a GST fusion protein (GST:Cds1190) in a
Mus81:HA strain. GST:Cds1190 proteins were purified and
detected with amido black or immunoblotted with antibodies to HA.
Mus81:HA coprecipitated with the wild-type but not with the mutant
FHA domain. (C) Mus81 is a phosphoprotein. A Mus81:13myc strain was
treated with HU (+) or not treated. (
phosphatase (+) or not treated,
(Cds1 regulates Mus81 phosphorylation in vivo. Immunoblot analysis detected Mus81:myc as a closely migrating doublet (Fig. 2A). HU treatment caused Mus81:myc to migrate as a lower-mobility species, indicative of phosphorylation, as confirmed by phosphatase treatment (Fig. 2C). HU-induced phosphorylation of Mus81 was abolished in cds1 deletion, cds1-fha*, and cds1-kd (kinase-dead) strains (Fig. 2A). The lower-mobility form of Mus81:myc observed in asynchronous cells was also absent in the cds1 mutants. This species appears to be a phosphorylated form of Mus81 that is partially resistant to dephosphorylation by lambda phosphatase. A change in Mus81 mobility was not apparent for UV-irradiated cells (M. N. Boddy and P. Russell, unpublished data), but this result might be explained by the low activation rate of Cds1 in response to UV radiation compared to that in response to HU treatment (26).
Mus81 is important for DNA damage tolerance.
A
mus81 deletion strain was viable but sensitive to UV
irradiation (Fig. 3A). There are
conflicting reports on the UV sensitivity of cds1 cells
(26, 28). As previously observed (28), we have
found that Cds1 mutant cells are not significantly sensitive to UV
irradiation (M. N. Boddy and P. Russell, unpublished data). However, it is evident that Cds1 inactivation greatly enhances UV
sensitivity in a chk1 mutant background (26)
(Fig. 3A). Chk1 is essential for the G2-M DNA damage
checkpoint (43); thus, the chk1 mutation
highlights the importance of the DNA damage tolerance response during S
phase (26). The mus81 and cds1
mutations reproducibly enhanced UV sensitivity in a chk1
background (Fig. 3A). Inactivation of mus81+ did
not increase UV sensitivity in cds1 chk1 cells (Fig. 3A). The lack of synergy between mus81 and cds1
mutations indicated that Mus81 and Cds1 function in a related pathway.
The enhanced UV sensitivity of cds1 chk1 cells relative to
mus81 chk1 cells indicated that Mus81 is not the sole target
of Cds1. The mus81 rad13 and mus81 uve1 mutants
were more UV sensitive than any single mutant (Fig. 3B and C).
Furthermore, the mus81 mutation enhanced the UV sensitivity
of rad13 uve1 cells (Fig. 3D). These data implicated Mus81 in DNA damage tolerance. The mus81 mutation did not
enhance the UV sensitivity in a cds1 rad13 uve1
background (Fig. 3E). As we observed in a chk1
background, the lack of synergy between mus81 and
cds1 mutations in a rad13 uve1 background
supported the conclusion that Cds1 and Mus81 function in a similar
pathway. The Mus81 tolerance function appears to be checkpoint
dependent, because mus81 did not exacerbate the UV
sensitivity of a checkpoint-defective rad3 strain (Fig. 3F).
Rad3 is required for Cds1 activity (8, 26).
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or 
, but not 
,
exhibited strong genetic interactions with mus81 (Fig. 4C). A similar genetic interaction was reported for cds1 and
DNA polymerase mutations (6). No interaction was
observed between mus81 and cdc10-129 or
cdc25-22, mutations that arrest cell cycle progression in
G1 or G2, respectively.
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Mus81 is essential for viability in the absence of Rqh1. The RecQ family of DNA helicases includes Bloom's syndrome protein in humans and Rqh1 in fission yeast. These proteins are important for maintaining genome integrity. Fission yeast rqh1 mutants have replication abnormalities that cause elevated recombination and sensitivity to HU (31, 40). It has been proposed that Rqh1 is also important for coping with stalled or collapsed replication forks (10). We found that rqh1 mus81 spores germinated but were inviable. These findings support the notion that Mus81 is important for correcting abnormal DNA structures that arise during replication. It is interesting that rqh1 rhp51 double mutants are viable (31). These results distinguish the functions of Mus81 and Rhp51 and suggest that the proteins may function both in partially dependent pathways, as in the case of UV damage tolerance, and independently, as indicated by their genetic interactions with rqh1 mutations.
