Two modes of DNA
double-strand break repair
are reciprocally regulated
through the fission yeast
Miguel Godinho Ferreira
and Julia Promisel Cooper1
Telomere Biology Laboratory, Cancer Research UK,
London WC2A 3PX, UK
Several considerations suggest that levels of the two ma-
jor modes of double-strand break (DSB) repair, homolo-
gous recombination (HR), and nonhomologous end join-
ing (NHEJ), are regulated through the cell cycle. How-
ever, this idea has not been explicitly tested. In the
absence of the telomere-binding protein Taz1, fission
yeast undergo lethal telomere fusions via NHEJ. These
fusions occur only during periods of nitrogen starvation
and fail to accumulate during logarithmic growth, when
the majority of cells are in G2. We show that G1 arrest is
the specific nitrogen starvation-induced event that pro-
motes NHEJ between taz1−telomeres. Furthermore, the
general levels of NHEJ and HR are reciprocally regulated
through the cell cycle, so that NHEJ is 10-fold higher in
early G1 than in other cell cycle stages; the reverse is
true for HR. Whereas NHEJ is known to be dispensable
for survival of DSBs in cycling cells, we find that it is
critical for repair and survival of DSBs arising during G1.
Received July 1, 2004; revised version accepted July 27, 2004.
DNA double-strand breaks (DSBs) are among the most
deleterious types of damage with which cellular DNA
repair systems must contend. If a DSB is left unrepaired
in a dividing cell, the portion of the chromosome that is
left unconnected to the centromere is unable to segre-
gate to the daughter cell, giving rise to chromosome de-
letions. If incorrectly repaired, DSBs may lead to chro-
mosome translocations and other aberrations (Pierce et
Two major pathways repair DSBs, nonhomologous end
joining (NHEJ), and homologous recombination (HR; van
Gent et al. 2001). NHEJ joins two DNA ends irrespective
of their sequence, thereby generating errors if the two
ends are unrelated or inaccurately processed. Alterna-
tively, DSBs can be joined via HR processes that use
homologous DNA sequences (usually in the sister chro-
matid) as templates for repairing broken ends, thus pro-
viding error-free repair. HR is the pathway of choice in
budding and fission yeast, as NHEJ mutants are resistant
to ?-radiation, whereas HR mutations severely compro-
mise survival of ?-radiation (Siede et al. 1996; Manolis et
Remarkably, although cells can detect and respond to
a single DSB generated by DNA damage (Sandell and
Zakian 1993), they are perfectly capable of recognizing
the numerous ends of chromosomes (e.g., 184 in a G2
human somatic cell) as nondeleterious structures. To
conserve genome stability, telomeres have developed the
property of being refractory to DNA repair processes. Re-
cent studies have uncovered some of the components
underlying this property (Ferreira et al. 2004). In humans,
the TRF2 protein binds telomeres and protects them
from inappropriate repair reactions; interfering with
TRF2 function leads to chromosome end fusions, cell
cycle arrest, and apoptosis (Karlseder et al. 1999).
Taz1 is the fission yeast ortholog of both TRF2 and the
other human telomeric DNA-binding protein, TRF1
(Cooper et al. 1997; Li et al. 2000; Ferreira and Cooper
2001). In the absence of Taz1, several telomere functions
are disrupted, but cells are still viable in unperturbed cell
cycles (Cooper et al. 1997, 1998). We have shown that
Taz1 loss renders telomeres vulnerable to the two DSB
repair pathways, but that end fusions, which form via
NHEJ, only occur as cells arrest in G1 during nitrogen
starvation (Ferreira and Cooper 2001). During logarith-
mic growth, fission yeast are mainly in G2 and taz1−
telomere fusions are absent. However, NHEJ-mediated
taz1−telomere fusions arise during logarithmic growth
in rad22−HR-deficient cells, suggesting that HR protects
dysfunctional (taz1−) telomeres from NHEJ. These re-
sults led us to propose that NHEJ becomes prominent in
G1-arrested cells, whereas HR dominates during loga-
rithmic (mainly postreplicative) growth.
