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: firstname.lastname@example.org.
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
10314 RAMEY 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 studySRH014 MATa 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 INSTABILITY 10315
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
strains. We found that the extent of Rad53p activation in asf1?
cells is very similar to that observed previously in asf1 mutant
cells (24) and is similar to the extent of Rad53p activation that
occurs in a rad52? strain that is unable to perform any homol-
ogous recombination (Fig. 3A). This result indicates that
Rad53p is activated in asf1? cells at levels similar to cells that
are unable to repair spontaneous double-strand DNA damage.
As expected, we found that deletion of RAD24, MEC1, or
RAD9, but not deletion of MRC1, prevented activation of
Rad53p due to endogenous DNA damage in asf1? cells (Fig.
3A and data not shown). This lack of requirement for Mrc1p
for Rad53p activation reflects the fact that Rad9p is recruited
to stalled replication forks in mrc1 mutants, resulting in
Rad53p activation (25). To address whether replicational
stress is activating the DNA damage checkpoint, we examined
whether Mrc1p is phosphorylated in asf1 mutants, where
Mrc1p phosphorylation is a specific response to replicational
stress. We observed a shift upward of the Mrc1p band corre-
sponding to phosphorylation in the asf1? strains that was ab-
sent in the wild-type strain (Fig. 3B). This result indicates that
the DNA damage checkpoint is activated in response to in-
creased replicational stress in cells lacking Asf1p.
To investigate whether activation of the DNA damage
checkpoint is necessary to protect asf1? cells from exiting
mitosis with catastrophic DNA damage, we examined micro-
colony formation. We found that deletion of both ASF1 and
the DNA damage checkpoint gene RAD53, MEC1, or RAD9
greatly reduces viability compared to each individual mutation.
An example of the reduced viability of cells deleted for both
ASF1 and a DNA damage checkpoint component is shown in
Fig. 3C for the Rad53p checkpoint kinase. We also observed
greatly reduced viability when we deleted ASF1 in combination
with a mutation in histone H2A that prevents its phosphory-
lation in response to DNA damage (Fig. 3D). The low viability
of the asf1? H2AS129A double mutant indicates that phos-
phorylation of H2A in response to DNA damage is important
in the absence of Asf1p. Our results indicate that the absence
of Asf1p increases the requirement for the DNA damage
checkpoint and that failure to arrest at the DNA damage
checkpoint leads to increased cell death in asf1 mutants.
asf1 mutants are sensitive to drugs and DNA polymerase
mutants that induce replicational stress. To investigate
whether the activation of the DNA damage checkpoint in asf1
mutants is a consequence of DNA replication stress, we exam-
ined their sensitivity to agents that induce replicational stress.
Consistent with this idea, we have previously observed that asf1
mutants are sensitive to hydroxyurea, which depletes the de-
oxynucleoside triphosphate pools and leads to stalling of rep-
lication forks (59). We found that loss of Asf1p also greatly
increases sensitivity to camptothecin (Fig. 4A). Camptothecin
inhibits the ability of topoisomerase 1 to religate the single-
strand breaks that it creates to relieve tension ahead of the
replication fork, leading to double-strand DNA damage during
DNA replication (23). Similarly, asf1 mutants are extremely
sensitive to cisplatin (Fig. 4B), which generates intrastrand
DNA cross-links that cause the replication fork to stall (13).
The problems generated by hydroxyurea, camptothecin, and
cisplatin during DNA replication can be fixed eventually by
DNA repair mechanisms. Therefore, to address directly
whether asf1 mutants have DNA replication problems rather
than DNA repair problems, we examined mutations of DNA
polymerase. We found that deletion of ASF1 lowered the re-
strictive temperature of the DNA polymerase alpha mutants,
pol1-1 and cdc17-1 (Fig. 4C). These results indicate that the
absence of Asf1p exasperates DNA replication problems.
During the course of our analyses, we generated a mutant
with both ASF1 and RAD52 deleted, where the Rad52 protein
is an essential component of the homologous recombination
machinery. We found that this double mutant was very sick
compared to either single mutant (Fig. 4D). This result sug-
gests that in the absence of Asf1p, cells have an increased
FIG. 4. Cells lacking Asf1p are sensitive to replicational stress.
(A) asf1 mutants are sensitive to camptothecin. Cultures of JKT0040
(WT) and JKT0041 (asf1?) were serial diluted (10-fold), spotted onto
YPD and YPD plus 1 or 2.5 mg of camptothecin/ml, and grown for 3
days at 30°C. (B) asf1 mutants are sensitive to cisplatin. Cultures of
JKT0040 (WT) and JKT0041 (asf1?) were serial diluted (10-fold),
spotted onto YPD and YPD plus 0.5 or 1 mg of cisplatin/ml, and grown
for 3 days at 30°C. (C) Deletion of ASF1 reduces the permissive
temperature of polymerase alpha mutants. Cultures of JKT0040 (WT),
BOB461 (pol1-17ts), JRY009 (asf1? pol1-17), BOB460 (cdc17-1),
JRY016 (asf1? cdc17-1), and JKT0041 (asf1?) were serial diluted
(10-fold), spotted onto YPD, and incubated at 27, 30, or 34°C for 3 to
4 days. (D) Synthetic sickness upon deletion of both ASF1 and RAD52.
Strains JKT0010 (WT), JKT0001 (asf1?), JKT0005 (asf1? rad52?),
and JKT0004 (rad52?) were struck out, grown at 30°C for 3 days, and
10318 RAMEY ET AL.MOL. CELL. BIOL.
requirement for the homologous recombinational machinery.
This result is particularly interesting because it is the homol-
ogous recombinational machinery that rescues cells that have
experienced replication problems.
The cell cycle defect of yeast lacking Asf1p is not due to
incomplete DNA replication. Next, we sought to determine
whether the metaphase-anaphase transition defect in cells
lacking Asf1p is a consequence of incomplete DNA replica-
tion. This question was relevant because we have now shown
that asf1? cells are sensitized to DNA replication problems,
and it was possible that the accumulation of asynchronous
asf1? cells with a 2C DNA content may reflect late-S-phase
cells rather than metaphase cells. To determine whether asf1
mutants have completed DNA replication, we added the DNA
synthesis inhibitor hydroxyurea to a yeast strain that had its
endogenous copy of ASF1 under the control of the galactose-
inducible GAL1 promoter. The addition of hydroxyurea to
cells that had been arrested before completion of replication,
such as cdc13-1 mutants, causes immediate arrest with a 2C
DNA content upon switching to the permissive temperature
(62) (Fig. 5A). In contrast, the addition of hydroxyurea to
metaphase-arrested cells with completely replicated chromo-
somes, such as cdc16-123 mutants, allows progression through
mitosis to the subsequent S phase upon switching to the per-
missive temperature (Fig. 5B). Addition of hydroxyurea to
cells lacking Asf1p at the same time that Asf1p was induced by
the addition of galactose permitted the cells that had accumu-
lated with a 2C DNA content to exit mitosis and arrest as they
entered the subsequent S phase (Fig. 5C). This experiment
indicated that the accumulation of cells lacking Asf1p with a
2C DNA content is not due to incomplete DNA replication
and therefore reflects arrest in metaphase rather than late S
phase. No significant difference was seen whether Asf1p was
induced or not at the time of hydroxyurea addition (Fig. 5C
and D) or in cells where ASF1 was deleted (data not shown),
demonstrating that Asf1p is not required for passage from
metaphase to the subsequent S phase. By a process of elimi-
nation, these data indicate that the functions of Asf1p during S
phase and/or G2phase of the cell cycle leads to metaphase
The cell cycle defect of asf1 mutants is due to the accumu-
lation of defects. To gain further insight into Asf1p function,
we investigated how rapidly or slowly the problems associated
with loss of Asf1p lead to the cell cycle defect. To do this, we
used a yeast strain in which an unstable copy of Asf1p is
expressed from the galactose-inducible and glucose-repressible
pGAL1 promoter that results in the disappearance of any
detectable Asf1p within minutes of its transcriptional repres-
sion (S. Zabaronick and J. Tyler, submitted for publication).
