From the Department of Radiation Oncology and the Winship Cancer Institute, Emory University School of Medicine, Atlanta, Georgia 30322
Received for publication, January 3, 2003
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ABSTRACT |
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The recognition of DNA double-stranded
breaks or single-stranded DNA gaps as a precondition for cell cycle
checkpoint arrest has been well established. However, how bulky base
damage such as UV-induced pyrimidine dimers elicits a checkpoint
response has remained elusive. Nucleotide excision repair represents
the main pathway for UV dimer removal that results in strand
interruptions. However, we demonstrate here that Rad53p
hyperphosphorylation, an early event of checkpoint signaling in
Saccharomyces cerevisiae, is independent of nucleotide
excision repair (NER), even if replication as a source of
secondary DNA damage is excluded. Thus, our data hint at primary base
damage or at UV damage (primary or secondary) that does not need to be
processed by NER as the relevant substrate of damage-sensing checkpoint proteins.
Checkpoints arrest cell cycle progression in the presence of DNA
damage to facilitate DNA repair and prevent genetic instability (1-3).
There is now a considerable amount of information available that
characterizes signal transmission mechanisms and downstream targets in
lower and higher eukaryotic cells. However, the precise roles of the
initial damage-sensing proteins and their relevant DNA or chromatin
substrates have remained largely elusive. Characterization of candidate
proteins supports the notion that the underlying mechanisms are
conserved throughout eukaryotic evolution (4-7). Consequently, the
yeast Saccharomyces cerevisiae will continue to play an
important role as a relevant model system.
A damage sensor function has been suggested for the
PCNA1-like complex composed
of the budding yeast proteins Rad17p, Ddc1p, and Mec3p, also termed
9-1-1 complex (following the nomenclature adapted for human cells and
Schizosaccharomyces pombe) (8-11). Although
direct evidence is still missing, it is likely that this ring-shaped
complex (12) encircles DNA and is loaded onto DNA by a replication
factor C-related complex in which the largest replication factor C
subunit is replaced by the checkpoint protein Rad24p (13). The
phosphoinositol kinase-related protein Mec1p (an ortholog of the human
ATR protein) has been identified as an independent damage-sensor
candidate (9, 11). Mec1p forms a complex with Lcd1p, a protein with an
affinity for DNA double-stranded breaks, and thus Mec1p kinase activity
can be targeted to sites of DNA damage (14). Mec1p and its downstream
target Rad53p are involved in virtually all DNA damage and replication-
related checkpoints.
Other potential sensor proteins responding to double-stranded breaks
are represented by the Rad50p·Xrs2p·Mre11p complex. This complex plays a well established role in non-homologous endjoining and
homologous recombination, whereas a function in checkpoint arrest
following treatment with strand break-inducing agents has only recently
been uncovered (15, 16).
Although such putative damage recognition complexes appear to be formed
independently from each other, their combined action is frequently
necessary for the generation of a downstream signal. A key player in
signal transmission is the Rad53p kinase that is phosphorylated in a
Mec1p-dependent fashion in response to DNA damage (17). A
pool of hyperphosphorylated Rad53p is generated through
autophosphorylation in trans, mediated through
complex formation with Rad9p following Mec1p-dependent
Rad9p phosphorylation (18).
In higher and lower eukaryotes, several studies have indicated that
even a single DNA double strand break can result in checkpoint activation if not immediately repaired (19-21). Other observations point to the significance of single-stranded DNA tracts (21, 22).
Surprisingly however, little is known regarding the recognition of base
damage that is not directly associated with double strand breaks, even
for such a generally well studied case as UV damage. Exposure of DNA to
ultraviolet light (mainly UV-C) results in the dimerization of adjacent
pyrimidine residues, giving rise to cyclobutane-type dimers and 6-4 photoproducts (23). These types of bulky DNA damage are readily
recognized and repaired by nucleotide excision repair (NER), which
functions by the introduction of single strand breaks bracketing the
lesion, followed by the removal of a single-stranded DNA fragment (24).
A global NER system and a repair system that is targeted by inhibition
of transcription to the site of the lesion can be distinguished. The
established correlation of double-stranded breaks and possibly
single-stranded DNA with checkpoint arrest could hint at a NER
intermediate involving free DNA ends as the effective recognized lesion
following UV treatment. However, any single-stranded gaps arising
directly from NER will be small and short-lived.
