MOLECULAR AND CELLULAR BIOLOGY, Dec. 2004, p. 10313–10327
0270-7306/04/$08.00?0 DOI: 10.1128/MCB.24.23.10313–10327.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 24, No. 23
Activation of the DNA Damage Checkpoint in Yeast Lacking the
Histone Chaperone Anti-Silencing Function 1
Christopher Josh Ramey, Susan Howar, Melissa Adkins, Jeffrey Linger,
Judson Spicer, and Jessica K. Tyler*
Department of Biochemistry and Molecular Genetics, University of Colorado Health
Sciences Center at Fitzsimons, Aurora, Colorado
Received 3 August 2004/Accepted 2 September 2004
The packaging of the eukaryotic genome into chromatin is likely to be important for the maintenance of
genomic integrity. Chromatin structures are assembled onto newly synthesized DNA by the action of chromatin
assembly factors, including anti-silencing function 1 (ASF1). To investigate the role of chromatin structure in
the maintenance of genomic integrity, we examined budding yeast lacking the histone chaperone Asf1p. We
found that yeast lacking Asf1p accumulate in metaphase of the cell cycle due to activation of the DNA damage
checkpoint. Furthermore, yeast lacking Asf1p are highly sensitive to mutations in DNA polymerase alpha and
to DNA replicational stresses. Although yeast lacking Asf1p do complete DNA replication, they have greatly
elevated rates of DNA damage occurring during DNA replication, as indicated by spontaneous Ddc2p-green
fluorescent protein foci. The presence of elevated levels of spontaneous DNA damage in asf1 mutants is due to
increased DNA damage, rather than the failure to repair double-strand DNA breaks, because asf1 mutants are
fully functional for double-strand DNA repair. Our data indicate that the altered chromatin structure in asf1
mutants leads to elevated rates of spontaneous recombination, mutation, and DNA damage foci formation
arising during DNA replication, which in turn activates cell cycle checkpoints that respond to DNA damage.
The eukaryotic genome is packaged into a nucleoprotein
structure known as chromatin. The basic repeating unit of
chromatin, the nucleosome, consists of approximately two
turns of DNA wrapped around an octamer of core histone
proteins (34). This association plays a fundamental role in
regulating gene expression (for recent reviews, see references
42 and 58). Chromatin structure modulates the access of pro-
teins to DNA and is therefore likely to regulate other aspects
of DNA processing, including the repair of double-strand
DNA damage and DNA replication (15, 36, 40).
Cell survival and maintenance of genomic integrity are de-
pendent on the efficient and accurate repair of DNA double-
stand breaks. Double-strand breaks occur when DNA replica-
tion forks stall (7), in response to exogenous DNA-damaging
agents or as a programmed event during growth or develop-
ment (19, 47). The repair of double-strand breaks depends on
the DNA damage checkpoint that detects and signals the pres-
ence of DNA damage and arrests cell cycle progression until
the damage is repaired (67). In budding yeast Saccharomyces
cerevisiae the DNA damage checkpoint is initiated by the in-
dependent localization of two checkpoint complexes to sites of
DNA damage. Rad24p forms an RFC-like complex with
Rfc2p-5p and loads the PCNA-like complex of Rad17p,
Mec3p, and Ddc1p at the site of the DNA lesion (27, 39, 48).
Independently, the PI3-family kinase ATR homolog Mec1p
and its binding partner, the ATRIP homolog Ddc2p, are re-
cruited to the DNA lesion in an RPA-dependent manner (27,
39, 69). Mec1p recruitment leads to the phosphorylation of
histone H2A (or histone variant H2AX in mammals) on serine
129 in the chromatin flanking the lesion (10). Once recruited to
the DNA, Mec1p phosphorylates the mediator kinase Mrc1p
in response to DNA replication stress and Rad9p in response
to double-strand DNA lesions (3, 14, 43). The key downstream
target of Mrc1p and Rad9p is the effector kinase Rad53p (3,
14). Rad53p is important for maintaining nucleotide levels
necessary for replication, stabilizing stalled replication forks
and preventing the degradation of Pds1p (which leads to cell
cycle arrest at the metaphase to anaphase transition) (44).
