From St. Vincent's Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065, Australia
Received for publication, October 19, 2000, and in revised form, January 17, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Forkhead-associated (FHA) domains are
multifunctional phosphopeptide-binding modules and are the hallmark of
the conserved family of Rad53-like checkpoint protein kinases.
Rad53-like kinases, including the human tumor suppressor protein Chk2,
play crucial roles in cell cycle arrest and activation of repair
processes following DNA damage and replication blocks. Here we show
that ectopic expression of the N-terminal FHA domain (FHA1) of the yeast Rad53 kinase causes a growth defect by arresting the cell cycle
in G1. This phenotype was highly specific for the
Rad53-FHA1 domain and not observed with the similar Rad53-FHA2,
Dun1-FHA, and Chk2-FHA domains, and it was abrogated by mutations that
abolished binding to a phosphothreonine-containing peptide in
vitro. Furthermore, replacement of the RAD53 gene
with alleles containing amino acid substitutions in the FHA1 domain
resulted in an increased DNA damage sensitivity in vivo.
Taken together, these data demonstrate that the FHA1 domain contributes
to the checkpoint function of Rad53, possibly by associating with a
phosphorylated target protein in response to DNA damage in
G1.
Checkpoint signaling pathways are crucial for maintaining genome
integrity by delaying the cell cycle in response to DNA damage, replicational stress or mitotic spindle assembly defects, and are
highly conserved throughout evolution (1, 2). Chk2-like cell cycle
checkpoint protein kinases are characterized by the presence of
noncatalytic FHA1 domains and
play crucial roles in the cellular response to DNA damage and
replication blocks (3, 4). For example, the human tumor suppressor
kinase Chk2 phosphorylates p53 (5-7), Cdc25C (8), and BRCA1 (9) in
response to DNA double strand breaks, leading to cell cycle arrest in
G1, G2/M, and S phase, respectively. Mutations
within the Chk2-FHA domain or truncations that disrupt the kinase
domain but leave the FHA domain intact are the cause of a subset of
cases of the Li-Fraumeni syndrome (usually caused by loss of p53) where
patients suffer from multiple independent primary tumors, typically at
a young age (10).
Budding yeast, Saccharomyces cerevisiae, contains three FHA
domain-containing protein kinases. Rad53 (11, 12) and Dun1 (13)
function in the mitotic checkpoint response to DNA damage and
replication blocks, and Mek1 plays an important role in meiosis (14).
In contrast to other family members that contain a single FHA domain in
the N terminus, Rad53 is the only checkpoint kinase with an additional
FHA domain in the C terminus. RAD53 arrests the cell cycle
in G1 and G2/M in response to DNA damage and in S phase in response to replication blocks (12, 15). In addition, it
regulates the transcriptional induction as well as the subcellular localization and activity of proteins involved in DNA damage repair (12, 16, 17). RAD53 also has an essential, DNA
damage-independent function by regulating the levels and activity of
ribonucleotide reductase, and thereby the availability of dNTPs, during
S phase (18, 19). Several RAD53-dependent
checkpoint pathways, but few definitive kinase substrates, have been
identified. Following DNA damage in G1, Rad53
phosphorylates the Swi6 transcription factor, thereby contributing to
the repression of the CLN1 and CLN2 cyclin genes
and delaying transition into S phase (20). In response to replication
blocks and DNA damage in S phase, Rad53 inhibits the "firing" of
late replication origins (21) by negatively regulating the Dbf4/Cdc7
kinase complex (22-24). As part of the G2/M DNA damage
checkpoint, Rad53 prevents anaphase entry and mitotic exit by
inhibition of the polo-like kinase Cdc5 (25).
FHA domains are highly diverse protein-protein interaction modules
characterized by a 55-75-residue motif with only 7 residues that are
>65% conserved among 120 family members (26). However, intact FHA
domains are much larger than the core motif detected in computer
comparisons and contain up to 180 amino acid residues (27, 28). FHA
domains form an 11-stranded We have recently determined the Rad53 and Dun1 FHA domain boundaries
and demonstrated that overexpression of the Dun1-FHA domain in yeast
can compete the Dun1-dependent transcriptional induction of
ribonucleotide reductase 2 (RNR2) following replication blocks (28). To gain insight into the function of the Rad53-FHA1 domain, we have used a similar approach in the present study. We show
that overexpression of the FHA1 domain can mimic the DNA damage-dependent G1 arrest function of the
intact Rad53 kinase, and that this phenotype is abrogated by mutation
of residues that are involved in phosphopeptide binding. Moreover, we
show that introduction of amino acid changes, that disrupt this
overexpression phenotype, into the RAD53 gene results in an
increased sensitivity of yeast to DNA damage. These data indicate that
FHA1 domain association with a threonine-phosphorylated target protein
may be involved in the G1 checkpoint function of Rad53.
Generation of FHA Domain Constructs--
FHA domain constructs
were generated by polymerase chain reaction (PCR) using
synthetic oligonucleotides (all oligonucleotide sequences are available
as on-line supplemental materials) as described (28). For bacterial
expression, cDNAs were cloned into the NcoI and
BglII sites of pQE60 (Qiagen). For yeast expression, constructs were ligated to the Gal4 DNA binding domain for nuclear targeting by cloning into the constitutive expression vector pAS2 (CLONTECH), followed by ligation of the resulting
HindIII-SalI restriction fragments into the
inducible vector p416GAL1 (32). Sequences of all PCR
generated fragments were confirmed by automated DNA sequence analysis (ABI).
Site-directed and Random Mutagenesis--
Site-directed FHA1
mutants were generated by PCR using synthetic oligonucleotides (see
supplemental materials available in on-line version of this article)
and mutations were confirmed by automated DNA sequence analysis. Random
mutagenesis of the Rad53-FHA1 was performed by low fidelity PCR
essentially as described (33), except that 20 µM
manganese chloride was found to give the highest yield of viable yeast
colonies with only single amino acid substitutions. The resulting
library in pAS2 consisted of ~1950 independent clones.