In S. cerevisiae, two studies have analyzed in some detail
checkpoint responses at the G1/S transition in
NER-deficient cells (25, 26). An S-phase delay was observed in
UV-irradiated NER-deficient cells at much lower doses than in the
isogenic wild type (25). However, this delay was not influenced
by Rad9p, a protein necessary for both G1 and
G2/M checkpoint arrest in the wild type. More detailed analysis indicated that UV-irradiated NER-deficient cells do
not arrest upstream of START as the wild type, albeit at higher doses
(25). At the molecular level, it was independently confirmed that this
low dose arrest response observed in NER-deficient cells depends on
S-phase entry and requires the checkpoint kinases Mec1p and Rad53p but
not other checkpoint proteins including putative damage sensors (such
as Rad9p, Rad17p, Rad24p, Mec3p, or Ddc1p) (26). Nevertheless, these
proteins are needed for efficient UV-induced mutagenesis in
NER-deficient cells, and a subset is also required for UV-induced
sister chromatid exchange (27).
These observations neither prove nor disprove the view that damage
processing by NER is indispensable for checkpoint responses to UV
damage outside of S-phase. It needs to be taken into account that the
effects analyzed so far have been triggered by much lower doses of UV
than usually given to wild-type cells. If primary unprocessed UV damage
is the relevant trigger of checkpoint signaling, the dose range
required for wild-type responses will have to be applied. However, any
interpretation will have to consider the high lethality of NER mutants
in such dose range.
This study addresses primarily the requirement of NER for events that
precede cell cycle checkpoint arrest. While avoiding complications
because of secondary lesions arising from the interference of
photodimers with replication, we have concentrated on the analysis of
NER-deficient cells that were synchronized and UV-treated in G1- and M-phase. We conclude that in these cell cycle
stages NER is not required for Rad53p hyperphosphorylation, an
indispensable step of arrest signal transmission. However, such an
independence of NER is only uncovered when approximately the same UV
dose range is applied that is required for Rad53p activation in the
wild type. Although the resulting high lethality ultimately precludes a
meaningful interpretation of the actual cell cycle effects, our data
support models that assume recognition of UV-induced initial base
damage and not of NER-dependent DNA repair intermediates by
sensor proteins as the essential step. Alternatively, primary or
secondary non-dimer DNA damage may be critical as recognized lesions.
Yeast Strains--
The haploid S. cerevisiae strain
SX46A MATa ade2 ura3-52 trp1-289 his3-532 was
used as wild type (source: E. C. Friedberg, originally from J. Rine). In this strain background, a pronounced G1
arrest following treatment with UV or ionizing radiation is observed
(28). Construction of the NER-deficient
rad14 Cell Synchronization and UV Treatment--
For cell culture and
media recipes, we followed standard procedures of yeast genetics and
cell biology (29). Cells were synchronized in M-phase by resuspending a
mid-logarithmic phase culture in fresh YPD (1% yeast extract, 2%
peptone, 2% dextrose) at 1 × 107 cells/ml and by
adding nocodazole (U. S. Biological) to 10 µg/ml. A second aliquot
was added after 60 min of incubation at 30 °C, and the culture was
used following another 75 min of incubation. Cells were synchronized in
G1 with the yeast pheromone Cell Cycle Analysis--
For FACS analysis, ~1 × 107 cells were withdrawn at each time point, spun down,
washed, and fixed in 90% ethanol. Staining with propidium iodide has
been described elsewhere (30). Samples were analyzed on a BD
Biosciences FACScan using the CellQuest program suite.
Detection of Rad53p--
A sample of 1 × 108
cells was used for each time point. Trichloroacetic acid protein
extracts were prepared using zirconium beads (Biospec Products)
essentially as described (31). Approximately one-third of total protein
extract was loaded per lane of an 8% PAGE gel (1:80
bisacrylamide:acrylamide ratio). Separated proteins were transferred to
nitrocellulose by semidry electroblotting, and immunoblot analysis was
performed according to standard techniques (32). S. cerevisiae Rad53p antibody (Sc6749, raised in goat, Santa Cruz
Biotechnology) was used at a 1:300 dilution, secondary anti-goat
horseradish peroxidase-conjugated antibody (Santa Cruz Biotechnology)
at a 1:3000 dilution. SuperSignal West Pico kit (Pierce) was used for
chemiluminescence signal detection.