Rad53p has also been implicated in maintaining proper his-
tone levels during DNA replication, providing a link between
chromatin assembly and DNA replication (17).
Replication defects are the major source of spontaneous
genomic instability in the cell and the DNA damage check-
point is the principal defense against such instability (44, 65).
When a replication fork pauses or stalls, it is either stabilized
and restarted by proteins involved in the DNA damage check-
point response or the stalled replication fork reverses to form
so-called “chicken-feet” structures that may lead to deleterious
recombination events (44, 65). A balance exists between pro-
cessing stalled replication forks via restarting or via recombi-
nation. This is seen by the fact that the mutation of proteins
that stabilize stalled replication forks or that promote progres-
sion of replication forks leads to elevated levels of recombina-
tion, which is dependent on the RAD52 epistasis group of
The chromatin assembly factors that package histones and
DNA together into nucleosomes have recently been linked to
DNA repair and the DNA damage response. The histone
chaperones known as anti-silencing function 1 (ASF1) and
chromatin assembly factor 1 (CAF-1) deposit histones H3 and
H4 onto newly replicated DNA in vitro (51, 59). Yeast with
* Corresponding author. Mailing address: Department of Biochem-
istry and Molecular Genetics, University of Colorado Health Sciences
Center at Fitzsimons, P.O. Box 6511, Aurora, CO 80045. Phone: (303)
724-3224. Fax: (303) 724-3221. E-mail: email@example.com.
ASF1 deleted are highly sensitive to double-strand DNA-dam-
aging agents (5, 28, 59), whereas yeast with CAF-1 deleted are
highly sensitive to UV irradiation (26). The increased sensitiv-
ity of chromatin assembly factor mutants to DNA-damaging
agents may reflect a direct role for these factors in modulating
chromatin structure during DNA repair. For example, human
Asf1p cooperates with CAF-1 to assemble nucleosomes after
nucleotide excision repair in vitro (38). In addition, a molec-
ular connection between Asf1p and genomic stability has been
provided by the identification of a dynamic interaction be-
tween Asf1p and the Rad53p DNA damage checkpoint protein
(11, 24). The interaction between Asf1p and Rad53p down-
regulates the chromatin assembly activity of Asf1p. Upon
phosphorylation of Rad53p by the DNA damage checkpoint,
Asf1p is released, allowing it to bind histones and modify the
chromatin structure (11, 24). These results suggest that the
activation of Asf1p may be an important cellular response to
DNA damage and replicational stress (11, 24).
Chromatin assembly factors have also been implicated in the
maintenance of genomic integrity during normal growth. For
example, expression of a dominant-negative form of the largest
subunit of CAF-1 in human cells leads to DNA damage and
activation of the S-phase checkpoint (64). Similarly, deletion of
ASF1 or the genes encoding CAF-1 increases gross chromo-
somal rearrangements in yeast (41). These results taken to-
gether suggest that defects in chromatin assembly induce dou-
ble-strand DNA damage. Despite the evidence linking the
chromatin assembly factor Asf1p to genomic stability, the
mechanism whereby chromatin structure affects genomic in-
tegrity is unclear. Here we show that the slow growth rate of
yeast lacking Asf1p is due to activation of the DNA damage
checkpoint as a consequence of increased levels of spontane-
ous DNA damage and recombination occurring during DNA
replication. We propose that the altered chromatin structure in
asf1 mutants causes genomic instability as a consequence of
elevated levels of DNA repair events occurring during S phase.
MATERIALS AND METHODS
Yeast molecular genetics. The yeast strains examined here are listed in Table
1. All yeast strains were haploid derivatives of W303-1 except strains BOB461
and JRY009, which are A364A, and BOB852 and NW020, which are S288C. The
endogenous DDC2 gene product was epitope tagged at its C terminus with an
enhanced green fluorescent protein (EGFP) cassette by using a previously de-
scribed approach (33). Cells were grown in complete media at 30°C to mid log
phase prior to all analyses unless indicated otherwise. Standard yeast genetic
manipulations and media were used.