After repression of ASF1, the passage of 20 h was required
before the growth rate of yeast lacking Asf1p switched from
the growth rate of wild-type yeast to that of asf1? yeast (Fig.
5B). This distinct switch in growth rate at 20 h is apparent from
comparison of the culture growth between each time point
(Fig. 5C). These data indicate that a threshold of molecular
defects has to be reached before the slow-growth phenotype
occurs after removal of Asf1p.
Loss of Asf1p leads to genomic instability. To better under-
stand the role of Asf1p in chromosomal integrity, we wanted to
distinguish between activation of the DNA damage checkpoint
in asf1? cells as a consequence of altered chromatin structure
or by DNA lesions. Activation of the DNA damage checkpoint
by disrupted chromatin structure in human cells is not accom-
panied by the formation of DNA repair foci (4). Therefore, we
examined whether asf1 mutants have more spontaneous DNA
repair foci than wild-type cells by looking for foci of Ddc2p
tagged with GFP (39). DNA repair foci are “factories” in
which multiple DNA lesions, DNA damage checkpoint pro-
teins, and DNA repair proteins come together to mediate
repair (31, 32). We observed that 5% of wild-type cells have
spontaneous Ddc2p-GFP foci, a finding consistent with pub-
lished data (39) (Fig. 6A and B). In contrast, 20% of asf1?
yeast had spontaneous Ddc2p-GFP foci (Fig. 6B). For refer-
ence, 15% of rad52? cells that are incapable of repairing dam-
age by homologous recombination had Ddc2p-GFP foci (Fig.
6B). The Ddc2p foci in asf1? cells were found primarily in
small budded (S-phase) (45%) or large budded (G2/M-phase)
(55%) cells (Fig. 6C). These data demonstrate that asf1? cells
have elevated amounts of DNA lesions due to problems during
Asf1p is not required for repair of double-strand DNA dam-
age. To determine whether the increased number of DNA
damage foci in asf1? cells is due to the failure of asf1? cells to
repair DNA damage or due to increased incidence of DNA
lesions, we examined the ability of asf1? cells to perform dou-
ble-strand DNA repair. First, we analyzed the integrity of chro-
mosomes isolated from asf1? and wild-type cells at increasing
times after removal of methyl methanesulfonate (MMS). MMS
is a DNA alkylating agent that results in both single and dou-
ble-strand DNA breaks (49). After a short treatment (5 min)
with MMS, double-strand DNA damage was apparent from
the smearing of the chromosomes, as detected by pulsed-field
electrophoresis (Fig. 7A). The chromosomes of both wild-type
and asf1? cells were distinct bands again after 1 and 3 h after
the removal of MMS, indicating repair of double-strand DNA
lesions (Fig. 7A). In parallel, we measured the viability of the
cells that were used for chromosome isolation and found no
significant changes in viability (as determined by growth on
YPD) for each of the wild-type and asf1? samples before,
during, or after MMS treatment (data not shown). This result
indicates that the reappearance of the intact chromosomes
(Fig. 7A) was indeed due to DNA repair. Therefore, we con-
clude that Asf1p is not required for religation of double-strand
breaks on a chromosomal scale.
In order to examine the repair of a defined double-strand
break, we sought to determine whether Asf1p was required for
the repair of an HO lesion at the MAT locus by homologous
recombination (Fig. 7B). At increasing times after induction of
the HO endonuclease (under the control of the GAL1 pro-
moter), cells were plated onto glucose to repress the HO en-
donuclease and their viability was determined. Viability is de-
pendent on the repair of the HO lesion by homologous
recombination. As a control for a strain that is defective for
repair of the HO lesion, the viability of rad52? cells was greatly
reduced (Fig. 7B). We found no significant difference in cell
viability between asf1 mutants and wild-type cells after HO
induction, suggesting that asf1? cells are competent to repair
the HO lesion (Fig. 7B).
To evaluate the ability of yeast lacking Asf1p to perform
homologous recombination at the molecular level, we moni-
VOL. 24, 2004 ALTERED CHROMATIN STRUCTURE CAUSES GENOMIC INSTABILITY10319
tored repair of the HO lesion. To do this, we used a quanti-
tative PCR-based analysis of the MAT region to monitor the
cutting and repair of the HO lesion (Fig. 8A) (B. Tamburini
and J. Tyler, submitted for publication). We used a primer pair
that would produce a product unique to MAT that would differ
by 0.1 kb between MATa and MAT? cells (Fig. 8A). A MAT
locus with an HO lesion will yield no MAT PCR product. In
general, MATa cells are repaired by using the information at
HML? to yield MAT? yeast, whereas MAT? yeast are repaired
by using the information at HMRa to yield MATa yeast (Fig.