Several other lines of evidence support the idea that
the two major modes of DSB repair are cell cycle regu-
lated. From a teleological standpoint, cells might prefer
error-free HR whenever possible, that is, when template
copies, preferably sister chromatids, are available. Con-
versely, during G1, error-prone NHEJ would be neces-
sary, as sister chromatid templates are unavailable. Al-
though diploid cells possess homologous chromosomes
during G1, mechanisms exist to disfavor mitotic recom-
bination between homologous chromosomes and the po-
tential ensuing loss of heterozygosity in somatic mam-
malian cells (Moynahan and Jasin 1997). In chicken
DT40 lymphocytes, NHEJ mutants (ku70−/−and DNA-
PK−/−) are particularly sensitive to ionizing radiation dur-
ing G1, whereas Rad54−/−HR mutants are more sensi-
tive during S and G2 phases (Takata et al. 1998). Further-
more, levels of plasmid recircularization via NHEJ
increase during G1 in budding yeast (Karathanasis and
Wilson 2002), whereas the formation of DSB-induced
Rad52 HR foci is reduced in G1 cells (Lisby et al. 2001).
Here, we demonstrate that HR and NHEJ are recipro-
cally regulated through the cell cycle. By changing cell
cycle profiles, we can modulate the appearance of NHEJ-
mediated telomere fusions during both vegetative
growth and nitrogen starvation. Furthermore, by directly
measuring repair, we show that levels of the two modes
of repair vary through the cell cycle by a factor of 10,
with NHEJ being higher in G1 and HR being higher
[Keywords: Cell cycle; DNA repair; HR; NHEJ; Taz1; telomere]
E-MAIL Julie.Cooper@cancer.org.uk; FAX 44-20-7269-3258.
Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/
GENES & DEVELOPMENT 18:2249–2254 © 2004 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/04; www.genesdev.org 2249
Results and Discussion
Occurrence of pre-START G1 during logarithmic
growth induces taz1−telomere fusions
Logarithmically growing fission yeast increase in size
during the G2 phase of the cell cycle, and upon comple-
tion of mitosis, cells generally possess sufficient mass to
proceed directly to S phase. Thus, the vegetative cell
cycle lacks a pre-START G1 phase. However, nitrogen-
starved cells arrest in G1. As fission yeast prefers hap-
loidy, these G1 cells contain only one copy of each chro-
taz1−cells accumulate telomere fusions when sub-
jected to nitrogen starvation, yet these same cells lack
telomere fusions, and indeed show virtually wild-type
levels of viability, when grown in rich medium (Ferreira
and Cooper 2001). To determine whether the taz1−telo-
mere fusions are a consequence of the G1 arrest induced
by nitrogen starvation, we investigated cells in which a
pre-START G1 phase occurs during vegetative growth.
smaller cells than wild type. These cells cannot initiate
replication until sufficient cell mass is attained, and re-
main in G1 for extended periods with low levels of Cdc2
kinase activity (Russell and Nurse 1987). wee1-50 taz1−
cultures were grown at the semipermissive temperature
of 32°C and divided into halves. One half was starved for
nitrogen for 24 h at 32°C, whereas the other was grown
in nitrogen-rich medium; aliquots of both were taken for
FACS and PFGE analyses. Southern blotting with telo-
mere probes revealed that telomere fusions were present
not only in the nitrogen-starved culture, but also in veg-
etatively growing wee1-50 taz1−cells (Fig. 1B). Thus, by
recreating pre-START G1 during the vegetative cell
cycle, we induce telomere fusions in taz1−cells growing
in nitrogen-rich medium.