Yeast Strains and Cultures--
Unless otherwise indicated,
experiments were performed in the K699 genetic background
(MATa, ade2-1, trp1-1,
leu2-3,112, his3-11,15, ura3,
can1-100). rad9
In synchronization experiments, cells were diluted to 0.2 optical
density units and treated twice for 2 h with 20 µg/ml
Flow Cytometry--
For cell cycle analyses, 1-ml aliquots were
removed from yeast cultures and fixed in 70% ethanol. 200 µl of
cells were washed with 50 mM sodium citrate, pH 7, and
incubated overnight with 0.1 mg/ml RNase A in 50 mM sodium
citrate at 37 °C. Cells were sonicated, treated with 4 µg/ml
propidium iodide, and analyzed using a Becton Dickinson FACScan and
CellQuest software.
RAD53 Gene Disruptions and Allele
Replacements--
RAD53 gene disruptions and allele
replacements were generated in the sml1 Bacterial Expression, Protein Purification, and in Vitro Binding
Assays--
Recombinant proteins were expressed and purified as
described (28), dialyzed against 20 mM Hepes, pH 7, 1 mM dithiothreitol, concentrated to 5 mg/ml using Centricon
membranes (Amicon), and stored at 4 °C until use. For enzyme-linked
immunosorbent assays (ELISAs), microtiter plates were coated overnight
with 20 µg/ml synthetic p53-derived phosphothreonine-containing
peptide (p53(pT18): APPLSQEpTFSDLWKL) diluted in 0.2 M
sodium carbonate buffer, pH 9.6. Plates were blocked in 5% BSA in PBS,
0.1% Tween 20, incubated with purified FHA domains in 1% BSA in PBS,
probed with 0.2 µg/ml anti-His4 antibody (Qiagen),
followed by secondary antibody conjugated with horseradish peroxidase.
200 µl of 10 mM
2,2'-azino-bis[3-ethylbenzthiazoline-6-sulfonic acid], 0.3%
H2O2 in 5.35 mM trisodium citrate,
3.8 mM citric acid, pH 4.5, was used for color development,
measured at 405 nm. Data from four experiments using three different
protein preparations were normalized to the fitted maximum of binding
by the wild-type FHA1 domain.
Protein Extracts and Immunoblot Analysis--
Protein extracts
were prepared by resuspending yeast pellets from 10 ml cultures in 200 µl of buffer (8 M urea, 5% SDS, 40 mM
Tris-HCl, pH 6.8, 0.1 mM EDTA, 0.4 mg/ml bromphenol blue,
10 µg/ml aprotinin, 5 µg/ml leupeptin, 10 µg/ml soybean trypsin
inhibitor, 2 mM PMSF, 10 mM benzamidine, 1 mM sodium vanadate, 50 mM sodium fluoride, 1%
Overexpression of the Rad53-FHA1 Domain Causes a Growth
Defect in Yeast--
We used a galactose-inducible overexpression
system to identify phenotypes that might provide clues to the function
of the N-terminal FHA domain (FHA1) of the yeast Rad53 kinase.
Transformed yeast strains had normal growth properties when the
expression of these constructs (fused to the Gal4-DNA binding domain
for nuclear targeting) was repressed in media lacking galactose (Fig. 1A). However, strains
overexpressing the entire Rad53 N terminus (residues 1-199 including
the FHA1 domain) or expressing a construct containing only the
biochemically defined FHA1 domain (residues 20-164) had a dramatic
growth defect and failed to form visible colonies on solid media
containing galactose (Fig. 1A). This growth defect was
highly specific for the Rad53-FHA1 domain as it was not observed with
the related Rad53-FHA2, Dun1-FHA, and Chk2-FHA domains (Fig.
1A) that were expressed at similar protein levels (Fig.
1B). In addition, this phenotype was dependent on the
integrity of the FHA1 domain as it was abrogated by double-alanine
substitution of the highly conserved residues Ser-85 and His-88, as
well as truncation of just 15 residues at either end of the
proteolytically defined domain boundaries (Fig. 1A).
Although overexpression of the C-terminal Rad53-FHA2 domain did
not affect normal cell growth, it caused a considerably increased DNA
damage sensitivity and decreased viability on plates containing galactose and the radiomimetic compound MMS (Fig. 1A,
right panel). This phenotype is consistent with
genetic evidence that the Rad53-FHA2 domain is involved in the DNA
damage-dependent activation of Rad53 (30). Likewise, the
Dun1-FHA domain construct used here can suppress the
Dun1-dependent transcriptional induction of RNR2 following replication blocks (28) and can also compete the activation of Dun1 kinase by replication blocks or DNA damage.2
Therefore, the Rad53-FHA2 and Dun1-FHA domains are fully functional in
this system, and the fact that only the FHA1 domain causes a growth
defect underscores the specificity of this phenotype.
The FHA1 Growth Defect Is Unrelated to the Essential Function of
RAD53--
RAD53has an essential DNA damage-independent function by
regulating the availability of dNTPs during S phase, that can be suppressed by deletion of the RNR inhibitor SML1
(18). To test if the FHA1 domain causes the growth defect by competing
with the RAD53 essential function, we analyzed its effect on
the growth of a sml1
Another possible reason for the growth defect could be that FHA1
overexpression generates a checkpoint signal that results in cell cycle
arrest. The FHA1 domain can bind to phosphorylated Rad9 in
vitro (29). To test if the growth defect is the result of an
interaction of the FHA1 domain with Rad9, overexpression experiments
were repeated in a rad9 deletion strain. Fig. 2A
shows that overexpression of the Rad53-FHA1 domain, but not the vector and double-mutant controls, again caused a dramatic growth defect in
the rad9 FHA1 Overexpression Arrests the Cell Cycle in
G1--
To assess the FHA1 phenotype in more detail,
overexpression effects were examined in liquid cultures. Fig.
3A shows that
FHA1-overexpressing cultures had considerably slower growth kinetics
than the vector control, which became apparent after ~8 h in
galactose-containing media. To determine whether overexpression of the
FHA1 domain affects cell viability, aliquots of these cultures were
plated on dextrose-containing plates and tested for their ability to form colonies. Colony formation by the FHA1-overexpressing strain was
directly proportional to its optical density throughout the 24-h
experiment, and colony numbers per optical density unit were very
similar for the FHA1 and control cultures (Fig. 3A,
inset). This demonstrates that the
FHA1-dependent growth defect is reversible and not the
result of increased lethality.