8A) (63). This PCR-based approach generated the same re-
sults as Southern analysis (data not shown) but was more
convenient and easier to internally control. To analyze HO
cleavage and repair, HO endonuclease was induced at 0 h by
addition of galactose, followed by the addition of glucose at 2 h
to repress HO endonuclease. HO cleavage and repair was
apparent in the wild-type strain (Fig. 8B and C). Both mating
types are generated during repair because immediate recleav-
FIG. 5. Cells lacking Asf1p complete DNA replication. (A) cdc13-1 mutation prevents the completion of replication. An asynchronous culture
of logarithmically growing strain BOB463 with a temperature-sensitive cdc13-1 allele was shifted to the nonpermissive temperature (37°C) for 2 h,
leading to arrest in late S phase. Hydroxyurea was added to a 200 mM final concentration at the same time the cells were shifted to a permissive
temperature (23°C) to induce functional Cdc13p. A sample was taken for flow cytometry analysis of the cell cycle distribution immediately (“0
min”) and at every 15 min thereafter, as shown. (B) A cdc16-123 mutation allows completion of replication. The identical manipulations were
performed in parallel as for panel A, with strain 1825-1B carrying the temperature-sensitive cdc16-123 allele. (C) The 2C accumulation of asf1
mutants is not due to incomplete replication. Hydroxyurea was added to 200 mM to strain SRH014 carrying the ASF1 gene under control of the
pGAL1 promoter growing in raffinose medium at 23°C. At the same time as the hydroxyurea addition, galactose was added (1%) to induce Asf1p
expression. No Asf1p is detected during growth of this strain in raffinose, and 1% galactose was empirically determined to induce Asf1p to the same
levels as the endogenous protein. The induction of Asf1p was assayed during this experiment by Western analysis, in which synthesis of Asf1p was
detected within 15 min, and was fully induced by 45 min (data not shown). A sample was taken for flow cytometry analysis of the cell cycle
distribution immediately (“0 min”) and at every 15 min thereafter. (D) Asf1p is not required for passage from metaphase to S phase. The
experiment was performed as described for panel C but without the inclusion of 1% galactose. The flow cytometry profiles of DNA content are
shown, and the black profiles indicate a time point where the result is most apparent between the strains. (E) The growth defect in asf1 mutants
is a result of an accumulation of defects. Strains SRH015 (WT), SRH014 (Cond. ASF1), and SRH016 (asf1?) were grown in YEPR plus 1%
galactose to a Klett reading of 30 and then switched to YPD medium to repress ASF1 transcription in the Cond. ASF1 strain. The Asf1 protein
was not detectable 5 min after the switch to YPD medium. 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 ?100 to keep the cells in mid-log phase. Dilution factors were multiplied back into the
Klett readings to obtain the Klett units plotted above. (F) Growth ratio switch analysis. To determine the time at which the growth rate after
degradation of Asf1p (Cond. ASF1) switches from the growth rate of wild-type yeast (ASF1) to that of asf1 mutant yeast (asf1?), the growth ratio
for each culture was plotted every hour after Asf1p degradation in panel E. The growth ratio is the Klett reading at any given time divided by the
Klett reading at the previous time, reflecting how much the culture grew in each 1-h period. The growth ratio is not absolutely constant, since the
cultures spend a short amount of time outside their ideal growth conditions during the hour that dilution occurred (the time points with the lowest
growth ratios). It is clear that the growth rate after degradation of Asf1p in the Cond. ASF1 strain very closely followed that of the wild-type (ASF1)
strain, until 20 h after degradation of Asf1p, wherein the growth ratio drops to that of the asf1 mutant (asf1?).
10320 RAMEY ET AL.MOL. CELL. BIOL.
age and repair occurs on some templates during the 2 h induc-
tion of HO endonuclease. As a control for a strain that cannot
perform homologous recombination, we examined repair in a
rad52? strain (Fig. 8D and E). Minimal repair occurred in the
rad52? strain and presumably the slight increase in repaired
MAT? product is a result of repair by simple ligation via non-
homologous end joining. We found that the asf1? strain was
able to repair its HO lesion with similar kinetics to the wild-
type strain and with no less than 50% the efficiency of repair
seen in the wild-type strain (Fig. 8F and G). This finding
indicates that Asf1p is not required for homologous recombi-
nation, since homologous recombination can clearly occur in
the absence of Asf1p.
The accessibility of the chromatin structure at the donor
locus is known to influence the strand invasion step of homol-
ogous recombination (53). To investigate whether the chroma-
tin assembly factor Asf1p influences the chromatin structure at
the donor locus, we analyzed the efficiency and intermediates
of HO mating type switching (Fig. 8H). The HO site was
efficiently cut in asf1?, wild-type, and rad52? strains (Fig. 8I).
After transcriptionally repressing the HO endonuclease with
glucose, we found that the asf1? and wild-type cells were
equally capable of mating-type switching (by repairing the HO
lesion at MATa with the HML? sequences), where ca. 50% of
the cells of each strain had switched to MAT? by 2 h. As a
positive control for a gene required for mating-type switching,
the rad52? strain failed to detectably switch to MAT? (Fig. 8I).
Instead, the rad52? strain exhibited extensive formation of
single-strand DNA, where the double-strand break end is re-
sected. The asf1? strain exhibited no greater resection than the
wild-type strain. These results show that Asf1p does not no-
ticeably affect the rate or extent of mating-type switching and
indicate that there is no enhancement or reduction in the
ability of the broken end to invade the chromatin of the donor
sequences in asf1? cells. All of these analyses taken together
demonstrate that Asf1p is not required for the repair of dou-
ble-strand breaks via homologous recombination and, by in-
ference, the increased DNA damage foci in asf1? cells reflect
an increased incidence of DNA damage rather than a failure to
Yeast lacking Asf1p have an elevated rate of spontaneous
mutation and SCE. To determine whether Asf1p and conse-
quently, chromatin structure, are required for genomic stabil-
ity, we determined the spontaneous mutation rates for yeast
disrupted or wild type for ASF1. The approach was to measure
the mutation rate of the endogenous CAN1 gene that encodes
an arginine permease. The product of the wild-type CAN1
gene allows uptake of arginine and the closely related meta-
bolic poison canavanine. Therefore, only cells that have de-
leted or inactivated their CAN1 gene by mutation will be viable
on canavanine plates (16, 35). We found that CAN1 was con-
sistently inactivated in asf1? yeast at a 2.5-fold-higher rate
(1.58 ? 10?7? 1.24 ? 10?7) than CAN1 inactivation in wild-
type yeast (6.39 ? 10?8? 2.24 ? 10?8). Therefore, there is a
significant (P ? 2.03 ? 10?4) increase in mutation rate upon
loss of Asf1p. As a positive control for an extreme example of
an elevated spontaneous mutation rate, we determined in par-
allel the mutation rate of yeast deleted for the MSH2 gene that
is required for mismatch repair. The msh2? strain exhibited a
spontaneous mutation rate of 1.04 ? 10?5—163 times higher
than wild-type cells. To determine what types of mutations had
occurred to increase the mutation rate of asf1? strains, Canr
colonies were assayed for the presence of the CAN1 locus by
PCR. We found that none of the Canrasf1? colonies were due
to deletion of the CAN1 locus. Sequencing of the entire CAN1
gene from multiple independent Canrasf1? colonies indicated
that the spontaneous mutations were nucleotide substitutions
and single nucleotide deletions (data not shown). From this
analysis, we conclude that loss of Asf1p results in an increase
in the rate of spontaneous DNA mutation.
To gain further evidence for a requirement for Asf1p in
maintaining genomic stability after DNA replication, we mea-
sured the rate of spontaneous recombination occurring in
asf1? cells. We used an assay to measure SCE, a recombina-
tion event that is initiated in S phase (2, 9). Wild-type and asf1
mutant strains containing a 5? fragment of HIS3 next to a 3?
fragment of HIS3 were used to quantitate spontaneous SCE by
the formation of a functional HIS3 gene, as described previ-
ously (12). We consistently found that asf1 mutants performed
SCE 2.5 times more frequently (1.71 ? 10?7? 7.42 ? 10?9)
than wild-type yeast (6.96 ? 10?8? 2.5 ? 10?8). Therefore,
there is a significant (P ? 1.20 ? 10?4) increase in recombi-
nation rate upon loss of Asf1p. These data indicate that asf1
mutants have an elevated rate of recombination, suggesting
that abnormal chromatin structures lead to increased recom-
bination during replication.