The telomere fusions seen in vegetatively growing
wee1-50 taz1−cells are primarily intramolecular (i.e., re-
sulting in self-circularization), whereas both inter- and
intramolecular fusions are seen in nitrogen starved taz1−
cells (Fig. 1B). Intrachromosomal fusions also predomi-
nate in logarithmically growing taz1−cells impaired in
HR (Ferreira and Cooper 2001). Schizzosaccaromyces
pombe cells possessing circular chromosomes are viable
(Naito et al. 1998; Nakamura et al. 1998), whereas inter-
chromosomal fusions generate lethal dicentric chromo-
somes and are only maintained in nondividing (i.e., G1-
arrested) cultures. Therefore, the predominance of intra-
chromosomal fusions in wee1-50 taz1−cells supports the
idea that the fusions occur while the cells are actively
dividing in rich medium.
Absence of G1 arrest during nitrogen starvation
prevents telomere fusions
If prolonging the G1 period during vegetative growth can
induce taz1−telomere fusions, then the absence of G1
arrest during nitrogen starvation might prevent telomere
fusions. To test this, we generated taz1−strains lacking
the CDK inhibitor Rum1. Upon nitrogen starvation,
rum1−cells are unable to restrain S phase and arrest with
replicated chromosomes and high levels of Cdc2 kinase
activity. Nonetheless, rum1−mutants do activate the
Ste11 transcription factor and its targets in response to
nitrogen starvation (Stern and Nurse 1998). As expected,
neither rum1−nor rum1−taz1−strains were able to arrest
in G1, even after prolonged nitrogen starvation and
maintained 2C DNA content (Fig. 1C). Remarkably,
rum1−taz1−cells exhibit no telomere fusions during ni-
trogen starvation, (Fig. 1C) indicating that the Rum1-
dependent G1 arrest, and not some event downstream of
Ste11 activation, activates the pathway that results in
taz1−telomere fusions. Thus, by indirectly manipulating
the activity of the Cdc2 kinase, we can manipulate the
susceptibility of taz1−telomeres to undergo NHEJ-in-
duced fusions in a manner independent of nutritional
status, with low Cdc2 activity inducing telomere fusions
and high Cdc2 activity preventing them.
Telomeric 3? overhangs persist in G1 cells
taz1−telomeres possess extensive 3? G-strand overhangs
during logarithmic growth (Tomita et al. 2003). In prin-
ciple, such overhangs should promote the strand inva-
sions that initiate HR, whereas NHEJ would require
overhang removal. Indeed, upon inhibition of TRF2 func-
tion in human cells, NHEJ-dependent telomere fusions
are accompanied by a reduction in 3?-overhang signal
(van Steensel et al. 1998; Smogorzewska et al. 2002). To
investigate whether elevated NHEJ stems from a global
loss of 3? overhangs in G1 taz1−cells, we used native
in-gel hybridization analysis. Telomeric restriction frag-
ments from asynchronous and nitrogen-starved cultures
were electrophoresed under nondenaturing conditions
and hybridized to a G-strand telomere probe. Both cy-
cling and G1-blocked taz1−cells show intense hybridiza-
tion (Fig. 2A), whereas no signal is detectable using a
complementary C-rich strand probe (data not shown).
The overhang signal also persists in taz1−cells lacking
sustain telomere fusions. (A) Diagram of telomeric NotI restriction
fragments on chromosome I and II (C, I, L, and M); chromosome III
lacks NotI restriction sites. (B,C) Southern blot analysis of genomic
DNA digested with NotI, separated by PFGE, and probed with a
telomeric oligonucleotide. Telomeric restriction fragments are in-
dicated. C* comprises the C restriction fragment as well as the
C + L, C + M, and C + I fusions that cannot be resolved under these
conditions. (B) Inactivation of wee1+extends early G1 and induces
fusions of taz1−telomeres during logarithmic growth. The most
prominent fusion band (top arrow) corresponds to circularized chro-
mosome I. Prolonged growth of wee1tscells at the semipermissive
temperature typically distorts the FACS profile due to delayed cy-
tokinesis in small cells. (C) rum1+deletion prevents G1 arrest dur-
ing nitrogen starvation, and prevents starvation from inducing fu-
sion between taz1−telomeres.
The stage of the cell cycle determines whether taz1−cells
Ferreira and Cooper
2250GENES & DEVELOPMENT
telomere fusions (via pku70−and lig4−deletion). A ca-
veat of the native hybridization method is that it can
only address global changes in the abundance of over-
hanging DNA and does not address individual telomeres.