To examine if the FHA1 domain reduces cell growth in a specific phase
of the cell cycle, aliquots of liquid cultures were analyzed by flow
cytometry. Starting cultures were adjusted to give equal cell densities
(~0.5 optical density units) at the end of this experiment to
minimize nonspecific effects on the cell cycle profile. In this
experiment, the majority of vector control cells had a 2n DNA content
(corresponding to G2/M) before, and 6 and 12 h after
addition of galactose (Fig. 3B). The cell cycle profile of
the FHA1 strain without, and 6 h after addition of galactose
(i.e. at time points when the growth defect was not apparent) was essentially identical to the vector control. However, induction of FHA1 expression for 12 h resulted in a marked
increase in cells with a 1n DNA content (Fig. 3B). To
determine whether this accumulation of cells in G1 was the
result of a cell cycle arrest or just a slower G1-S
transition, FHA1-overexpressing and vector control cultures were
synchronized in G1 using Dependence of the FHA1 Phenotype on Its Phosphopeptide-binding
Ability--
Only seven residues are highly conserved among FHA
domains (26). To find out if these highly conserved residues are
important for FHA1 domain function, we performed an alanine-screening
mutational analysis in a constitutive expression system. The
top panel in Fig.
4A shows that the FHA1
phenotype could be reproduced in this system; expression of the
wild-type FHA1 domain again prevented colony formation, whereas equal
amounts of yeast transformed with equal amounts of vector or
double-mutant (S85A/H88A) control DNAs gave rise to numerous colonies.
Fig. 4A shows that single alanine substitutions of each of
the >80% consensus residues (Gly-69, Arg-70, Ser-85, His-88) as well
as Asn-107 and Gly-108, that are conserved in >65% of all FHA
domains, abrogated the overexpression phenotype and resulted in
numerous viable colonies. In contrast, alanine substitution of the
>65% conserved residue Asn-112 did not affect the phenotype,
indicating that this residue is functionally neutral in the context of
the FHA1 domain. Immunoblot analyses of viable colonies demonstrated
that three of the alanine substitutions (G69A, H88A, G108A) resulted in
considerably reduced FHA1 protein levels, indicating that these
residues may be important for proper domain folding (Fig.
4B). In contrast, FHA1 domains with substitutions of Arg-70,
Ser-85, or Asn-107 were still expressed at high levels (Fig.
4B) that were similar to the galactose-inducible wild-type FHA1 preventing cell growth (Fig. 4C). The abrogation of the
growth defect therefore indicates that these residues may be directly involved in the binding to a target protein that is crucial for the
G1 arrest phenotype.
To test these hypotheses, His6-tagged recombinant FHA1
domains were expressed in E. coli. Immunoblots of
fractionated bacterial lysates (Fig.
5A) revealed that the
solubility of these recombinant proteins mimicked their expression
levels in vivo. Mutants that were expressed at low levels in
yeast (G69A, H88A, G108A) were almost exclusively insoluble in
vitro, supporting the notion that these domains are misfolded. In
contrast, the wild-type FHA1 domain and the mutants with high
expression levels in vivo (R70A, S85A, N107A) were
predominantly soluble in vitro. Although the N112A mutant
had an unperturbed phenotype in vivo, indicating that it is
properly folded in yeast, a large fraction was insoluble when expressed
in bacteria. However, the relative solubility of this domain was much
greater than that of the G69A, H88A, and G108A mutants, and once
purified its solubility did not differ from the recombinant wild-type
FHA1 domain (data not shown).
Soluble FHA1 domains, as well as the Dun1-FHA domain, were purified
(Fig. 5B) and tested in an ELISA for their ability to bind
to a phosphothreonine-containing peptide, that has previously been
shown to interact with an extended Rad53-FHA1 domain-containing GST
fusion protein (residues 2-279) (29). In this assay (Fig. 5C), the wild-type FHA1 domain bound to the phosphopeptide
with high affinity (half-maximal binding at 6 µg/ml, ~350
nM). Although the dose-response curve of the N112A mutant,
that had an unperturbed phenotype in vivo, was shifted to
about 1 order of magnitude higher concentrations (EC50 ~ 66 µg/ml) it still bound reasonably well with a profile similar to
the Dun1-FHA domain (EC50 ~ 33 µg/ml). In contrast, the
mutants with an abrogated in vivo phenotype bound either
very poorly (S85A, 35% maximal binding at 1 mg/ml) or not at all
(R70A, N107A).
Taken together, these data indicate that the ability to bind
phosphopeptide target sequences is a critical determinant of the growth
defect caused by the FHA1 domain. However, there are clearly additional
specificity requirements for the FHA1-induced G1 arrest, as
the Dun1-FHA domain had an in vitro phosphopeptide-binding profile similar to the N112A mutant without causing the same phenotype.
Identification of Crucial Nonconserved Residues in an in Vivo
Random Mutagenesis Screen--
The strong growth-defect phenotype of
the overexpressed FHA1 domain provided us with an opportunity to
identify additional functionally important residues in a random
mutagenesis screen. For this purpose, yeast were transformed with a
library of randomly mutagenized FHA1 domains. Relative to the plating
efficiency of the double-mutant (S85A/H88A) control, ~8% of the FHA1
sequences in the library contained mutations that abrogated the
phenotype and gave rise to viable colonies (data not shown). 264 viable clones were counterscreened by immunoblotting to select only
full-length FHA1 domains for sequence analysis. A representative blot
is shown in Fig. 6A. Only 47 of the clones screened expressed the full-length FHA1 domain, and 45 of
these contained single point mutations, while the majority contained
truncated domains, presumably due to the introduction of stop-codons or
frameshifts during the mutagenesis step. Altogether, 13 different
mutations with amino acid substitutions in four conserved (G69S, R70K,
N107I, N107K, N107S, G108A) and six nonconserved residues (C34R, V36D,
L78S, S105C, S105T, L124P, L143P) were identified (Fig. 6B).
These mutants differed somewhat in their expression levels, but with
the exception of L124P all were expressed at considerably higher levels
than the random G108A mutant (Fig. 6B) that was expressed at
low levels similar to its counterpart generated by site-directed
mutagenesis (Fig. 4B).