FIG. 6. Loss of Asf1p increases Ddc2p-GFP foci formation. (A) A typical metaphase asf1? cell with the Ddc2p-GFP focus indicated by the
arrow. A single focus is the site of repair of multiple DNA lesions and the majority of cells with Ddc2p-GFP foci in this analysis contained a single
focus. (B) Proportion of asynchronous cells containing Ddc2p-GFP foci in strain JRY006 (WT), JRY007 (asf1?), and JRY008 (rad52?). (C) Cell
cycle distribution of asf1? cells containing Ddc2p-GFP foci, as determined by bud morphology, where “none,” “small,” and “large” refer to the
VOL. 24, 2004 ALTERED CHROMATIN STRUCTURE CAUSES GENOMIC INSTABILITY10321
We have established that the slow growth of yeast lacking
the Asf1p chromatin assembly factor is due to the activation of
their DNA damage checkpoint. The activation of the DNA
damage checkpoint in asf1 mutants is a consequence of in-
creased genomic instability, as indicated by elevated probabil-
ity of DNA damage foci, elevated recombination rates, and
elevated mutation rates; events that are all initiated during S
phase. We have found that Asf1p is not required for DNA
repair per se, and therefore our data indicate that the altered
chromatin structure in asf1 mutants leads to increased genomic
instability after DNA replication.
Cells lacking Asf1p have increased DNA damage, even
though they can repair DNA damage. Spontaneous DNA dam-
age occurs during DNA replication and is repaired by homol-
ogous recombination. There are two possible models to explain
the striking increase in spontaneous DNA repair foci seen in
asf1 mutants (Fig. 6). First, asf1 mutants may not be capable of
repairing endogenous damage as seen in rad52 mutants or,
second, the loss of Asf1p may increase the occurrence of en-
dogenous DNA damage. Initially, the first possibility seemed
likely because yeast lacking Asf1p are clearly inviable when
plated onto medium containing double-strand DNA-damaging
agents (28, 59). However, we have not been able to detect a
significant requirement for Asf1p in the extent or efficiency of
repair of a defined HO lesion at the MAT locus by homologous
recombination (Fig. 8). It is possible that the recombinational
repair of the HO lesion is not truly representative of general
double-strand break repair since the donor loci at HMR/HML
are present in an inaccessible chromatin structure (19). The
cell may have evolved specialized machinery to gain access to
the buried donor DNA sequences at HMR and HML, such that
repair of the HO lesion at MAT may be less affected by the loss
of Asf1p than double-strand breaks elsewhere in the genome.
However, this does not appear to be the case, since the repair
of MMS-induced double-strand breaks is indistinguishable be-
tween wild-type cells and cells with ASF1 deleted on the chro-
mosomal scale in our pulsed-field gel electrophoresis analyses
Further evidence for a lack of requirement for Asf1p in the
repair of an HO lesion has been provided by Qin and Parthun
(46). Consistent with our results showing that homologous
recombinational repair is intact in asf1 mutant cells, the study
of Qin and Parthun showed at worst a 50% efficiency of HO
repair in an asf1 mutant compared to wild-type cells upon
inspection of their data. Clearly, the subtle, if any, defect in
homologous recombination in asf1 mutants cannot explain the
elevated occurrence of DNA damage foci in the absence of
Asf1p, because even higher levels of DNA damage foci were
observed in an asf1 mutant compared to a rad52 mutant that is
totally defective in homologous recombination (Fig. 6). In sup-
port of this finding, a recent report demonstrated that asf1
mutant cells have an elevated spontaneous rate of occurrence
of Rad52 DNA repair foci in budded cells (45). Therefore, the
elevated occurrence of DNA damage foci in the absence of
Asf1p is likely to reflect a higher incidence of DNA damage
rather than the failure to repair DNA damage. The fact that
the DNA damage checkpoint is activated in cells lacking Asf1p
also supports the idea that these cells have a higher incidence
of DNA damage. As such, the reduced viability of asf1? yeast
upon inactivation of the DNA damage checkpoint (Fig. 3) is
probably a consequence of precocious progression through
mitosis with damaged DNA.
Yeast lacking Asf1p grow slowly as a consequence of acti-
vating the DNA damage checkpoint; however, this slow growth
rate is not apparent until 20 h after Asf1p removal (Fig. 5).
FIG. 7. Asf1p does not appear to be required for double-strand
break repair. (A) asf1 mutants are competent at repairing gross chro-
mosomal damage. Cells from cultures of ROY1172 (WT) and
ROY1170 (asf1?) were arrested for 2 h with nocodazole, followed by
a 5-min treatment with 0.048% MMS. The MMS was then removed,
and the cells were recovered for 1, 3, or 18 h at 30°C. Chromosomes
were isolated after the arrest (pre), after MMS treatment (MMS), and
at 1, 3, or 18 h after removal of the MMS (1, 3, and 18 h, respectively)
and were resolved by pulsed-field agarose gel electrophoresis, followed
by ethidium bromide staining. The characteristic pattern of S. cerevi-
siae chromosomes is seen in the marker lane. Induction and repair of
double-strand breaks is most apparent by monitoring the disappear-
ance and reappearance of the two largest chromosomes (indicated by
the asterisks), the majority of which appears to be damaged at this level
of MMS exposure. Double-strand breaks are seen as a reduction in the
intensity of a chromosome and an increase in chromosome ladder
smearing. Identical results were obtained with or without nocodazole
arrest (data not shown). (B) asf1 mutants are competent for repair of
a defined HO lesion by homologous recombination. Plasmid yECP50
carrying the pGAL1:HO gene was introduced into strains JRY2334
(WT), YM004 (asf1?), and JKT0004 (rad52?), and HO endonuclease
was induced by the addition of galactose to the medium. Identical
amounts of dilutions were plated onto glucose medium at increasing
times after HO induction, grown for 3 days at 30°C, and viability was
determined by colony counting. Viability for each strain prior to in-
duction of the HO endonuclease was designated 100%. The data are
the average and standard deviation of three independent experiments.
10322RAMEY ET AL.MOL. CELL. BIOL.
FIG. 8. Molecular analysis of DNA repair in asf1 mutants. (A) Schematic of the mating-type loci in yeast. The locations of PCR primers are
shown. If the mating type is “a,” then the PCR product is 1.0 kb; if it is “?,” then the product is 1.1 kb. (B) Cutting and repair of the HO lesion
in wild-type yeast. HO endonuclease was induced at t ? 0 in strain BAT009 (WT) by the addition of galactose and was repressed at 2 h by the
addition of glucose. Samples were analyzed throughout the time course with the primers shown in panel A and control primers. (C) Quantitation
of DNA cutting and repair in WT yeast. The MATa and ? products were quantified from the gel in panel B and were normalized to the control
product. The amount of MAT product at t ? 0 was normalized to 1. (D) As in panel B, but with strain BAT022 (rad52?). (E) Same as in panel
C, but with strain BAT022 (rad52?). (F) Same as in panel B, but with strain YM004 (asf1?). (G) Same as in panel C, but with strain YM004 (asf1?).
(H) Schematic of the mating-type loci used to investigate resection during repair of an HO induced double-strand break. (I) Analysis of resection
by Southern hybridization after HO induction. Genomic DNA from mating type a strains JRY2334 (WT), YM004 (asf1?) and JKT004 (rad52?)
was isolated from asynchronous cultures before induction of the HO nuclease “Glu” at 0.5 and 1 h after the addition of galactose and then at 1
and 2 h after the subsequent addition of glucose to repress the HO endonuclease and allow repair of the HO lesion by mating-type switching.