Flow cytometry analysis. For cell cycle analysis, ?5 ? 106cells for each sample
were stained with propidium iodide as described previously (52). 10,000 cells
from each sample were scanned with a Beckman Coulter XL-MCL machine.
Micromanipulation of yeast. Strains were grown overnight to mid-log phase in
yeast extract-peptone-dextrose (YPD) media. Cultures were diluted 20-fold and
spotted onto YPD plates. Individual cells representing various stages of the cell
cycle were micromanipulated onto a grid. Growth and division were visualized
microscopically over a period of 48 h at 23°C by using a ?20 magnification
Western blot analysis of checkpoint proteins. Western analysis of checkpoints
proteins was performed as described previously (8). Antibodies for Rad53p and
Rad9p were kindly provided by Noel Lowndes.
Growth curve analysis. Yeast cultures were grown in yeast extract-peptone-
raffinose (YEPR) plus 1% galactose to a Klett reading of 30 to induce expression
of Asf1p in the conditional ASF1 and then switched to YPD medium to repress
expression of Asf1p. Klett readings were taken every hour, and the cultures were
diluted back to a Klett reading of 30 whenever any culture grew to a reading of
more than 100 to keep the cells in mid-log phase. Dilution factors were multi-
plied back into the Klett readings to obtain the final Klett units. Growth readings
were taken by using a Klett-Summersen photoelectric colorimeter (model 800-3).
Analysis of Ddc2p foci formation. Logarithmically growing cells were fixed and
processed for GFP fluorescence as described previously (39). Images were cap-
tured with a Nikon E800 epifluorescence microscope with a Gene-Snap cooled
charge-coupled device camera and Metamorph imaging software.
Pulsed-field gel electrophoresis analysis. Chromosome-sized DNA fragments
were immobilized in low-melting-point agarose and processed for resolution by
pulsed-field gel electrophoresis as described previously (18).
HO repair viability assay. Induction of HO in liquid culture was achieved by
growing cells in YEPR at 30°C and adding galactose (final concentration of 2%).
Samples were removed at various times after galactose addition, sonicated and
then counted on a hemocytometer to determine cell concentrations, followed by
dilution and spreading of ca. 400 cells onto YEP-glucose to repress HO expres-
sion and determine the number of viable cells. The plates were scored after 3
days growth at 30°C.
PCR analysis of mating-type switching. Primers flanking the HO site in the
MAT locus were used to determine mating type by PCR amplification (primer
sequences are available upon request). Cultures were grown overnight in raffin-
ose-containing medium. Galactose and then glucose were added to 2% at the
times indicated in the figure legends. Primers to the RAD27 gene were included
in the multiplex PCR as an internal control. The number of PCR cycles to
produce amplification in the linear range was determined empirically. The ratio
of the MAT product to the control product was quantified by using Labworks
(GelPro4.0; Media Cybernetics, LP).
Analysis of resection during homologous recombination. Measurement of
mating-type switching was performed as described previously (53). Briefly, pu-
rified genomic DNA was digested with StyI, and fragments were resolved on an
alkaline denaturing gel. The Southern blot was probed with a fragment that
overlaps the StyI fragment containing the HO cleavage site of MAT and the next
most distal fragment.
Analysis of mutation frequency. The rate of accumulation of Canrmutants in
cell populations was determined by fluctuation analysis by using the method of
the median (29) as described previously (35). Each fluctuation test was repeated
independently at least two times. Independent Canrmutants were isolated by
streaking strains JKM179 (WT), YM001 (asf1?), and YM003 (msh2?) to single
colonies on YPD plates. Then, single colonies were resuspended in water and
diluted as appropriate onto YPD and ?ARG?canavanine plates; mutation of
the CAN1 gene allows growth on ?ARG?canavanine plates. The results are
expressed as the mean ? the standard deviation of multiple independently
derived mutation rates.