Increased DNA Damage Sensitivity of rad53 Alleles with Amino Acid
Substitutions in the FHA1 Domain--
To test if mutations that
abrogate the FHA1 overexpression phenotype are relevant for Rad53
function at physiological protein levels, one of the site-directed and
one of the random mutations were each introduced into the
RAD53 gene using a modified allele-replacement procedure. To
avoid secondary effects, the R70A and N107K mutations were selected for
this purpose because their high overexpression protein levels (Figs. 4
and 6) indicated that they would not compromise Rad53 folding and
stability. For comparison, we also generated a kinase-defective
rad53-K227A allele (40) and a rad53
All rad53 strains constructed were viable and, except for
the deletion strain, had Rad53 protein levels similar to wild-type RAD53 (Fig. 7). To test if the
FHA1 domain contributes to the DNA damage response function of Rad53,
logarithmically growing cultures were treated for 3 h with MMS and
then plated on YPD medium to determine survival rates. In these
experiments, both FHA1 mutations had almost identical effects on cell
viability, and relative to the RAD53 allele led to a
2.5-4-fold increased lethality after DNA damage (Fig. 7B).
This confirms that these FHA1 residues are important for the function
of Rad53.
Our results show that overexpression of the Rad53-FHA1 domain
causes a dramatic growth defect (Fig. 1) by arresting yeast in the
G1 phase of the cell cycle (Fig. 3). This phenotype is specific for the FHA1 and not observed with three similar domains (Rad53-FHA2, Dun1-FHA, Chk2-FHA; Fig. 1) and mutational analyses indicate that it involves binding to a phosphorylated protein (Figs. 4
and 5). The FHA1-induced growth defect was also apparent in strains
containing the sml1 deletion as a suppressor of the essential function of RAD53, as well as in a
sml1-1 strain lacking RAD53 altogether,
demonstrating that the overexpression phenotype is not
dominant-negative or competitive with Rad53 (Fig. 2). One of the
numerous checkpoint functions of the Rad53 kinase is to arrest the cell
cycle prior to START in response to DNA damage during G1
(20). Therefore, FHA1 domain overexpression seems to mimic this
established checkpoint function of intact Rad53 even in the absence of
DNA damage. Interestingly, the same FHA1 point mutations that abolish
the overexpression phenotype (Figs. 4 and 6) also lead to an increased
DNA damage sensitivity in mutant rad53 alleles (Fig. 7). The
simplest explanation to combine our observations is that the FHA1
domain is involved in the G1 checkpoint function of Rad53,
and that the overexpression phenotype is caused by the binding of the
FHA1 to a protein it usually only interacts with in response to DNA
damage. In this model, the FHA1 domain normally interacts with other
parts of the intact kinase but upon Rad53 activation in response to DNA
damage becomes accessible and tethers a protein that is required for
transition into S phase. This interaction is terminated when intact
Rad53 phosphorylates this target, which maintains the G1
arrest by a different mechanism and at the same time lowers its
affinity for FHA1, and as a result the transient FHA1/target complex
dissociates. In the ectopic overexpression scenario, the target remains
trapped as it can not be phosphorylated by Rad53, and the continued
sequestration of this protein causes an extended G1 arrest.
The increased DNA damage sensitivity of the rad53 alleles
with point mutations in the FHA1 domain (Fig. 7) clearly demonstrates that this domain contributes to the DNA damage response function of
Rad53. However, whereas the rad53-R70A and
rad53-N107K mutations reduced cell survival after DNA damage
by up to 4-fold, DNA damage sensitivities of the "kinase-dead"
rad53-K227A allele and the deletion mutant were an order of
magnitude higher (Fig. 7). The similar DNA damage sensitivity of
kinase-defective and deletion mutants indicates that the protein kinase
catalytic domain is the most critical component of the Rad53 effector
function. Rad53 has complex DNA damage regulated functions with
multiple effectors in all phases of the cell cycle (16, 17, 20, 24, 25, 41). Possible reasons for the lower DNA damage sensitivity of the
rad53-FHA1 mutants relative to the kinase-dead Rad53 could be that this domain functions only in a specific phase of the cell
cycle, or that it targets the kinase domain only to a specific subset
of Rad53 effectors. This functional restriction would be similar to the
Rad53-FHA2 domain. Deletions of the FHA2 do not increase lethality
after UV-dependent DNA damage (42), but point mutations
that abolish its interaction with Rad9 almost completely abrogate the
G2/M cell cycle arrest after
cdc13-dependent DNA damage (30).
Our overexpression results are somewhat similar to earlier reports
showing that overexpression of intact Rad53 results in slower cell
growth (11) by delaying G1-S transition by ~60 min (42)
and, when fused to green fluorescent protein, overexpressed Rad53 can
severely impair the ability of yeast to form colonies on solid media
(43). The green fluorescent protein-Rad53 phenotype can be suppressed
by simultaneous overexpression of the Ptc2 protein phosphatase (43).
However, simultaneous overexpression of Ptc2 had no effect on our FHA1
overexpression phenotype (data not shown), indicating that the FHA1
domain causes the G1 arrest by a different mechanism. Our
results are also consistent with a previous report that deletion of
large parts of the FHA1 domain from Rad53
(rad53- The strong growth-defect phenotype of the FHA1 domain (Fig. 1) makes
this system an ideal model for the structure-function analysis of
individual FHA residues in vivo, particularly as it is
abrogated by the same mutations that increase the DNA damage sensitivity of rad53 alleles under physiological expression
conditions (Fig. 7). Limited proteolysis experiments recently
demonstrated that the FHA1 domain is contained within residues 24-156
of Rad53 (28). This assignment is confirmed by results of the present study, showing first that truncation of the FHA1 domain to residues 40-141 does not elicit the growth defect of the intact domain (Fig.
1A), and second that mutation of Cys-34 or Leu-143 in the random-mutagenesis screen also abolishes the phenotype (Fig. 6). This
assignment is consistent with the recently solved NMR and x-ray
crystallographic structures of the FHA1 domain (44, 45).
Critical residues characterized in this study are highlighted in the
apo-structure of the FHA1 domain (44) in Fig.