Cell Cycle Response in UV-Treated M-phase Cells--
In
S. cerevisiae, the influence of NER on UV-induced
cell cycle arrest and associated responses has not been investigated in
cell cycle stages other than G1/S. We regarded studying
UV-treated M-phase cells as an attractive approach because a checkpoint
response can be determined that is not influenced by the generation of secondary DNA damage in S-phase. To this end, cells synchronized in
metaphase by treatment with the tubulin inhibitor nocodazole were
UV-irradiated in suspension and released in fresh medium without
inhibitor. The macrocolony survival resulting from this and all further
treatment protocols is shown in Fig. 1.
Cell cycle progression was analyzed by FACS analysis. In
repair-competent wild-type cells, a dose of 20 J/m2
resulted in only a brief M-phase arrest during the first cycle, whereas
treatment with 120 J/m2 caused an arrest of at least 2 h (Fig. 2A). In NER-deficient rad14 Rad53p Hyperphosphorylation in UV-Treated M-phase Cells--
We
reasoned that a rapid signaling response such as Rad53p
hyperphosphorylation that is more proximal to the damage recognition process might be less affected by the lethally damaged state of the
studied mutant cells. Thus, we analyzed in the samples shown in Fig. 2
the extent of Rad53p hyperphosphorylation in the form of slower moving
protein bands visualized by immunoblotting (Fig. 3). Following treatment with the lower UV
dose (20 J/m2), only a small fraction of Rad53p was found
to be transiently phosphorylated, whereas the higher dose resulted in a
very pronounced Rad53p modification. Most importantly, these results
were essentially the same in both the wild type and NER-deficient
rad14 mutant (Fig. 3). The only difference between the
strains appeared to be the diminishing level of hyperphosphorylated
Rad53p that is observed after 3 h of incubation in the wild type
but not in the mutant. Thus, the phosphorylation level correlates with
exit from M-phase arrest (Fig. 2) and may reflect successful DNA damage repair. As expected, UV-induced Rad53p hyperphosphorylation was found
to be strictly dependent on RAD9 (Fig. 3).
Rad53p Hyperphosphorylation in UV-treated G1-arrested
Cells--
Because these data indicate that Rad53p phosphorylation is
not significantly influenced by the status of NER in UV-irradiated M-phase cells, we decided to re-evaluate the influence of NER on
checkpoint signaling in G1 cells over a range of UV doses. We synchronized the identical strains with the yeast pheromone
Additionally, we prevented S-phase entry in G1-synchronized
UV-treated cells by the use of the thermoconditional cdc4-1
mutation (Fig. 5). RAD cdc4-1
and rad14 A considerable amount of information is available on the highly
sensitive and specific initiation of cell cycle checkpoint arrests and
associated responses following the occurrence of DNA double-stranded
breaks (1-7). Surprisingly, little is known regarding the initiating
molecular events that accompany such responses in cells exposed to
base-damaging agents that by themselves do not introduce DNA
double-stranded breaks or single-stranded gaps. Here, we have
concentrated on the analysis of the effects of UV-C because the nature
of the induced base damage (i.e. pyrimidine dimers) and its
repair are well understood (23, 24). Ultraviolet radiation is a potent
trigger of checkpoint arrest and transcriptional responses in lower and
higher eukaryotes (23, 33-36).
At the outset, several mechanisms of damage recognition can be
envisioned. First, a specific type of primary DNA lesion may be
directly recognized by the binding of one or more sensor proteins. Second, the conversion of primary damage into a DNA repair intermediate or the formation of a DNA-repair protein complex may create a substrate
for damage-sensing components of the checkpoint pathways. Third, the
interference of damage with cellular processes such as DNA replication
or transcription may elicit the response. This may include the
formation of secondary structural DNA damage such as single-stranded
gaps during DNA synthesis. Lastly, the possibility should not be
dismissed that non-DNA targets of the applied agent play essential
roles in checkpoint activation. The identification of protein kinase
p38 as an important component of UV-induced G2 arrest in
mammalian cells may provide such an example (37).
Because no data on G2/M-phase regulation in NER-deficient
budding yeast were available, we first characterized UV-induced M-phase
arrest in cells synchronized at the metaphase/anaphase transition. In
response to DNA double strand breaks, a bifurcated pathway regulates
checkpoint arrest at or downstream of this stage with both branches
being dependent on Mec1p kinase (38-41). One subpathway regulates the
securin Pds1p through the Chk1p kinase, whereas the other
involves the kinases Rad53p and Dun1p. However, Chk1p appears be
dispensable for cell cycle arrest after UV (39, 42), and it remains to
be demonstrated that the arrest pathways for UV damage are similarly organized.