Recombination assay. The rate of sister chromatid exchange (SCE) in cell
populations was determined by fluctuation analysis by using the method of the
median (29) as described previously (35). Each fluctuation test was repeated
independently at least two times. A strain containing the SCE substrate of
3??-his3 5??his3 that forms a functional HIS3 upon recombination was kindly
provided by Michael Fasullo. Yeast that had undergone independent recombi-
nation events were isolated by streaking strains YD122 (WT) and JRY013
(asf1?) to single colonies on YPD plates. Single colonies were then resuspended
in water and diluted as appropriate on YPD and plates lacking histidine. The
results are expressed as the mean ? the standard deviation of multiple indepen-
dently derived mutation rates.
asf1 mutants have a metaphase-anaphase cell cycle defect.
We set out to determine why yeast lacking Asf1p grow slowly
(59). Flow cytometry analysis demonstrated that asynchronous
cultures of yeast deleted for the ASF1 gene (asf1?) accumulate
with a G2/M DNA content compared to an isogenic wild-type
culture (Fig. 1A). To determine the stage in G2/M at which
asf1 mutant cells arrest, we performed microscopic analysis of
the DNA and bud size on a population basis. We found that
deletion of ASF1 increases the proportion of cells in meta-
phase, defined as large budded cells with one mass of DNA at
the bud neck, from 15% in wild-type cultures to ca. 40% of the
population in asynchronous asf1? cultures (Fig. 1B). These
cells also had short mitotic spindles pointing toward the bud
neck, as determined by immunofluorescence analysis with an
10314RAMEY ET AL.MOL. CELL. BIOL.
anti-tubulin antisera (data not shown), a finding consistent
with being in metaphase. Taken together, these results dem-
onstrate that asf1 mutants accumulate in metaphase of the cell
To gain further evidence of a cell cycle defect in cells lacking
Asf1p, we examined the growth of individual cells. Cells were
micromanipulated onto a grid on an agar plate, and cell growth
and division were observed (Fig. 1C). Wild-type cells had pro-
gressed through multiple divisions by 18 h. In contrast, asf1?
cells had only progressed through three to five divisions by
18 h. Notably, many of the asf1? cells were greatly enlarged
compared to the wild type (Fig. 1C), where enlarged cells
reflect continued growth during cell cycle arrest. The enlarged
size of cells lacking Asf1p is also apparent from the broadening
of the DNA peaks in our flow cytometry analyses (Fig. 1A).
Taken together, these data clearly indicate that loss of Asf1p
leads to cell cycle defects.
Loss of Asf1p activates the DNA damage checkpoint. We
had previously observed that asf1 mutants are highly sensitive
to double-strand DNA-damaging agents (59). To determine
TABLE 1. Yeast strains used in this study
MATa ade2-1 can1-100 his3-11 leu2-3,112 trp1-1 ura3-1 GAL ade3::Gal10:HO
MATa trp1-1 ura3-1 can1-100 ADE bar::LEU2 his3-11 GAL rad52::TRP1 [pGAL-HO URA3]
MATa ?lys2 asf1::his5?bar1::LEU2 TELVIIL::URA3 trp1-1 his3-11 leu2-3,112 can1-100
MATa rad52::TRP1 trp1-1 ura3-1 can1-100 ADE bar1::LEU2 his3-11
MATa asf1::his5?rad52::TRP bar1::LEU2 leu2-3,112 his3-11 can1-100 ?lys2 TELVIIL::URA3 trp1-1
MATa his3-11 leu2-3,112 lys2 trp1-1 ura3-1 bar1::LEU2
MATa his3-11 leu2-3,112 lys2 trp1-1 ura3-1 bar1::LEU2 pds1::18MYC-TRP
MATa his3-11 leu2-3,112 lys2 trp1-1 ura3-1 bar1::LEU2 pds1::18MYC-TRP asf1::his5?