8. This model shows that the six
conserved residues whose mutation abrogates the phenotype (shown in
red) are clustered in close proximity. Interestingly,
Gly-69, His-88, and Gly-108 are mostly buried with little surface
exposure, indicating a role in domain folding rather than target
binding. This is consistent with the low expression levels of the
respective alanine mutants in vivo and their insolubility in
a recombinant expression system in vitro. In contrast,
Arg-70, Ser-85, and Asn-107, are surface-exposed in the cluster of
conserved residues, consistent with roles in directly binding to
targets. Among these residues, Arg-70 seems to be most crucial, as even the highly conservative R70K substitution identified in the random mutagenesis screen disrupts the growth defect phenotype (Fig. 6B). Asn-112 (shown in yellow) is unique among
the conserved residues as its substitution by alanine does not disrupt
the phenotype. This is reflected in the structure where it is not part
of the cluster of conserved residues. Interestingly, large parts of
this residue are buried underneath adjacent residues in the structure, which explains why the alanine substitution of this residue leads to a
partial solubility problem when expressed in bacteria (Fig. 5A).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sandwich that is similar to the fold of
the C-terminal domain of the smad tumor suppressor/transcription factor
family (27). In addition to checkpoint kinases, FHA domains are also
found in several other signaling molecules including Forkhead-like
transcription factors, from which their name originated. Recent reports
indicate that FHA domains bind directly to threonine-phosphorylated
residues in target proteins (29). In the case of Rad53, it has been
demonstrated that association of the C-terminal FHA2 domain with
phosphorylated Rad9 is essential for Rad53 activation and
G2/M arrest following DNA damage (30). Interestingly, the
Rad53-FHA2 domain can also bind directly to phosphotyrosine-containing
peptides in vitro, indicating that FHA domains may be
bifunctional phosphothreonine/phosphotyrosine-binding domains (31).
In vitro, the N-terminal FHA1 domain of Rad53 also binds to
phosphorylated Rad9 as well as to phosphothreonine-containing synthetic
peptides (29), but its physiological function is presently unclear.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and rad17
strains (34) were in the BY4741 background (MATa,
leu2
0, his3
1,
met15
0, ura3
0) and obtained
from Research Genetics. The sml1
and
rad53
sml1-1 strains were in the W303-1A
genetic background (MATa, ade2-1, can1-100,
his3-11,15, leu2-3, trp1-1, ura3-1) (18). A K699
dun1
strain was generated using a PCR fragment containing
50 base pairs of the DUN1 gene flanking the LEU2
selectable marker, and the deletion was confirmed by PCR and
immunoblotting.2 Some
experiments were performed in K699 strains containing
galactose-inducible cyclin genes (LEU2::GAL1-CLN1
and LEU2::GAL1-CLN2 (35)) or vectors for Ptc2
overexpression (36), respectively. Start cultures for galactose-inducible experiments were grown in selective medium containing 2% sucrose. For induction of protein expression, cultures were grown in selective medium containing 2% sucrose and 4%
galactose. For growth rate studies, aliquots were removed from
exponentially growing cultures and optical densities were measured at
600 nm. Cell viability at the same time points was assayed by plating aliquots on 1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) glucose (YPD) medium and counting colonies after 3 days of incubation at 30 °C.
-factor. Cells were released from
-factor arrest by three washes
in selective medium containing 2% sucrose and 4% galactose and
resuspended in fresh medium.
strain that is
viable without Rad53. RAD53 was disrupted using a PCR
product containing the URA3 selectable marker flanked by a
45-base pair RAD53 genomic sequence (see supplemental information on-line). RAD53 allele replacements (R70A,
N107K, and K227A) were performed using a PCR-based strategy similar to the one described by Erdeniz et al. (37), except that the
S. cerevisiae rather than the Kluyveromyces lactis
URA3 gene was used as the tagged selectable marker and that fusion
fragments corresponded only to the targeted domains rather than the
entire coding sequence. Strains containing mutated rad53
alleles were identified by colony PCR, using primers outside the
PCR-generated targeting sequences, followed by hybridization with
discriminatory oligonucleotides. The correct mutations and absence of
secondary mutations were confirmed by automated DNA sequence analysis.
For survival assays, aliquots were removed from log phase cultures immediately before and 3 h after addition of MMS, and spread onto YPD plates.
-mercaptoethanol) and vigorous vortexing in the presence of 100 µl
of glass beads. Proteins were transferred onto Polyscreen polyvinyl
difluoride membranes (PerkinElmer Life Sciences), and probed
with 0.8 µg/ml polyclonal anti-Rad53 antiserum (Santa Cruz
Biotechnology), 0.1 µg/ml monoclonal anti-Gal4 DNA binding domain
antibody (Santa Cruz Biotechnology), 0.1 µg/ml monoclonal anti-actin
antibody (Roche), or 0.2 µg/ml anti-His4 monoclonal antibody (Qiagen), using secondary antibodies conjugated with horseradish peroxidase and ECLTM reagents (Amersham Pharmacia Biotech) for detection.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (61K):
[in a new window]
Fig. 1.
FHA domain overexpression phenotypes.
A, yeast strains containing vector-control, Rad53 N
terminus, Rad53-FHA1, Rad53-FHA1 mutant (20-164 with substitutions
S85A/H88A), truncated Rad53-FHA1, Rad53-FHA2, Chk2-FHA, and the
Dun1-FHA p416GAL1 constructs were plated on 2% sucrose
(left), 2% sucrose + 4% galactose to induce FHA domain
expression (center), and on 2% sucrose + 4% galactose + 0.007% MMS (only the relevant half of the MMS plate is shown).
B, immunoblot analysis of protein extracts of the different
strains after 6-h induction with 4% galactose. FHA domain fragments
were detected using an anti-Gal4 antibody, and samples were reprobed
using an anti-actin antibody as a loading control.
strain (Fig.
2). In this experiment, expression of the
FHA1 domain again inhibited colony formation in the sml1
strain to the same extent as in the wild-type strain. Similar results
were also obtained in a rad53
strain (viable because of a
sml1-1 mutation; Fig. 2). This indicates that the FHA1
overexpression phenotype is unrelated to the essential function of
RAD53.
View larger version (71K):
[in a new window]
Fig. 2.
Maintenance of the FHA1 overexpression
phenotype in checkpoint-defective strains and an extragenic suppressor
strain of the RAD53 essential function.
A, vector-control, Rad53-FHA1, and Rad53-FHA1 mutant
(S85A/H88A) p416GAL1 constructs in wild-type, rad9 , and
rad17
strains in the BY4741 background were plated on 2%
sucrose (top), and on 2% sucrose + 4% galactose.
B, cultures of vector-control (V) and
Rad53-FHA1 (F) constructs in the W303-1A background
(wild-type, rad53
sml1-1, sml1
),
and the K699 background (wild-type, dun1
) (from
left to right), were adjusted to 0.05 optical
density units and 2 µl plated in a series of 10-fold dilutions on 2%
sucrose (top) and on 2% sucrose + 4% galactose.