Genomic DNA was digested with StyI, and the resulting Southern blots were hybridized by using the probe indicated in panel A. The HO-cut
fragment appeared between 0.5 and 1 h after HO induction with galactose, followed by the appearance of the MAT? product in wild-type and asf1?
strains, 2 h after repression of the HO endonuclease by glucose addition. The MAT? product cannot be observed in the rad52? strain due to the
inability to perform homologous recombinational repair of the HO lesion. Arrows indicate the formation of single-stranded DNA tails that result
from the inability of StyI to cleave the single-stranded DNA, which is due to resection beyond one or more distal StyI sites.
VOL. 24, 2004 ALTERED CHROMATIN STRUCTURE CAUSES GENOMIC INSTABILITY10323
This rules out the possibility that the loss of Asf1p disregulates
transcription of genes important for cell cycle progression be-
cause we would expect to see a growth defect upon degrada-
tion of Asf1p sooner than 20 h since the average half-life of
mRNA is a mere 19 min (22). Also, there is no disregulation of
obvious candidates for alteration of the cell cycle in our mi-
croarray analyses of asf1 mutants (Zabaronick and Tyler, sub-
mitted). A more likely model to explain the delayed growth
defect upon degradation of Asf1p is that chromatin structure
gradually becomes more perturbed upon loss of Asf1p until a
threshold is reached that affects cellular processes enough to
activate the DNA damage checkpoint. Taken together, the
available data indicate that Asf1p is not required for DNA
repair per se but, in its absence, the accumulation of altered
chromatin structures eventually triggers the DNA damage
checkpoint. Consistent with this idea, Asf1p is dispensable for
the recovery from DNA damage checkpoint-induced meta-
phase arrest (Fig. 5). This result is important because it indi-
cates that Asf1p is not required for the repair events that the
DNA damage checkpoint is stalling the cell cycle to allow.
Rather, the absence of Asf1p is causing more damage events
that provide opportunities for inaccurate repair.
If asf1 mutants can repair double-strand DNA damage, why
are they inviable on plates containing double-strand DNA-
damaging agents? An important clue to the answer comes from
the fact that transient exposure to high doses of DNA-damag-
ing agents does not reduce the viability of asf1 mutants. We
have found that an event after religation of the DNA lesion,
perhaps the repair of the chromatin structure, does not occur
efficiently in asf1 mutant cells, leading to a delay in turning off
or recovery from the DNA damage checkpoint (S. Howar and
J. Tyler, unpublished data). Constant exposure of asf1 mutants
to DNA-damaging agents therefore would never give the cells
an opportunity to grow because of the delayed recovery from
the DNA damage checkpoint.
Yeast lacking Asf1p are mutators and have increased rates
of recombination. Consistent with the idea that asf1 mutants
have elevated rates of spontaneous DNA damage foci, the loss
of Asf1p leads to a moderate but consistently elevated rate of
spontaneous mutation and recombination. This finding was
confirmed in a recent report that also found elevated rates of
sister chromatid exchange in asf1 mutant cells (45). If Asf1p
were actively involved in the repair of DNA damage, we would
expect the mutation rate to increase in proportion to the
amount of lesions induced in cells lacking Asf1p. However,
treatment of asf1? cultures with MMS did not increase the
mutation rate any more than treatment of wild-type cells with
MMS (data not shown), a finding consistent with an indirect
influence of Asf1p on the fidelity of DNA repair. This indirect
influence is most likely via an affect of Asf1p during or after
DNA replication, since mutations result when errors made by
cellular replicases go unrepaired or are repaired in an error-
prone fashion. Similarly, the loss of Asf1p leads to elevated
rates of SCE, which is also initiated during DNA replication.
Therefore, we propose that the elevated mutation rate and
recombination rate upon deletion of ASF1 is a consequence of
the loss of Asf1p’s function in maintaining normal chromatin
structure (59), leading to problems during or after DNA rep-
Asf1p protects against genomic instability during DNA rep-
lication. The weight of evidence suggests that the absence of
Asf1p leads to problems during S phase that result in genomic
instability. This evidence includes the fact that asf1 mutants
activate the Mrc1p kinase that responds to replication prob-
lems and are sensitive to both DNA replication stresses and
mutation of DNA polymerase alpha. Deletion of ASF1 also
leads to gross chromosomal rearrangements (41), increased
spontaneous DNA damage foci during S phase (45; the present
study), and increased recombination (45; the present study)
and mutation rates—events that are all triggered during DNA
replication. There are also many genetic interactions that
strongly suggest a mechanistic link between Asf1p and the
processing of stalled replication forks. For example, deletion of
ASF1 is synthetically lethal with deletion of the SGS1 Rec-Q
helicase that regulates recombination at stalled replication
forks (56). Similarly, deletion of ASF1 is synthetically lethal
with deletion of the MGS1 helicase that facilitates fork pro-
gression through alternative damage avoidance mechanisms
(57). Deletion of ASF1 is also synthetically lethal with deletion
of MMS4, which encodes part of an endonuclease complex
involved in processing of stalled replication forks (57). We
noted a similar relationship between Rad52p and Asf1p in that
rad52?asf1? cells have a greatly reduced viability compared to
either single mutant (Fig. 3). Rad52p is required for the for-
mation of Holliday junctions during S phase, an intermediate
of the rescue of stalled DNA replication forks (68). Similarly,
deletion of both ASF1 and the RAD50 gene, whose product is
required for the processing of stalled replication forks by ho-
mologous recombination, results in inviability (57).
There are several potential mechanisms whereby loss of
Asf1p may function to influence genomic stability during S
phase: (i) the altered chromatin structure in asf1 mutants may
lead to elevated rates of replicational stalling, which results in
elevated rates of homologous recombinational repair to re-
solve the stalled forks; or (ii) replication forks stall at the
normal frequency in asf1 mutants, but the abnormal chromatin
structure in asf1 mutants leads to preferential processing of the
stalled forks by homologous recombination rather than restart-
ing. Neither model would absolutely require Asf1p to be func-
tioning to assemble chromatin at the DNA replication fork,
although this certainly is possible. There is evidence that Asf1p
is involved in both replication-independent and replication-
dependent chromatin assembly, as well as replication-indepen-
dent chromatin disassembly in vivo (1). Evidence for a repli-
cation-independent chromatin assembly role for Asf1p in vivo
includes the fact that Asf1 exists in a complex with the histone
variant H3.3 that is assembled into highly transcribed genes
and the histone chaperone HirA that is required for replica-
tion-independent chromatin assembly (55). Evidence for a rep-
lication-dependent chromatin assembly role for Asf1p in vivo
includes the facts that Asf1 was discovered in a complex with
newly synthesized histones that are assembled onto newly rep-
licated DNA (59), that Asf1 exists in a complex with the his-
tone variant H3.1 that is assembled only onto newly replicated
DNA (55), and that Asf1 interacts with the replication-specific
histone chaperone CAF-1 (55, 59). As such, the altered global
chromatin structure that exists in asf1 mutants (1a) that is
leading to genomic instability during S phase could result from
either the replication-dependent or replication-independent
10324 RAMEY ET AL.MOL. CELL. BIOL.
functions of Asf1p in maintaining normal chromatin structure
because half of the chromatin on each newly replicated daugh-
ter DNA strand is inherited from the old chromatin and half is
newly assembled chromatin.