MATa his3-11 leu2-3,112 lys2 trp1-1 ura3-1 bar1::LEU2 pds1::18MYC-TRP rad9::URA3
MATa his3-11 leu2-3,112 lys2 trp1-1 ura3-1 bar1::LEU2 pds1::18MYC-TRP rad24::Kan
MATa his3-11 leu2-3,112 lys2 trp1-1 ura3-1 bar1::LEU2 pds1::18MYC-TRP asf1::his5?rad9::kan
MATa his3-11 leu2-3,112 lys2 trp1-1 ura3-1 bar1::LEU2 pds1::18MYC-TRP rad24::Kan asf1::his5?
MATa his3-11 leu2-3,112 lys2 trp1-1 ura3-1 bar1::LEU2 pds1::18MYC-TRP asf1::his5?rad9::URA3 mrc1::kan
MATa his3-11 leu2-3,112 lys2 trp1-1 ura3-1 bar1::LEU2 pds1::18MYC-TRP rad9::URA3 mrc1::kan
MATa his3-11 leu2-3,112 lys2 trp1-1 ura3-1 bar1::LEU2 pds1::18MYC-TRP asf1::his5?mrc1::kan
MATa his3-11 leu2-3,112 lys2 trp1-1 ura3-1 bar1::LEU2 pds1::18MYC-TRP mrc1::kan
MATa his3-11 leu2-3,112 lys2 trp1-1 ura3-1 bar1::LEU2 sml1::HPH rad53::kan asf1::his5?
MATa his3-11 leu2-3,112 lys2 trp1-1 ura3-1 bar1::LEU2 sml1::HPH mec1::kan
MATa his3-11 leu2-3,112 lys2 trp1-1 ura3-1 bar1::LEU2 sml1::HPH mec1::kan asf1::his5?
MAT? ?hmla::ADE1 ?HO::ADE1 ?hmr::ADE1 ade1 leu2-3,112 lys5 ura3-52 ade3::Gal10:HO
MATa his3-11 leu2-3,112 lys2 trp1-1 ura3-1 bar1::LEU2 sml1::HPH
MATa his3-11 leu2-3,112 lys2 trp1-1 ura3-1 bar1::LEU2 sml1::HPH asf1::his5?
MATa his3-11 leu2-3,112 lys2 trp1-1 ura3-1 bar1::LEU2 sml1::HPH rad53::kan
MATa his3-11 leu2-3,112 lys2 trp1-1 ura3-1 bar1::LEU2 ddc2::GFP-kan
MATa his3-11 leu2-3,112 lys2 trp1-1 ura3-1 bar1::LEU2 ddc2::GFP-kan asf1::his5?
MATa his3-11 leu2-3,112 lys2 trp1-1 ura3-1 bar1::LEU2 ddc2::GFP-kan rad52::TRP
MAT? leu2 trp1 lys2 pol1-17 asf1::kan
MATa his3-11 leu2-3,112 lys2 trp1-1 ura3-1 bar1::LEU2 mrc1::13myc-Kan
MATa his3-11 leu2-3,112 lys2 trp1-1 ura3-1 bar1::LEU2 mrc1::13myc-Kan asf1::his5?