Hence, some telomeres may lose overhangs, whereas
others gain them, generating no net change in signal
strength. Nevertheless, we speculate that the NHEJ ma-
chinery can engage taz1−overhang-containing telomeres
as substrates, and that overhang removal is concomitant
with the end-joining reaction. Furthermore, our observa-
tion that NHEJ-dependent telomere fusions do occur in
logarithmically growing taz1−cells that lack Rad22 (Fer-
reira and Cooper 2001) argues against the idea that G2
taz1−telomeres are incompetent for NHEJ.
DSB repair pathways are cell cycle regulated
taz1−rad22−cells suggested that HR precludes NHEJ at
dysfunctional telomeres during vegetative growth (Fer-
reira and Cooper 2000). Thus, unprotected telomeres
may be recipients of whatever DNA repair pathway pre-
dominates generally in the cell at a given cell cycle stage.
This idea implies that cell cycle regulation of DNA re-
pair would be seen not only at telomeres, but also
throughout the genome. To test this, we directly mea-
sured the two main modes of DSB repair in different
stages of the cell cycle. We used nitrogen-starved cells as
representative of the G1 phase and logarithmically grow-
ing cells as representative of the S, G2, and M phases.
FACS analysis indicated that upon nitrogen starvation,
60%–70% of cells arrest in G1, whereas ∼100% of the
appearanceof telomerefusions inlog-phase
cells in logarithmically growing cultures exhibit only a
2C DNA peak.
Plasmid-based assays have been used in both fission
and budding yeasts to investigate the genetic require-
ments for NHEJ and HR (Orr-Weaver et al. 1981; Keeney
and Boeke 1994; Boulton and Jackson 1996; Muris et al.
1997; Baumann and Cech 2000; Manolis et al. 2001). To
assess NHEJ, a plasmid containing a replication origin is
linearized within sequences that lack homology to the
yeast genome, and then transformed into yeast cells. The
uncut plasmid is transformed in parallel to normalize for
strains. Logarithmically growing S. pombe cells display a
low plasmid end-joining efficiency (Fig. 3A) and, as ob-
served previously, this end-joining is almost entirely de-
pendent on pku70+(Baumann and Cech 2000; Manolis et
al. 2001). Strikingly, end-joining levels were seven- to
10-fold higher in nitrogen-starved G1 cells (Fig. 3A).
However, this elevation of NHEJ was absent in nitrogen-
starved rum1−cells that are unable to undergo G1 arrest
(Fig. 3A). Thus, Ku-dependent DSB repair is up-regulated
in G1-arrested cells, consistent with our hypothesis that
taz1−telomeres fuse during G1 arrest, because NHEJ is
generally elevated under these conditions. Moreover, the
genetic requirement of rum1+for elevated NHEJ sug-
gests that Rum1-dependent down-regulation of Cdc2 ki-
nase is a key event in the switch between the two modes
A straightforward model of direct competition be-
tween NHEJ and HR pathways would predict that, in the
absence of HR, NHEJ levels would rise during logarith-
mic growth. However, we did not detect an increase in
plasmid end-joining efficiency in logarithmically grow-
ing HR-deficient rad22−or rhp51−mutants (Fig. 3A).
This suggests that the two pathways are regulated inde-
pendently, such that in the absence of one, the other
mode of repair is still subject to cell cycle control. Al-
ternatively, DNA ends that attempt HR in the absence of
rad22+or rhp51+may be reversed and channeled to
To measure HR during different stages of the cell
cycle, we used a plasmid-integration assay in which
leu1−strains are transformed with a plasmid that lacks a
replication origin and is linearized within the leu1+
auxotrophic marker. These strains must integrate the
cycle. (A) NHEJ is up-regulated in G1 (−N) arrested cells. Levels of
plasmid end joining are represented as the number of transformants
obtained with linear vs. circular plasmid. Cells lacking Rum1 fail to
arrest in G1 and NHEJ levels remain low. (B) HR is down-regulated
in G1 nitrogen-starved cells. Integration of a linear leu1+pJK148
plasmid into the genome is expressed as the ratio of tranformants for
the linearized vector (which lacks an origin of replication) vs. su-
percoiled pJK148-ars1. Rum1 is required for down-regulation of HR
during nitrogen starvation.