C, yeast strains induced to express vector-control,
Rad53-FHA1, and Rad53-FHA1 mutant (S85A/H88A) constructs were treated
for 1 h with 0.1% MMS (M), 150 mM
hydroxyurea (H), or left untreated (
). Protein extracts
were immunoblotted with anti-Rad53 and anti-actin antibodies.
strain; the FHA1 phenotype must therefore be
independent of its proposed interaction with Rad9. Similar results were
also obtained in a strain lacking RAD17 (Fig.
2A), a component of an alternative DNA damage-sensor pathway
for Rad53 activation (38), and in a strain lacking DUN1
(Fig. 2B), a Rad53 effector at the transducer level. The
maintenance of the FHA1 overexpression phenotype in rad9
,
rad17
, rad53
, and dun1
strains indicates that the growth defect does not result from an
FHA1-generated checkpoint signal at the sensor/transducer level,
e.g. by displacing Rad53 from inhibitory complexes. This
genetic evidence was confirmed by immunoblot analysis of the Rad53
activation state. Rad53 is activated by phosphorylation resulting in
slower migrating bands in SDS-polyacrylamide gels (39). Fig.
2C shows that expression of the FHA1 domain did not result
in shifted (i.e. activated) Rad53 under basal conditions,
nor did it inhibit Rad53 activation by MMS-induced DNA damage or
hydroxyurea-induced replication blocks.
View larger version (32K):
[in a new window]
Fig. 3.
Cell cycle analysis of the Rad53-FHA1
growth defect phenotype. A, yeast strains containing
vector-control (circles) and Rad53-FHA1 (squares)
p416GAL1 constructs were grown for 24 h in 2% sucrose + 4% galactose. At 2-h intervals, aliquots were removed and cell
growth monitored by optical density measurements (filled
symbols), and cell viability determined by plating on YPD
medium (open symbols). In the inset,
the same values are plotted as cell viability versus cell
growth for a linear fit (vector-control, broken
line; FHA1, solid line). B,
1-ml aliquots were removed from exponentially growing vector-control
and Rad53-FHA1 containing cultures at the time points indicated, and
analyzed by flow cytometry. C, vector-control and FHA1
p416GAL1 constructs induced for 10 h in 4% galactose
were diluted to 0.2 optical density units, synchronized in
G1 by -factor, and released into galactose medium.
Aliquots removed at the times indicated were analyzed by flow
cytometry.
-factor and then released into
fresh galactose-containing medium. Fig. 3C shows that the
majority of the control culture had shifted to G2/M within
75 min after
-factor release. In contrast, the majority of
FHA1-overexpressing cells were still in G1 150 min after
release from the pheromone (Fig. 3C).
-Factor arrest
kinetics (before the release) were similar for the FHA1 and control
culture (data not shown), indicating that other phases of the cell
cycle were largely unaffected. Therefore, cell cycle arrest in the
G1 phase is the major reason for the inability of the
FHA1-overexpressing strain to form visible colonies on solid medium.
View larger version (50K):
[in a new window]
Fig. 4.
In vivo mutational analysis of
highly conserved FHA residues. A, colony formation of yeast
constitutively overexpressing Rad53-FHA1 (WT), double mutant
Rad53-FHA1(S85A/H88A), and FHA1 with individual alanine substitutions
of the highly conserved FHA domain residues (middle
panel, >80% consensus; bottom panel,
>65% consensus). B, FHA1 expression levels were analyzed
by immunoblot analysis of viable colonies with an anti-Gal4 DNA binding
domain antibody. C, comparison of constitutive expression
levels of R70A, S85A, and N107A FHA1 mutants with inducible wild-type
FHA1 levels preventing cell growth.
View larger version (25K):
[in a new window]
Fig. 5.
Biochemical characterization of highly
conserved FHA domain residues. A, immunoblot analysis
of the solubility of bacterially expressed Rad53-FHA1 and FHA1 domain
mutants using an anti-His4 antibody (P,
particulate fraction; S, soluble fraction). B,
Coomassie Blue-stained SDS-polyacrylamide gel containing 1 µg/lane of
purified recombinant soluble FHA1, Dun1-FHA, and FHA1 domain mutants.
Mass standards in the leftmost lane are (from
bottom to top) 6.5, 14, 21, 31, 43, 69, and
97/112/200 kDa. C, ELISA of FHA domains tested for their
ability to bind a phosphothreonine-containing peptide. FHA1 data points
represent the means ± S.E. of four experiments; only a single
experiment was performed with the Dun1-FHA. WT, wild-type
FHA1.
View larger version (83K):
[in a new window]
Fig. 6.
Identification of crucial nonconserved FHA1
residues in a random mutagenesis screen. A, a
representative immunoblot of phenotypic revertants screened for
full-length FHA1 clones. Full-length clones identified here contain
single amino acid substitutions of N107K (lane
4), R70K (lane 9), and N107I
(lane 13). Truncated fragments with different
masses than shown here were detected in other blots (data not shown).
Note that no FHA1 fragments were detected in lanes
2, 3, 6, 7, 11,
and 12. Lane C shows a double-mutant
(FHA1-S85A/H88A) control. B, immunoblot analysis comparing
protein levels of the 13 different random mutations identified in the
random mutagenesis screen. Note that more than one substitution of
Ser-105 or Asn-107 abrogated the phenotype.
mutant. All of these alleles were generated in a strain containing the sml1
mutation as an extragenic suppressor of the
essential viability function of RAD53.
View larger version (26K):
[in a new window]
Fig. 7.
Replacement of the RAD53
gene with alleles containing amino acid substitutions in the FHA1
and investigation of DNA damage sensitivity. A,
schematic diagram of Rad53 showing the location of amino acid
substitutions resulting from allele replacement. B, cell
survival of RAD53, rad53-R70A, rad53-N107K,
rad53-K227A, and rad53 strains after 3-h treatment
with 0.03% MMS (solid bars) or 0.04% MMS
(open bars), expressed as percentage of viable
cells relative to untreated controls. The bars represent the
mean ± standard error of six experiments. All strains are
sml1
. C, immunoblot analysis of Rad53
expression levels in these strains. The same samples were also probed
with an anti-actin antibody as a loading control.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
51-165 and
rad53-
99-161) results in increased UV
sensitivity (42). In that case, FHA1-deleted rad53 mutants
expressed from a centromeric plasmid in a rad53
background failed to protect from UV-dependent lethality in
contrast to wild-type and FHA2-deleted constructs. The more moderate
DNA damage sensitivity in our approach could have two reasons. First,
the DNA damage mechanism of UV irradiation (preferentially DNA adducts)
is different from MMS treatment (preferentially double-strand breaks).