MATa trp1::his3-?3? his3?5? URA3 asf1::KanMX
MATa hta1-htb1::LEU2 hta2-htb2::TRP1 leu2-1 ura3-52 his3-200 (hta1S129A-HTB1 CEN-HIS3) asf1::Kan
MATa hta1-htb1::LEU2 hta2-htb2::TRP1 leu2-1 ura3-52 his3-200 asf1::Kan (HTA1-HTB1 CEN-HIS3)
MAT? leu2 trp1 his7 cdc17-1 asf1::Kan
MATa ade2-1 can1-100 his3-11 leu2-3,112 trp1-1 ura3-1
This studySRH014MATa kanMX6-PGAL1-ASF1-3HA-PEST-HIS3 bar1::LEU2 can1-100 gal1::hisG his3-11 leu2-3,112 ?lys2
MATa ASF1-3HA-kanMX6 bar1::LEU2 can1-100 gal1::hisG his3-11 leu2-3,112 ?lys2 trp1-1 ura3-1
MATa asf1::kanMX6 bar1::LEU2 can1-100 gal1::hisG his3-11 leu2-3,112 ?lys2 trp1-1 ura3-1
MAT? ade2-1 LYS2 leu2-3,112 his3-11 trp1-1 ura3-1 asf1::his5? HMRa::ADE2 TELVIIL::URA3 can1-100
MAT? ade2-1 LYS2 leu2-3,112 his3-11 trp1-1 ura3-1 HMRa::ADE2 TELVIIL::URA3 can1-100
MAT? ?hmla::ADE1 ?HO::ADE1 ?hmr::ADE1 ade1 leu2-3,112 lys5 ura3-52 ade3::Gal10:HO asf1::Kan
MAT? ?hmla::ADE1 ?HO::ADE1 ?hmr::ADE1 ade1 leu2-3,112 lys5 ura3-ade3::Gal10:HO msh2::Kan
MATa ade2-1 can1-100 his3-11 leu2-3,112 trp1-1 ura3-1 asf1::Kan
MAT? leu2 trp1 his7 cdc17-1
MAT? leu2 trp1 lys2 pol1-17
MATa cdc13 ura3 his3 can1 trp1
MAT? met15 leu2 ura3 his3
MAT? met15 leu2 ura3 his3 asf1:kanMX6
MATa trp1::his3-?3? his3-?5? URA3
MAT? cdc16-123 bar1 trp1-1 can1-100 his3-11,15 leu2-3,112 ura3 GAL
MATa ade2-1 can1-100 his3-11 leu2-3,112 trp1-1 ura3-1 ?asf1::KanMX
MATa hta1-htb1::LEU2 hta2-htb2::TRP1 leu2-1 ura3-52 his3-200 (HTA1-HTB1 CEN-HIS3)
MATa hta1-htb1::LEU2 hta2-htb2::TRP1 leu2-1 ura3-52 his3-200 (hta1S129A-HTB1 CEN-HIS3)
Tyler et al. (59)
Tyler et al. (59)
VOL. 24, 2004ALTERED CHROMATIN STRUCTURE CAUSES GENOMIC INSTABILITY10315
whether the metaphase-anaphase delay of yeast lacking Asf1p
is due to activation of the DNA damage checkpoint, we mea-
sured the bud index for yeast deleted for ASF1 in combination
with DNA damage checkpoint mutants. Deletion of MEC1 and
RAD53, the central kinases in all genomic integrity checkpoint
pathways, rescued the accumulation of asf1? cells in meta-
phase (Fig. 2A). Note that SML1 is also deleted in these strains
to suppress the requirement for Rad53p and Mec1p in regu-
lating nucleotide levels (66). Deletion of the gene encoding the
Rad24p DNA damage checkpoint protein from asf1? cells also
prevented the accumulation in metaphase (Fig. 2B). Similarly,
deletion of the RAD9 gene encoding, the mediator kinase that
is activated in response to double-strand lesions, from asf1?
cells prevented the accumulation in metaphase (Fig. 2B).
These results indicate that the delay in the metaphase to an-
aphase transition in asf1 mutants is dependent on DNA dam-
age checkpoint proteins.
The accumulation of yeast lacking Asf1p in metaphase ap-
pears to be wholly due to activation of the DNA damage
checkpoint. Activation of the spindle assembly checkpoint can
also leads to metaphase arrest (30), and yet deletion of the
MAD2 component of the spindle assembly checkpoint failed to
rescue the metaphase accumulation defect of asf1? cells (data
not shown). Furthermore, by using a GFP-tagged minichromo-
FIG. 1. Yeast with ASF1 deleted have a metaphase-anaphase transition defect. (A) asf1 mutants accumulate in G2/M phase. Results from flow
cytometry analysis of DNA content of JKT0040 (WT) and JKT0041 (asf1?) asynchronous cultures are shown. (B) asf1 mutants accumulate in
metaphase. Budding indices of JKT0040 (WT) and JKT0041 (asf1?) asynchronous cultures are shown. The diagrams below each set of columns
indicate the relative size of the bud and position of the nuclear DNA. The data shown are the averages and standard deviations of three
independent blind counts of 100 cells for each strain. (C) asf1 mutant cells grow slowly. Individual cells from JKT0040 (WT) and JKT0041 (asf1?)
strains were micromanipulated onto a grid of agar and photographed immediately and 18 h later. The magnification was identical for all panels.