We do not favor the idea that all our results are due to
indirect effects of Asf1p on histone levels. Asf1p is known to
contribute to the cell cycle regulation of histone gene tran-
scription (54); however, the levels and stability of histone pro-
teins are not detectably different in wild-type and asf1 mutant
cells (1a, 17). Also, overexpression of histones H3 and H4
failed to restore the cell cycle and growth defects of asf1 mu-
tants (data not shown). Furthermore, there have not been any
reports of histone levels affecting genomic stability, and altered
histone levels do not appear to hamper the completion of
DNA replication (20).
There is previous evidence that chromatin structure can
regulate the movement and timing of the initiation of replica-
tion forks. For example, deletion of the histone deacetylase
Rpd3p creates a more open chromatin structure that leads to
an earlier firing of replication origins (61). It has also been
reported that mec1-4 temperature-sensitive mutants show
preferential break sites termed replication slow zones (RSZs)
that may occur due to specialized chromatin structures that are
difficult for replication forks to traverse, such as at the ribo-
somal DNA (6). By analogy to the situation at RSZs, we have
found that asf1 mutants have a more compact or overas-
sembled chromatin structure than normal (1a) that may pro-
vide an obstacle for the replication machinery leading to rep-
licational stalls. Strikingly, deletion of ASF1 results in an
absolute requirement for the product of the RRM3 gene that
mediates efficient replication through the compact chromatin
structure of ribosomal DNA (57). Conversely, overexpression
of Asf1p may generate a more open or underassembled chro-
matin structure that is easier to replicate through, as suggested
by the observation that overexpression of Asf1p can suppress
the requirement for Rad53p in stabilizing replication forks
during hydroxyurea treatment (24). It is possible that Asf1p
may be directly influencing the processivity of DNA replication
forks rather than indirectly through altered chromatin struc-
tures. For example, Asf1-mediated reassembly of chromatin
behind the DNA replication fork (59) may facilitate proper
timing of chromatin assembly to allow for the correct process-
ing of Okazaki fragments, preventing DNA damage. In sup-
port of this idea, deletion of both ASF1 and the gene encoding
Rad27p, a protein required for Okazaki fragment processing,
leads to synthetic lethality (57). Another possibility is that
Asf1-mediated disassembly of chromatin (1) ahead of the
DNA replication fork may facilitate smooth passage of the
replication machinery. However, if Asf1p is affecting the pro-
cessivity of replication forks, the effect is subtle because, de-
spite our extensive efforts, we have been unable to find any
evidence for increased replicational stalling in asf1 mutants by
either pulsed-field gel electrophoresis or two-dimensional rep-
licational gels (data not shown). Therefore, we favor the idea
that the altered chromatin structure in the absence of Asf1p
does not lead to more replication stalls per se but increases the
probability that the stalled replication forks will be resolved by
homologous recombination rather than be stabilized and re-
started. As such, our findings are consistent with the idea that
the chromatin structures formed by Asf1p stabilize stalled rep-
lication forks, facilitating their restarting.
The importance of proper chromatin modulation during
DNA replication has been demonstrated in human cells (64).
Overexpression of a dominant-negative form of the large sub-
unit (p150) of the chromatin assembly factor CAF-1 slowed
S-phase progression, induced double-strand breaks, and acti-
vated components of the DNA damage checkpoint that sensed
both DNA replication problems and DNA damage (64). How-
ever, depletion of CAF-1 p150 by siRNA only activated the
DNA damage checkpoint components that respond to DNA
replication problems and not to DNA damage (21). It is pos-
sible that the dominant-negative version of CAF-1 p150 in the
earlier study was acting by sequestering hAsf1 via its interac-
tion with the p60 subunit of CAF-1 (38, 60). As such, the DNA
damage and the activation of the DNA damage checkpoint
that result from overexpressing the dominant-negative CAF-1
(64) may be a consequence of disrupting the function of Asf1
in humans. We are investigating this possibility and the role of
Asf1 in genome stability in human cells.
In summary, Asf1p is required for the formation of proper
chromatin structures that are likely to be important for the
processing of stalled replication forks. The absence of Asf1p
leads to a greater requirement for resolution of stalled repli-
cation forks by homologous recombination, providing oppor-
tunities for inaccurate repair. As such, proper chromatin struc-
tures mediated by Asf1p have a novel and important role in
maintaining genomic integrity.
We thank Paul Megee for critical reading of the manuscript. We are
very grateful to Jim Haber, Rohinton Kamakaka, Paul Megee, Jocelyn
Krebs, Michael Fasullo, and Bob Sclafani for yeast strains; David
Toczyski for the Ddc2p-GFP plasmid; Noel Lowndes for antibodies to
Rad53 and Rad9; and Steven Jackson and Jocelyn Krebs for antibodies
to phosphorylated H2A. We thank Miguel Ferreria for stimulating
discussions and help and advice with pulsed-field gel electrophoresis
and Michelle Pham, Josh Carson, and Jack Milwid for technical assis-
tance. We are particularly grateful to the University of Colorado Can-
cer Center Flow Cytometry Facility for flow cytometry analyses.
This study was supported by an NIH award CA95641-01 to
J.K.T. J.K.T. is a scholar of the Leukemia and Lymphoma Society.
C.J.R. was supported by a predoctoral training grant in molecular
biology NIH T32GM 08730.
1. Adkins, M. W., S. R. Howar, and J. K. Tyler. 2004. Chromatin disassembly
mediated by the histone chaperone Asf1 is essential for transcriptional ac-
tivation of the yeast PHO5 and PHO8 genes. Mol. Cell 14:657–666.
1a.Adkins, M. W., and J. K. Tyler. The histone chaperone Asf1p mediates
global chromatin dissembly in vivo. J. Biol. Chem., in press.
2. Aguilera, A., S. Chavez, and F. Malagon. 2000. Mitotic recombination in
yeast: elements controlling its incidence. Yeast 16:731–754.
3. Alcasabas, A. A., A. J. Osborn, J. Bachant, F. Hu, P. J. Werler, K. Bousset,
K. Furuya, J. F. Diffley, A. M. Carr, and S. J. Elledge. 2001. Mrc1 transduces
signals of DNA replication stress to activate Rad53. Nat. Cell Biol. 3:958–
4. Bakkenist, C. J., and M. B. Kastan. 2003. DNA damage activates ATM
through intermolecular autophosphorylation and dimer dissociation. Nature
5. Bennett, C. B., L. K. Lewis, G. Karthikeyan, K. S. Lobachev, Y. H. Jin, J. F.
Sterling, J. R. Snipe, and M. A. Resnick. 2001. Genes required for ionizing
radiation resistance in yeast. Nat. Genet. 29:426–434.
6. Cha, R. S., and N. Kleckner. 2002. ATR homolog Mec1 promotes fork
7. Cox, M. M., M. F. Goodman, K. N. Kreuzer, D. J. Sherratt, S. J. Sandler,
and K. J. Marians. 2000. The importance of repairing stalled replication
forks. Nature 404:37–41.