Second, single amino acid substitutions in residues unlikely to be
involved in domain folding may be less disruptive to the overall Rad53
protein structure than partial domain deletions (one of the FHA1
deletion mutants resulted in clearly reduced Rad53 protein levels; Ref.
42), resulting in more specific phenotypes.
View larger version (44K):
[in a new window]
Fig. 8.
Position of important residues in the
Rad53-FHA1 apo-structure (44). Conserved residues are shown
in red if their mutation abrogates the FHA1 phenotype or in
yellow if their mutation does not affect the phenotype.
Residues shown in green abolished the phenotype in the
random mutagenesis screen. Views A, B, and
C are clockwise 90° rotations around a longitudinal
axis.
Functionally important nonconserved residues identified in the random mutagenesis screen are highlighted in green in Fig. 8. Cys-34, Val-36, Leu-124, and Leu-143 are buried in the core of the FHA1 domain, indicating that they are crucial for the proper domain fold. The other two critical nonconserved residues, Leu-78 and Ser-105, are very interesting as they directly interact with the cluster of conserved residues. Although the main chain residues of Leu-78 are surface-exposed and could interact with targets, the major function of its side chain is probably to correctly position the surface-exposed conserved residue Arg-70 that is crucial for target-binding (Fig. 5C) and with which it closely interacts. One of the phenotype-abrogating substitutions of Ser-105 (S105T) is highly conservative, underscoring the crucial role of this residue for the function of the FHA1 domain. In contrast to the conserved residues in the cluster that should have similar functions across all FHA domains, i.e. most likely direct interaction with the phosphate group in their targets, the nonconserved Ser-105 may therefore be a major secondary interaction site involved in target discrimination and binding specificity.
The FHA1 target that is involved in the G1 arrest
overexpression phenotype is presently unclear. Activated Cdc28/Cln
cyclin-dependent kinase complexes play a major role in the
transition from G1 into S phase (46). Although simultaneous
overexpression of the CLN1 or CLN2 G1
cyclin genes does not overcome the FHA1-dependent arrest phenotype (data not shown), Cdc28 or its modulator Cks1 remain potential components of the pathway affected by FHA1 overexpression. Our future work will be directed at identifying proteins involved in
the FHA1 overexpression phenotype and evaluating their role as
effectors in the G1 checkpoint function of Rad53.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Matthew O'Connell and Tony Tiganis for help with FACS analyses; Rodney Rothstein, Doris Germain, Leon Helfenbaum and Mary-Jane Gething for yeast strains and plasmids; Ming-Daw Tsai, Michael Yaffe and their coworkers for communicating data before publication; Carolyn McNees and Mark Schwartz for discussions; and Bruce Kemp, Carolyn McNees, Matthew O'Connell, Andy Poumbourios, and Tony Tiganis for critically reading the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the National Health and Medical Research Council of Australia (NHMRC), by an NHMRC R. D. Wright award (to J. H.), and by Australian postgraduate awards (to B. L. P. and A. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains Supplemental Table I.
To whom correspondence should be addressed. Tel.: 61-3-9288-2480;
Fax: 61-3-9416-2676; E-mail:
heier@ariel.its.unimelb.edu.au.
Published, JBC Papers in Press, January 18, 2001, DOI 10.1074/jbc.M009558200
2 A. Hammet and J. Heierhorst, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: FHA, Forkhead-associated; ELISA, enzyme-linked immunosorbent assay; MMS, methylmethane sulfonate; PCR, polymerase chain reaction; YPD, yeast extract/peptone/glucose.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Elledge, S. J.
(1996)
Science
274,
1664-1672 |
2. | Rhind, N., and Russell, P. (1998) Curr. Opin. Cell Biol. 10, 749-758[CrossRef][Medline] [Order article via Infotrieve] |
3. |
Matsuoka, S.,
Huang, M.,
and Elledge, S. J.
(1998)
Science
282,
1893-1897 |
4. |
Brown, A. L.,
Lee, C. H.,
Schwarz, J. K.,
Mitiku, N.,
Piwnica-Worms, H.,
and Chung, J. H.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
3745-3750 |
5. |
Chehab, N. H.,
Malikzay, A.,
Appel, M.,
and Halazonetis, T. D.
(2000)
Genes Dev.
14,
278-288 |
6. |
Hirao, A.,
Kong, Y. Y.,
Matsuoka, S.,
Wakeham, A.,
Ruland, J.,
Yoshida, H.,
Liu, D.,
Elledge, S. J.,
and Mak, T. W.
(2000)
Science
287,
1824-1827 |
7. |
Shieh, S. Y.,
Ahn, J.,
Tamai, K.,
Taya, Y.,
and Prives, C.
(2000)
Genes Dev.
14,
289-300 |
8. | Chaturvedi, P., Eng, W. K., Zhu, Y., Mattern, M. R., Mishra, R., Hurle, M. R., Zhang, X., Annan, R. S., Lu, Q., Faucette, L. F., Scott, G. F., Li, X., Carr, S. A., Johnson, R. K., Winkler, J. D., and Zhou, B. B. (1999) Oncogene 18, 4047-4054[CrossRef][Medline] [Order article via Infotrieve] |
9. | Lee, J. S., Collins, K. M., Brown, A. L., Lee, C. H., and Chung, J. H. (2000) Nature 404, 201-204[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Bell, D. W.,
Varley, J. M.,
Szydlo, T. E.,
Kang, D. H.,
Wahrer, D. C.,
Shannon, K. E.,
Lubratovich, M.,
Verselis, S. J.,
Isselbacher, K. J.,
Fraumeni, J. F.,
Birch, J. M.,
Li, F. P.,
Garber, J. E.,
and Haber, D. A.
(1999)
Science
286,
2528-2531 |
11. | Zheng, P., Fay, D. S., Burton, J., Xiao, H., Pinkham, J. L., and Stern, D. F. (1993) Mol. Cell. Biol. 13, 5829-5842[Abstract] |
12. | Allen, J. B., Zhou, Z., Siede, W., Friedberg, E. C., and Elledge, S. J. (1994) Genes Dev. 8, 2401-2415[Abstract] |
13. | Zhou, Z., and Elledge, S. J. (1993) Cell 75, 1119-1127[Medline] [Order article via Infotrieve] |
14. |
de los Santos, T.,
and Hollingsworth, N. M.