Role of Mus81 in mitotic and meiotic divisions. The mus81 deletion strain contained moderately elongated cells (Fig. 5C). This phenotype was reminiscent of rhp51, rhp54, and rhp55 recombination mutant cells that appear to trigger checkpoint arrest in the absence of extrinsic DNA-damaging agents (23, 29, 30). A mus81 rad3 culture had few elongated cells but frequent "cut" cells, in which DNA was unequally segregated to daughter cells, a phenotype that signals checkpoint failure (Fig. 5C). Hence, the mus81 mutation triggers a Rad3-dependent checkpoint delay of mitosis. It appears that Mus81, together with other recombinational repair enzymes, is important for the timely completion of DNA replication. Recombinational repair of collapsed replication forks that occur in the absence of DNA-damaging agents might explain why mus81 mutations trigger a Rad3-dependent checkpoint delay.
The role of Mus81 in meiosis was also investigated. In a mating of wild-type cells, approximately 80% of the resultant spores were viable (Fig. 5D). In contrast, approximately 0.1% of the spores from a mus81 × mus81 conjugation were viable (Fig. 5D). The effect of mus81 mutations on spore viability was even more severe than that observed in an rhp51 × rhp51 mating, in which approximately 2.5% of the spores germinated to produce colonies. The reason for the mus81 spore inviability has not been investigated, but it might arise from difficulties in either meiotic DNA replication or recombination.| |
DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Cds1 is best known and understood as a checkpoint kinase that delays mitosis when DNA synthesis is inhibited by HU (8, 26, 28). However, the S-M checkpoint activity of Cds1 is arguably not its most important function. This conclusion is based on the fact that cds1 cells exhibit very low viability when incubated in HU, even though mitosis is restrained by Chk1 (8, 26, 28). Thus, the recovery activity of Cds1 is centrally important for survival under replicational stress, but it is poorly understood. The function of Cds1 as a damage tolerance enzyme is even more mysterious. We undertook a screen to identify novel protein interactions involving Cds1, with the hope of ascertaining how Cds1 promotes damage tolerance and recovery from replicational stress. The central finding of this report is that Cds1 interacts physically with Mus81, a novel damage tolerance protein.
Cds1 is important for DNA damage tolerance. DNA damage tolerance refers to mechanisms that facilitate successful replication of damaged DNA (17). If inactivation of a gene diminishes the UV survival of cells that are unable to repair UV lesions, then this gene can be considered to have importance in DNA damage tolerance. Cds1 was hypothesized to be involved in UV-induced DNA damage tolerance, but formal proof of this point, as defined above, was lacking (31). Thus, at the outset of these studies, it was essential to test whether cds1 mutations impair the UV survival of cells that cannot repair UV lesions. An affirmative answer was obtained in epistasis studies carried out with mutants defective for NER and UVER. Hence, we have provided formal evidence that Cds1 is important for DNA damage tolerance.
Using the same approach, we established that a functional FHA1 domain is required for the damage tolerance function of Cds1. This result provided the rationale for a yeast two-hybrid screen carried out with FHA1, which led to the identification of a protein that is highly related to budding yeast Mus81. The striking similarity to budding yeast Mus81 induced us to give the same name to the fission yeast protein. We have no convincing evidence that the two proteins are functionally analogous, but the possibility seems quite likely. Mus81 mutation in both yeasts results in sensitivity to UV but not ionizing radiation (this study and reference 22). Mus81 shares homology with the XPF family of endonucleases, suggesting a possible enzymatic activity for Mus81. Such an activity is speculative and remains to be established with biochemical assays. Mus81 is phosphorylated in a Rad3- and Cds1-dependent manner following exposure to HU. However, UV treatment does not result in a visible mobility change in Mus81 (Fig. 2C and data not shown). This may be due to the weaker activation of Cds1 by UV radiation than by HU treatment (26). Although the phosphorylation state of Mus81 is dependent on Cds1, we have been unable to phosphorylate Mus81 with purified Cds1 in vitro (M. N. Boddy and P. Russell, unpublished data). This may be due to the absence of a cofactor or to the possibility that another Cds1-dependent kinase is responsible for the phosphorylation. At present, this precludes the mapping of phosphorylation sites on Mus81 and establishing a functional effect of such phosphorylation. It is interesting that the modification of Mus81 observed during an unperturbed cell cycle is S-phase specific and Cds1-Rad3 dependent (Fig. 2A and C) (M. N. Boddy and P. Russell, unpublished data). This strongly suggests that the replication checkpoint is activated by normal replication.Mutant mus81 cells display phenotypes similar to and distinct from those of recombination repair mutants. The role of recombination repair machinery in normal replication is becoming more apparent (19). In fission yeast, rhp54 (Rad54) cells exhibit a checkpoint-dependent delay in the cell cycle (30). This is true of other members of the Rad52 epistasis group of recombination repair proteins (42; M. N. Boddy and P. Russell, unpublished data). The defect in these cells appears to be manifested during replication. Mus81-defective cells, like recombination repair-defective mutants, show a checkpoint-dependent delay in the cell cycle. It is important to note that cds1 and rad3 cells, although slightly less viable than wild-type cells, show no profound cell cycle defect. This demonstrates that Mus81 has basal functions that are not dependent on the checkpoint proteins, including Cds1. Indeed, as also observed for recombination repair proteins, combining rad3 and mus81 mutations results in the accumulation of cut cells. This suggests that mus81 cells are defective in an aspect of DNA metabolism that, based on current data, is required for normal replication. Consistent with the similar phenotypes of Mus81 and recombination repair mutants, Mus81 appears to function in an Rhp51-dependent pathway for the tolerance of UV damage. The tolerance function may represent in part an augmentation of a mechanism that is required to repair stalled forks during normal replication. It is tempting to speculate that the Cds1-dependent phosphorylation of Mus81 stimulates or modulates this basal function.