In this study, we found that a significant dose-dependent
M-phase arrest can be induced by UV irradiation in cells released from
nocodazole arrest. Rad53p hyperphosphorylation correlates well with the
arrest response in the wild type. A similar analysis in a
rad14 Deliberately, we have not attempted to modify the UV dose according to
the much lower survival level of NER-deficient cells. However, we have
to consider the implications of working with a population of lethally
damaged cells that are most probably severely inhibited in all cellular
functions (such as transcription) shortly after treatment. A recent
study (45) has suggested a defect in UV-induced G2/M arrest
in NER-deficient cells solely based on budding analysis. However, we
doubt that a morphological marker is meaningful in lethally irradiated
cells. We argue that the observed early marker for checkpoint
activation, Rad53p hyperphosphorylation, represents a physiological
response to UV damage even in NER-deficient cells because the extent
and time course do not appear to be different from wild type. We are
less certain regarding the observed cell cycle effects. NER-deficient
cells lethally irradiated with 20 J/m2 are still capable of
M/G1 transition following a delay in M-phase. However, this
delay is not affected by an additional rad9 defect and thus
represents more likely a passive inhibitory effect of damage than an
actively regulated checkpoint response. Alternatively, a different
RAD9-independent checkpoint may have been triggered. A high
level of unrepaired DNA damage in mitosis may activate the
spindle-assembly checkpoint as described recently for human cells (46).
In our experiments, NER-deficient M-phase cells lethally irradiated
with 120 J/m2 remained and ultimately died in this stage.
Our observations suggest that checkpoint signaling through the Rad53p
pathway is independent of UV dimer incision and, thus, that a
damage-recognition mechanism that targets primary DNA base damage and
not repair-induced strand interruptions may be operating. However,
although the FACS profiles argue against massive DNA degradation, we
have not formally demonstrated the absence of DNA strand breakage.
Certain double-stranded break repair-deficient mutants in S. cerevisiae exhibit a pronounced UV sensitivity, and
double-stranded breaks are clearly detectable during post-irradiation growth of UV-treated yeast (47). Throughout our experiments, S-phase is
never entered and cannot be a source of secondary damage, but other
mechanisms are not excluded. Double-stranded breaks may arise from
recombinational repair of unrepaired UV damage in M-phase-synchronized
cells. In yeast and other systems, UV-induced recombination events were
indeed found to be independent of NER and replication (48-50).
However, such an explanation can hardly account for our observations in
haploid cells arrested in G1 where no homologous chromosome
is available.
The apparent lack of effect of NER on checkpoint pathway activation
prompted us to re-evaluate previous observations in G1 cells. An S-phase delay has been reported in NER-deficient strains at a
much lower dose level than in the wild type, and this response turned
out to be the result of RAD9-independent activation of a
MEC1-dependent checkpoint response in early
S-phase, most probably triggered by secondary damage or inhibition of
replication fork progression (25, 26). However,
G1-checkpoint arrest following treatment with DNA-damaging
agents occurs at or upstream of START in repair-proficient cells (25,
45, 51). The influence of NER remained to be analyzed at a dose level
required for such a response in the wild type. Obviously, the entry
into S-phase had to be prevented. Thus, we confined the analysis of
Rad53p phosphorylation to UV-treated cells that were kept arrested by Our data on cdc4-1 are in conflict with a previous study
(26) that demonstrated in a comparable approach a need for S-phase entry for Rad53p hyperphosphorylation in NER-deficient strains but not
in the wild type. Technical details of UV application cannot be
compared directly between the two studies. However, we were able to
verify our results over a range of doses. Differences in the used
antibody and the strain background could provide possible explanations
for the contrasting findings.
In summary, our results are compatible with models of UV damage
recognition for checkpoint activation outside of S-phase that do not
rely on the main pathway of dimer repair or on cell cycle progression.
These observations are consistent with two types of hypotheses. First,
the recognized DNA damage that triggers the checkpoint response is
primary DNA damage such as UV-induced pyrimidine dimers that are
recognized by checkpoint sensor proteins independent of NER. The extent
of the response will depend on the level and persistence of initial
damage and thus initially on the dose but subsequently also on cellular
repair capacity. The continued presence of unrepaired damage may
sensitize the response, and indeed, Rad53p phosphorylation is found in
G1 cells at lower doses than in the wild type. However,
this latter response is delayed and would not have prevented S-phase
entry if cells had not been kept artificially in G1. Such a
concept of initial damage recognition is supported by recent data on
human ATR protein, the homolog of yeast Mec1p (52). The observed
increased affinity of ATR protein for UV-dimer containing DNA indicates
a role as a sensor of primary UV damage.