VOL. 24, 2004 ALTERED CHROMATIN STRUCTURE CAUSES GENOMIC INSTABILITY10325
8. de la Torre-Ruiz, M. A., C. M. Green, and N. F. Lowndes. 1998. RAD9 and
RAD24 define two additive, interacting branches of the DNA damage check-
point pathway in budding yeast normally required for Rad53 modification
and activation. EMBO J. 17:2687–2698.
9. Dong, Z., and M. Fasullo. 2003. Multiple recombination pathways for sister
chromatid exchange in Saccharomyces cerevisiae: role of RAD1 and the
RAD52 epistasis group genes. Nucleic Acids Res. 31:2576–2585.
10. Downs, J. A., N. F. Lowndes, and S. P. Jackson. 2000. A role for Saccharo-
myces cerevisiae histone H2A in DNA repair. Nature 408:1001–1004.
11. Emili, A., D. M. Schieltz, J. R. Yates III, and L. H. Hartwell. 2001. Dynamic
interaction of DNA damage checkpoint protein Rad53 with chromatin as-
sembly factor Asf1. Mol. Cell 7:13–20.
12. Fasullo, M., J. Koudelik, P. AhChing, P. Giallanza, and C. Cera. 1999.
Radiosensitive and mitotic recombination phenotypes of the Saccharomyces
cerevisiae dun1 mutant defective in DNA damage-inducible gene expression.
13. Fuertesa, M. A., J. Castillab, C. Alonsoa, and J. M. Perez. 2003. Cisplatin
biochemical mechanism of action: from cytotoxicity to induction of cell death
through interconnections between apoptotic and necrotic pathways. Curr.
Med. Chem. 10:257–266.
14. Gilbert, C. S., C. M. Green, and N. F. Lowndes. 2001. Budding yeast Rad9
is an ATP-dependent Rad53 activating machine. Mol. Cell 8:129–136.
15. Gontijo, A. M., C. M. Green, and G. Almouzni. 2003. Repairing DNA
damage in chromatin. Biochimie 85:1133–1147.
16. Grenson, M., M. Mousset, J. M. Wiame, and J. Bechet. 1966. Multiplicity of
the amino acid permeases in Saccharomyces cerevisiae. I. Evidence for a
specific arginine-transporting system. Biochim. Biophys. Acta 127:325–338.
17. Gunjan, A., and A. Verreault. 2003. A Rad53 kinase-dependent surveillance
mechanism that regulates histone protein levels in Saccharomyces cerevisiae.
18. Guthrie, C. F., and G. R. Fink. 1991. Guide to yeast genetics and molecular
biology, vol. 194. Academic Press, New York, N.Y..
19. Haber, J. E. 1998. Mating-type gene switching in Saccharomyces cerevisiae.
Annu. Rev. Genet. 32:561–599.
20. Han, M., M. Chang, U. J. Kim, and M. Grunstein. 1987. Histone H2B
repression causes cell-cycle-specific arrest in yeast: effects on chromosomal
segregation, replication, and transcription. Cell 48:589–597.
21. Hoek, M., and B. Stillman. 2003. Chromatin assembly factor 1 is essential
and couples chromatin assembly to DNA replication in vivo. Proc. Natl.
Acad. Sci. USA 100:12183–12188.
22. Holstege, F. C., E. G. Jennings, J. J. Wyrick, T. I. Lee, C. J. Hengartner,
M. R. Green, T. R. Golub, E. S. Lander, and R. A. Young. 1998. Dissecting
the regulatory circuitry of a eukaryotic genome. Cell 95:717–728.
23. Hryciw, T., M. Tang, T. Fontanie, and W. Xiao. 2002. MMS1 protects against
replication-dependent DNA damage in Saccharomyces cerevisiae. Mol.
Genet. Genomics 266:848–857.
24. Hu, F., A. A. Alcasabas, and S. J. Elledge. 2001. Asf1 links Rad53 to control
of chromatin assembly. Genes Dev. 15:1061–1066.
25. Katou, Y., Y. Kanoh, M. Bando, H. Noguchi, H. Tanaka, T. Ashikari, K.
Sugimoto, and K. Shirahige. 2003. S-phase checkpoint proteins Tof1 and
Mrc1 form a stable replication-pausing complex. Nature 424:1078–1083.
26. Kaufman, P. D., R. Kobayashi, and B. Stillman. 1997. Ultraviolet radiation
sensitivity and reduction of telomeric silencing in Saccharomyces cerevisiae
cells lacking chromatin assembly factor-I. Genes Dev. 11:345–357.
27. Kondo, T., T. Wakayama, T. Naiki, K. Matsumoto, and K. Sugimoto. 2001.
Recruitment of Mec1 and Ddc1 checkpoint proteins to double-strand breaks
through distinct mechanisms. Science 294:867–870.
28. Le, S., C. Davis, J. B. Konopka, and R. Sternglanz. 1997. Two new S-phase-
specific genes from Saccharomyces cerevisiae. Yeast 13:1029–1042.
29. Lea, D. E., and C. A. Coulson. 1948. The distribution of the numbers of
mutants in bacterial populations. J. Genet. 49:264–268.
30. Lew, D. J., and D. J. Burke. 2003. The spindle assembly and spindle position
checkpoints. Annu. Rev. Genet. 37:251–282.
31. Lisby, M., A. Antunez de Mayolo, U. H. Mortensen, and R. Rothstein. 2003.
Cell cycle-regulated centers of DNA double-strand break repair. Cell Cycle
32. Lisby, M., U. H. Mortensen, and R. Rothstein. 2003. Colocalization of
multiple DNA double-strand breaks at a single Rad52 repair centre. Nat.
Cell Biol. 5:572–577.
33. Longtine, M. S., A. McKenzie III, D. J. Demarini, N. G. Shah, A. Wach, A.
Brachat, P. Philippsen, and J. R. Pringle. 1998. Additional modules for
versatile and economical PCR-based gene deletion and modification in Sac-
charomyces cerevisiae. Yeast 14:953–961.
34. Luger, K., A. W. Mader, R. K. Richmond, D. F. Sargent, and T. J. Richmond.
1997. Crystal structure of the nucleosome core particle at 2.8 A ˚resolution.
35. Marsischky, G. T., N. Filosi, M. F. Kane, and R. Kolodner. 1996. Redun-
dancy of Saccharomyces cerevisiae MSH3 and MSH6 in MSH2-dependent
mismatch repair. Genes Dev. 10:407–420.
36. McNairn, A. J., and D. M. Gilbert. 2003. Epigenomic replication: linking
epigenetics to DNA replication. Bioessays 25:647–656.
37. Megee, P. C., and D. Koshland. 1999. A functional assay for centromere-
associated sister chromatid cohesion. Science 285:254–257.
38. Mello, J. A., H. H. Sillje, D. M. Roche, D. B. Kirschner, E. A. Nigg, and G.
Almouzni. 2002. Human Asf1 and CAF-1 interact and synergize in a repair-
coupled nucleosome assembly pathway. EMBO Rep. 3:329–334.
39. Melo, J. A., J. Cohen, and D. P. Toczyski. 2001. Two checkpoint complexes
are independently recruited to sites of DNA damage in vivo. Genes Dev.