10316 RAMEY ET AL.MOL. CELL. BIOL.
some system (37), we found no increase in minichromosome
missegregation in asf1? cells compared to wild-type cells (data
not shown). Consistent with our results, yeast that are deleted
for both ASF1 and the gene encoding the large subunit of the
CAF-1 chromatin assembly factor have been reported to show
a G2/M arrest independent of spindle assembly checkpoint
activation and segregate a minichromosome at frequencies
similar to wild-type cells (50).
To gain molecular proof that the mitotic delay in cells lack-
ing Asf1p is due to activation of the DNA damage checkpoint
pathway, we looked for phosphorylation of DNA damage
checkpoint kinases. We confirmed that the Rad53p DNA dam-
age checkpoint protein is activated in the absence of DNA-
damaging agents in asf1? cells but not in wild-type cells (Fig.
3A), as published previously (24). As a reference for the
amount of Rad53p activation that occurs if the spontaneous
DNA damage occurring during DNA replication is not re-
paired, we compared Rad53p activation in asf1? and rad52?
FIG. 2. Loss of Asf1p activates the DNA damage checkpoint.
(A) Deletion of MEC1 or RAD53 fixes the metaphase defect of asf1?
cells. Budding index of JKT0040 (WT), JKT0041 (asf1?), JKT0186
(mec1?), JKT0187 (asf1? mec1?), JRY003 (rad53?), and JKT0185
(asf1? rad53?) strains was determined as described in Fig. 1B. (B) De-
letion of RAD24 or RAD9 fixes the metaphase defect of asf1? cells.
Budding index of JKT0040 (WT), JKT0041 (asf1?), JKT0042 (rad9?),
JKT0044 (asf1? rad9?), JKT0043 (rad24), and JKT0045 (asf1?
rad24?) were determined as described for Fig. 1B.
FIG. 3. The loss of Asf1p activates the DNA damage checkpoint.
(A) Rad53p is activated in asf1 mutants. Western blot analysis of the
DNA damage checkpoint kinase Rad53p was done in total protein
extracts derived from JKT0040 (WT), JKT0041 (asf1?), JKT0004
(rad52?), JRY003 (rad53?), and JKT0045 (rad24? asf1?) and strains,
and WT and asf1? strains treated with 0.2% MMS for 1 h. Activation
of Rad53p is determined by phosphorylation and is apparent by the
higher-molecular-weight bands that are indicated as “Rad53p-P.” (B)
Mrc1p is activated in asf1 mutants. Western blot analysis of the 13-myc
tagged DNA replication checkpoint protein Mrc1p was done in total
protein extracts derived from strains JRY010 (WT), JRY011 (asf1?),
a wild-type strain treated with 200 mM hydroxyurea for 2 h, and strain
JKT0004 (no tag). Activation of Mrc1p was determined by phosphor-
ylation and is apparent by the higher-molecular-weight bands that are
indicated as “Mrc1p-P.” (C) Deletion of both RAD53 and ASF1 leads
to severe growth defects. Strains JRY001 (sml1?), JRY003 (sml1?
rad53?), JRY002 (sml1? asf1?), and JKT0185 (sml1? asf1? rad53?)
were streaked onto YPD and grown for 3 days before photographing
them. (D) Mutation of serine-129 of H2A, together with deletion of
ASF1, leads to severe growth defects. Strains JKY001 (WT), JKY002
(H2AS129A), JRY015 (asf1?), and JRY014 (H2AS129A asf1?) were
streaked onto YPD and grown for 3 days before being photographed.
VOL. 24, 2004 ALTERED CHROMATIN STRUCTURE CAUSES GENOMIC INSTABILITY10317