(1999)
J. Biol. Chem.
274,
1783-1790 |
15. |
Gardner, R.,
Putnam, C. W.,
and Weinert, T.
(1999)
EMBO J.
18,
3173-3185 |
16. | Mills, K. D., Sinclair, D. A., and Guarente, L. (1999) Cell 97, 609-620[Medline] [Order article via Infotrieve] |
17. |
Bashkirov, V. I.,
King, J. S.,
Bashkirova, E. V.,
Schmuckli-Maurer, J.,
and Heyer, W. D.
(2000)
Mol. Cell. Biol.
20,
4393-4404 |
18. | Zhao, X., Muller, E. G., and Rothstein, R. (1998) Mol. Cell 2, 329-340[Medline] [Order article via Infotrieve] |
19. |
Desany, B. A.,
Alcasabas, A. A.,
Bachant, J. B.,
and Elledge, S. J.
(1998)
Genes Dev.
12,
2956-2970 |
20. |
Sidorova, J. M.,
and Breeden, L. L.
(1997)
Genes Dev.
11,
3032-3045 |
21. | Santocanale, C., and Diffley, J. F. (1998) Nature 395, 615-618[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Dohrmann, P. R.,
Oshiro, G.,
Tecklenburg, M.,
and Sclafani, R. A.
(1999)
Genetics
151,
965-977 |
23. |
Kihara, M.,
Nakai, W.,
Asano, S.,
Suzuki, A.,
Kitada, K.,
Kawasaki, Y.,
Johnston, L. H.,
and Sugino, A.
(2000)
J. Biol. Chem.
275,
35051-35062 |
24. |
Weinreich, M.,
and Stillman, B.
(1999)
EMBO J.
18,
5334-5346 |
25. |
Sanchez, Y.,
Bachant, J.,
Wang, H.,
Hu, F.,
Liu, D.,
Tetzlaff, M.,
and Elledge, S. J.
(1999)
Science
286,
1166-1171 |
26. | Hofmann, K., and Bucher, P. (1995) Trends Biochem. Sci. 20, 347-349[CrossRef][Medline] [Order article via Infotrieve] |
27. | Liao, H., Byeon, I. J., and Tsai, M. D. (1999) J. Mol. Biol. 294, 1041-1049[CrossRef][Medline] [Order article via Infotrieve] |
28. | Hammet, A., Pike, B. L., Mitchelhill, K. I., Teh, T., Kobe, B., House, C. M., Kemp, B. E., and Heierhorst, J. (2000) FEBS Lett. 471, 141-146[CrossRef][Medline] [Order article via Infotrieve] |
29. | Durocher, D., Henckel, J., Ferscht, A. R., and Jackson, S. P. (1999) Mol. Cell 4, 387-394[Medline] [Order article via Infotrieve] |
30. |
Sun, Z.,
Hsiao, J.,
Fay, D. S.,
and Stern, D. F.
(1998)
Science
281,
272-274 |
31. | Wang, P., Byeon, I. J., Liao, H., Beebe, K. D., Yongkiettrakul, S., Pei, D., and Tsai, M. D. (2000) J. Mol. Biol. 302, 927-940[CrossRef][Medline] [Order article via Infotrieve] |
32. | Mumberg, D., Müller, R., and Funk, M. (1994) Nucleic Acids Res. 22, 5767-5768[Medline] [Order article via Infotrieve] |
33. |
Wang, H.,
and Elledge, S. J.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
3824-3829 |
34. |
Winzeler, E. A.,
Shoemaker, D. D.,
Astromoff, A.,
Liang, H.,
Anderson, K.,
Andre, B.,
Bangham, R.,
Benito, R.,
Boeke, J. D.,
Bussey, H.,
Chu, A. M.,
Connelly, C.,
Davis, K.,
Dietrich, F.,
Dow, S. W.,
El Bakkoury, M.,
Foury, F.,
Friend, S. H.,
Gentalen, E.,
Giaever, G.,
Hegemann, J. H.,
Jones, T.,
Laub, M.,
Liao, H.,
et al..
(1999)
Science
285,
901-906 |
35. | Tyers, M., Tokiwa, G., and Futcher, B. (1993) EMBO J. 12, 1955-68[Abstract] |
36. |
Welihinda, A. A.,
Tirasophon, W.,
Green, S. R.,
and Kaufman, R. J.
(1998)
Mol. Cell. Biol.
18,
1967-1977 |
37. |
Erdeniz, N.,
Mortensen, U. H.,
and Rothstein, R.
(1997)
Genome Res.
7,
1174-1183 |
38. | Weinert, T. (1998) Cell 94, 555-558[Medline] [Order article via Infotrieve] |
39. | Sanchez, Y., Desany, B. A., Jones, W. J., Liu, Q., Wang, B., and Elledge, S. J. (1996) Science 271, 357-360[Abstract] |
40. | Sun, Z., Fay, D. S., Marini, F., Foiani, M., and Stern, D. F. (1996) Genes Dev. 10, 395-406[Abstract] |
41. |
Liberi, G.,
Chiolo, I.,
Pellicioli, A.,
Lopes, M.,
Plevani, P.,
Muzi-Falconi, M.,
and Foiani, M.
(2000)
EMBO J.
19,
5027-5038 |
42. | Fay, D. S., Sun, Z., and Stern, D. F. (1997) Curr. Genet. 31, 97-105[CrossRef][Medline] [Order article via Infotrieve] |
43. |
Marsolier, M. C.,
Roussel, P.,
Leroy, C.,
and Mann, C.
(2000)
Genetics
154,
1523-1532 |
44. | Liao, H., Yuan, C., Su, M. I., Yongkiettrakul, S., Qin, D., Li, H., Byeon, I. J., Pei, D., and Tsai, M. D. (2000) J. Mol. Biol. 304, 941-951[CrossRef][Medline] [Order article via Infotrieve] |
45. | Durocher, D., Taylor, I. A., Sarbassova, D., Haire, L. F., Westcott, S. L., Jackson, S. P., Smerdon, S. J., and Yaffe, M. B. (2000) Mol. Cell 6, 1169-1182[Medline] [Order article via Infotrieve] |
46. |
Reynard, G. J.,
Reynolds, W.,
Verma, R.,
and Deshaies, R. J.
(2000)
Mol. Cell. Biol.
20,
5858-5864 |