A profound meiotic defect was also observed in both mus81 and rhp51 cells. Double-strand breaks are generated during meiosis, and recombination repair machinery is used to heal the breaks. This fact alone can explain the defect of rhp51 mutants in meiosis; however, a defect in meiotic replication cannot be excluded as a contributory factor. Interestingly, unlike recombination repair-defective rhp51 cells, mus81 cells are not defective in the repair of double-strand breaks induced by gamma irradiation. Therefore, Rhp51 and Mus81 appear to have both overlapping and distinct functions. These facts suggest that the meiotic defect of mus81 cells is in some way different from that of rhp51 cells. In fact, mus81 mutants show poorer spore viability than rhp51 mutants. Mus81 appears to be important for the mitotic S phase; therefore, the mus81 meiotic defect may be due to a problem in premeiotic replication. A role for Mus81 in resolving aberrant meiotic recombination structures cannot be excluded.Genetic interactions with Rqh1 and DNA polymerase mutants. The bacterial DNA helicase RecQ shares homology with a number of important helicases found in eukaryotes (40). This family includes the Bloom syndrome (BLM) and Werner syndrome (WRN) helicases that are mutated in human diseases that predispose patients to cancer. Fission yeast Rqh1 is closely related to BLM and shows similarly elevated levels of mitotic recombination (40). More recently, Rqh1 mutant cells have been suggested to accumulate the recombination intermediate termed X-DNA (Holliday junctions) (13). Rqh1 is proposed to catalyze reverse branch migration to prevent X-DNA accumulation in a nonrecombinogenic manner. In the absence of Rqh1, cells may resolve X-DNA structures by cleavage, resulting in recombinants. Interestingly, we observed that mus81 rqh1 double mutants are not viable. It is therefore possible that Mus81 is required for a step in the resolution of Holliday junctions (or other abnormal DNA structures) that accumulate in rqh1 cells. That Mus81 exhibits homology to a family of endonucleases may be relevant in this context. Further, we found that the deletion of Mus81 in cells containing thermosensitive alleles of DNA polymerases alpha and delta significantly reduced their restrictive temperatures (Fig. 4C). We found no such genetic interaction with a thermosensitive allele of DNA polymerase epsilon (Fig. 4C). Interestingly, X-DNA accumulates in S. cerevisiae mutants of DNA polymerases alpha and delta but not epsilon (48). Extrapolation of these results to fission yeast would provide support for the role of Mus81 in the resolution of X-DNA structures. Studies are under way to address these possibilities.
Conclusions. We have uncovered a physical interaction between the evolutionarily conserved checkpoint kinase Cds1 and a novel damage tolerance protein, Mus81. Mus81 appears to function in a checkpoint- and recombination repair-dependent pathway for the tolerance of UV lesions. Mus81 is also required for normal cell cycle progression, potentially functioning during S phase to mitigate the recombinogenic properties of stalled replication forks. This basal role may be modified by interaction with Cds1 to promote the survival of lesions or conditions that impede the normal progression of replication forks, such as UV damage. It is noteworthy that human Cds1 is mutated in a subset of families that exhibit genetic inheritance of Li-Fraumeni cancer-prone syndrome (5). It will be important to determine if Cds1-defective cells derived from these patients exhibit the array of defects associated with inactivation of Cds1 in fission yeast and to consider the possibility that these defects might involve the human homolog of Mus81.
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ACKNOWLEDGMENTS |
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We thank members of the Russell laboratory and the Scripps Cell Cycle Groups for their help and support. Strains were kindly provided by Antony Carr and A. Yasui, and the yeast two-hybrid library was kindly provided by S. Elledge.
M.N.B. was supported by a Special Fellowship from the Leukemia and Lymphoma Society. Work in W.-D. Heyer's laboratory was supported by the Swiss National Science Foundation. This work was funded by a National Institutes of Health grant awarded to P.R.
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FOOTNOTES |
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* Corresponding author. Mailing address: Departments of Molecular Biology and Cell Biology, 10550 North Torrey Pines Rd., MB-3, The Scripps Research Institute, La Jolla, CA 92037. Phone: (858) 784-8273. Fax: (858) 784-2265. E-mail: prussell{at}scripps.edu.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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