Alternative hypotheses will rely on UV-induced DNA damage that is not
influenced by NER as the type of lesion relevant for checkpoint
activation. Such damage could constitute non-dimer type primary damage
or, alternatively, secondary lesions that originate from the
interference of damage with cellular processes. These include
transcription or non-NER repair, but our approach excludes an influence
of replication. Such damage may in fact be identical to persistent
double-stranded breaks, already known to be recognized by putative
damage sensor proteins of the checkpoint pathways. However, one
established double-stranded break sensor, the Rad50p·Xrs2p·Mre11p
complex, plays no role in UV-induced cell cycle arrest (15).
Agents of environmental relevance but also many anti-cancer drugs
introduce bulky base damage similar to UV. Checkpoint responses toward
these agents prevent genetic instability, and an understanding of the
molecular details involved in damage recognition will have many
implications for important issues related to human health and, in
particular to cancer, its prevention and its treatment.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
::HIS3 and
rad9
::URA3 derivatives have been
described elsewhere (25, 28). A cdc4 mutant (WS9110-3D
MATa cdc4-1 ade ura3-52 trp1-289) was derived
from the same background by crossing (25). In this strain, a
RAD14 deletion was introduced through homology-mediated trans-placement with a rad14
::KanMX4
PCR fragment that was originated from an existing null strain obtained
from a yeast strain repository (Euroscarf).
-factor (U. S. Biological).
A mid-logarithmic phase culture (~1 × 107 cells/ml)
was resuspended in fresh YPD, and
-factor was first added to 10 µg/ml. A second aliquot was added after 75 min of incubation at
30 °C or after 90 min at 25 °C in the case of cdc4 strains. Synchronization in G1 was complete after another
75 or 90 min in the case of cdc4 strains. Synchronized cells
were washed free of medium, resuspended in deionized water at a titer
of 2.5 × 107 cells/ml, sonicated briefly, and kept on
ice. 15- or 30-ml samples of the cell suspension were irradiated under
constant stirring in 100- or 150-mm-diameter plastic Petri dishes,
respectively. The used lamp delivered 254 nm of UV-C at a dose rate of
1 or 2 J/m2 × s
(Sanyo Denki Germicidal
Lamp G15T8). Appropriate dilutions were plated on solid YPD to
determine survival. Pelleted, irradiated, and mock-treated cells were
resuspended in fresh YPD at a titer of 1 × 107
cells/ml. Samples were taken during subsequent incubation at 30 °C
with the exception of cdc4 mutant cells, which were kept in
G1 by incubation at 37 °C. If required,
-factor-synchronized strains were kept arrested in G1
following irradiation by resuspension in their old pheromone-containing
medium and by the addition of further aliquots of
-factor
immediately and after 1 h of incubation.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cells, a similar brief arrest was found at the
lower dose (Fig. 2B). In contrast to the wild type, this
M-phase arrest was followed by a very pronounced arrest in a
stage with an apparent G1 DNA content. For the higher dose,
the NER-deficient cells remained arrested with a G2/M DNA
content throughout the experiment (Fig. 2B) and never
resumed cell cycle progression (data not shown). In the checkpoint and
NER-deficient rad9
rad14
double mutant, essentially the same results were found apart from a somewhat prolonged
M-phase arrest at the lower dose (Fig. 2C). However, it is
important to note that these experiments were performed with lethally
damaged cells (Fig. 1). For the lower UV dose, the colony survival of
both single mutant (rad14
) and double mutant (rad14
rad9) was already <0.01% (Fig. 1).
Therefore, it is difficult to interpret the observed cell cycle effects
as actively regulated, physiological checkpoint responses.
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Fig. 1.
Macrocolony survival of haploid yeast cells
treated with UV-C in M-phase or in G1-phase.
Cells were synchronized with nocodazole (filled symbols) or
with -factor (open symbols). The strains used were SX46A
RAD (
,
); rad14
(
,
) or
rad14
rad9
(
,
); and
WS9110-3D cdc4-1 RAD (
) or cdc4-1 rad14
(
).
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[in a new window]
Fig. 2.