40. Modesti, M., and R. Kanaar. 2001. DNA repair: spot(light)s on chromatin.
Curr. Biol. 11:R229–R232.
41. Myung, K., V. Pennaneach, E. S. Kats, and R. D. Kolodner. 2003. Saccha-
romyces cerevisiae chromatin-assembly factors that act during DNA replica-
tion function in the maintenance of genome stability. Proc. Natl. Acad. Sci.
42. Narlikar, G. J., H. Y. Fan, and R. E. Kingston. 2002. Cooperation between
complexes that regulate chromatin structure and transcription. Cell 108:475–
43. Osborn, A. J., and S. J. Elledge. 2003. Mrc1 is a replication fork component
whose phosphorylation in response to DNA replication stress activates
Rad53. Genes Dev. 17:1755–1767.
44. Pasero, P., K. Shimada, and B. P. Duncker. 2003. Multiple roles of replica-
tion forks in S phase checkpoints: sensors, effectors and targets. Cell Cycle
45. Prado, F., F. Cortes-Ledesma, and A. Aguilera. 2004. The absence of the
yeast chromatin assembly factor Asf1 increases genomic instability and sister
chromatid exchange. EMBO Rep. 5:497–502.
46. Qin, S., and M. R. Parthun. 2002. Histone H3 and the histone acetyltrans-
ferase Hat1p contribute to DNA double-strand break repair. Mol. Cell. Biol.
47. Roth, D. B., and S. Y. Roth. 2000. Unequal access: regulating V(D)J. re-
combination through chromatin remodeling. Cell 103:699–702.
48. Rouse, J., and S. P. Jackson. 2000. LCD1: an essential gene involved in
checkpoint control and regulation of the MEC1 signalling pathway in Sac-
charomyces cerevisiae. EMBO J. 19:5801–5812.
49. Schwartz, J. L. 1989. Monofunctional alkylating agent-induced S-phase-
dependent DNA damage. Mutat. Res. 216:111–118.
50. Sharp, J. A., A. A. Franco, M. A. Osley, and P. D. Kaufman. 2002. Chromatin
assembly factor I and Hir proteins contribute to building functional kineto-
chores in Saccharomyces cerevisiae. Genes Dev. 16:85–100.
51. Smith, S., and B. Stillman. 1989. Purification and characterization of CAF-I,
a human cell factor required for chromatin assembly during DNA replication
in vitro. Cell 58:15–25.
52. Stone, E. M., and L. Pillus. 1996. Activation of an MAP kinase cascade leads
to Sir3p hyperphosphorylation and strengthens transcriptional silencing.
J. Cell Biol. 135:571–583.
53. Sugawara, N., E. L. Ivanov, J. Fishman-Lobell, B. L. Ray, X. Wu, and J. E.
Haber. 1995. DNA structure-dependent requirements for yeast RAD genes
in gene conversion. Nature 373:84–86.
54. Sutton, A., J. Bucaria, M. A. Osley, and R. Sternglanz. 2001. Yeast asf1
protein is required for cell cycle regulation of histone gene transcription.
55. Tagami, H., D. Ray-Gallet, G. Almouzni, and Y. Nakatani. 2004. Histone
H3.1 and H3.3 complexes mediate nucleosome assembly pathways depen-
dent or independent of DNA synthesis. Cell 116:51–61.
56. Tong, A. H., M. Evangelista, A. B. Parsons, H. Xu, G. D. Bader, N. Page, M.
Robinson, S. Raghibizadeh, C. W. Hogue, H. Bussey, B. Andrews, M. Tyers,
and C. Boone. 2001. Systematic genetic analysis with ordered arrays of yeast
deletion mutants. Science 294:2364–2368.
57. Tong, A. H., G. Lesage, G. D. Bader, H. Ding, H. Xu, X. Xin, J. Young, G. F.
Berriz, R. L. Brost, M. Chang, Y. Chen, X. Cheng, G. Chua, H. Friesen, D. S.
Goldberg, J. Haynes, C. Humphries, G. He, S. Hussein, L. Ke, N. Krogan, Z.
Li, J. N. Levinson, H. Lu, P. Menard, C. Munyana, A. B. Parsons, O. Ryan,
R. Tonikian, T. Roberts, A. M. Sdicu, J. Shapiro, B. Sheikh, B. Suter, S. L.
Wong, L. V. Zhang, H. Zhu, C. G. Burd, S. Munro, C. Sander, J. Rine, J.
Greenblatt, M. Peter, A. Bretscher, G. Bell, F. P. Roth, G. W. Brown, B.
Andrews, H. Bussey, and C. Boone. 2004. Global mapping of the yeast
genetic interaction network. Science 303:808–813.
58. Turner, B. M. 2002. Cellular memory and the histone code. Cell 111:285–291.
59. Tyler, J. K., C. R. Adams, S. R. Chen, R. Kobayashi, R. T. Kamakaka, and
J. T. Kadonaga. 1999. The RCAF complex mediates chromatin assembly
during DNA replication and repair. Nature 402:555–560.
60. Tyler, J. K., K. A. Collins, J. Prasad-Sinha, E. Amiott, M. Bulger, P. J. Harte,
R. Kobayashi, and J. T. Kadonaga. 2001. Interaction between the Drosophila
CAF-1 and ASF1 chromatin assembly factors. Mol. Biol. Cell 21:6574–6584.
61. Vogelauer, M., L. Rubbi, I. Lucas, B. J. Brewer, and M. Grunstein. 2002.
Histone acetylation regulates the time of replication origin firing. Mol. Cell
62. Weinert, T. A., G. L. Kiser, and L. H. Hartwell. 1994. Mitotic checkpoint
genes in budding yeast and the dependence of mitosis on DNA replication
and repair. Genes Dev. 8:652–665.
63. Wu, X., J. K. Moore, and J. E. Haber. 1996. Mechanism of MAT alpha donor
10326 RAMEY ET AL.MOL. CELL. BIOL.
preference during mating-type switching of Saccharomyces cerevisiae. Mol.
Cell. Biol. 16:657–668.
64. Ye, X., A. A. Franco, H. Santos, D. M. Nelson, P. D. Kaufman, and P. D.
Adams. 2003. Defective S phase chromatin assembly causes DNA damage,
activation of the S phase checkpoint, and S phase arrest. Mol. Cell 11:341–351.
65. Zegerman, P., and J. F. Diffley. 2003. Lessons in how to hold a fork. Nat.
Struct. Biol. 10:778–779.
66. Zhao, X., E. G. Muller, and R. Rothstein. 1998. A suppressor of two essential
checkpoint genes identifies a novel protein that negatively affects dNTP
pools. Mol. Cell 2:329–340.
67. Zhou, B. B., and S. J. Elledge. 2000. The DNA damage response: putting
checkpoints in perspective. Nature 408:433–439.
68. Zou, H., and R. Rothstein. 1997. Holliday junctions accumulate in replica-
tion mutants via a RecA homolog-independent mechanism. Cell 90:87–96.
69. Zou, L., and S. J. Elledge. 2003. Sensing DNA damage through ATRIP
recognition of RPA-ssDNA complexes. Science 300:1542–1548.
VOL. 24, 2004 ALTERED CHROMATIN STRUCTURE CAUSES GENOMIC INSTABILITY 10327