Cell cycle distribution in cells synchronized
in metaphase by nocodazole and released from arrest following UV-C
treatment. Samples were withdrawn at the times indicated to the
left following irradiation with 0, 20, or 120 J/m2 UV, and DNA content was detected by FACS analysis. The
strains used were SX46A RAD, rad14 , and
rad14
rad9
.
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[in a new window]
Fig. 3.
Rad53p detection by immunoblotting in cells
synchronized in metaphase and released from arrest following UV-C
treatment. Samples were taken at the times indicated at the
top of the lanes following irradiation with 0, 20, or 120 J/m2 UV. The cell cycle distribution of each
sample was as shown in Fig. 2.
-factor in G1, treated with UV, and continued incubation
in the presence of
-factor to exclude replication as a source of
secondary DNA damage (Fig. 4).
Microscopic examination indicated continued "shmoo" appearance of
the cells, and FACS analysis confirmed G1 DNA content
throughout the experiment (Fig. 4B). As with M-phase cells,
we determined that the extent and dose response of UV-induced Rad53p
hyperphosphorylation were quite similar in wild type and rad14
mutant cells (Fig. 4A). Especially at
the lower dose, the mutant strain appeared to have a more pronounced
response than the wild type. Again, Rad53p modification was dependent
on functional Rad9p (Fig. 4A).
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Fig. 4.
Rad53p phosphorylation in cells synchronized
and kept arrested in G1 with
-factor following UV-C treatment.
A, Rad53p was detected by immunoblotting in cells irradiated
with 0, 10, or 60 J/m2 UV as indicated. Samples were taken
at the times noted at the top of the lanes. The
strains used were SX46A RAD, rad14
, and
rad14
rad9
. B, FACS profiles of
0- and 180-min samples indicate continued arrest in
G1.
cdc4-1 cells were treated with
-factor at
permissive temperature, irradiated with a range of UV doses, and
incubated at 37 °C to block cell cycle progression and entry into
S-phase. Continuous arrest in G1 was confirmed by FACS
analysis (data not shown). With the low doses of 4 and 8 J/m2, we observed some degree of Rad53p
hyperphosphorylation at the later time points in the NER-deficient
strain but not in the NER-proficient wild type. With the higher doses,
there was essentially no major difference in the extent and time course
of the observed Rad53p hyperphosphorylation between the strains (Fig.
5). Indicating successful repair, a reappearance of the original
non-modified Rad53p band was noted after 3 h of incubation in the
NER-proficient strain but not in the NER-deficient strain (20 and 40 J/m2).
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Fig. 5.
Rad53p phosphorylation in cdc4
cells synchronized in G1 with
-factor and kept in G1 by incubation at
the restrictive temperature following UV-C treatment. Cells were
kept arrested at 37 °C following treatment with 4, 8, 20, 40, and 60 J/m2 as indicated. Samples were taken at the times
indicated at the top of the figure. Strains WS9110-3D
cdc4-1 RAD and cdc4-1 rad14
are
compared.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mutant revealed that the extent, dose dependence, and time course of Rad53p modification remain essentially unaffected by
a NER defect. The Rad14 protein is necessary for early steps of UV
damage recognition, and deletion mutants are completely deficient in
the incision step (43, 44). We further demonstrated that Rad9p is
clearly required for UV-induced Rad53p phosphorylation, even in the
NER
background.
-factor or by incubation of cdc4-1 strains at the
restrictive temperature. In both cases, UV-induced hyperphosphorylation
of Rad53p was readily detectable. This is in contrast to studies indicating the inability of a single persistent double-stranded break
to trigger Rad53 hyperphosphorylation in G1 cells (20). Most importantly, the independence of UV-induced Rad53p phosphorylation from NER was again observed.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants CA87381 and ES01163.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.
Present address: Division of Biological Sciences, University of
Kansas, Haworth Hall, 1200 Sunnyside, Lawrence, KS 66045.
§ Present address: Dept. of Cell Biology and Genetics, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX 76107. To whom correspondence should be addressed. Tel.: 817-735-2048; Fax: 817-735-2610.
Published, JBC Papers in Press, January 8, 2003, DOI 10.1074/jbc.M300061200
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ABBREVIATIONS |
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The abbreviations used are: PCNA, proliferating cell nuclear antigen; ATR, ATM (ataxia telangiectasia-mutated) and Rad3-related; NER, nucleotide excision repair; FACS, fluorescence-activated cell sorting.
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