NHEJ and HR are reciprocally regulated through the cell
G1. (A) Telomere 3? overhangs are detected in nitrogen-starved
taz1−, pku70−taz1, lig4−taz1−cells. In-gel hybridization of EcoRI-
digested genomic DNA to a G-strand telomere oligonucleotide in
nondenaturing and denaturing conditions. (B) Quantitation of telo-
meric 3? overhangs. Hybridization in each lane was quantified, and
the ratio of nondenatured/denatured signal for each sample was nor-
malized against the ratio derived from logarithmically growing
taz1−cells. Error bars represent standard deviation for a minimum of
three independent experiments.
taz1−telomeres exhibit extensive ssDNA overhangs in
Cell cycle regulation of DSB repair
GENES & DEVELOPMENT 2251
plasmid to survive in medium lacking leucine. A circular
plasmid carrying an origin of replication and leu1+was
transformed in parallel to normalize for transformation
efficiencies. Levels of plasmid integration were high dur-
ing logarithmic growth and dependent on rhp51+(Fig.
3B), as previously reported (Muris et al. 1997). Deletion
of rad22+, the fission yeast RAD52 homolog, also led to
a severe reduction in HR, in agreement with van den
Bosch et al. (2001) and in contrast to the modest effect on
HR reported for a truncation of rad22+(Ostermann et al.
1993; Muris et al. 1997). Conversely to NHEJ, plasmid
integration levels were reduced 10- to 15-fold in G1-ar-
rested cells (Fig. 3B). Southern blotting demonstrated
that ∼90% of the integration events occurred at the chro-
mosomal leu1 locus (data not shown), confirming that
HR had been the pathway for integration. rum1−cells
failed to exhibit reduced HR during nitrogen starvation,
suggesting that down-regulation of HR depends upon G1
arrest. In agreement with a gene-targeting transfection
assay in mammalian cells (Pierce et al. 2001a), deletion
of pku70+did not lead to an elevation of HR (Fig. 3B).
Sister chromatid cohesion is a candidate regulator of
HR through the cell cycle (Jessberger 2002), as cohesion
is established during S phase and is required for efficient
DSB repair. Coupled with the observed preference for
sister chromatids over homologs as substrates for HR
(Kadyk and Hartwell 1992; Moynahan and Jasin 1997),
these observations suggest that HR dominates in G2
through the availability of a cohesed sister chromatid.
Hence, the prevalence of HR at taz1−telomeres during
G2 could simply reflect this availability. However, we
were unable to promote telomere fusions during loga-
rithmic growth by disrupting the cohesion complex in a
rad21tstaz1−double mutant (data not shown). Further-
more, the plasmid recircularization and integration
events that we used as measures of NHEJ and HR occur
in the absence of cohesion between damaged and undam-
Thus, the two major modes of DSB repair are regulated
reciprocally, such that during logarithmic growth, when
cells harbor two copies of each chromosome, HR domi-
nates over NHEJ. Conversely, in G1-arrested cells that
contain only one copy of each chromosome, the levels of
these two modes of repair are inverted, with lower HR
and higher NHEJ. These patterns of general repair levels
mirror the activities that we observe at taz1−telomeres,
and support the idea that cell cycle regulation of repair
directs the consequences of telomere dysfunction. The
telomere fusions that follow expression of dominant-
negative TRF2 in human cells can arise in both G1 and
G2 (Smogorzewska et al. 2002), although the latter may
reflect a requirement for passage through S phase to al-
low displacement of wild-type TRF2 by the dominant-
negative form. Nonetheless, a relative paucity of fusions
between dysfunctional telomeres during G2 in fission
yeast versus human cells is consistent with the idea that
mammalian cells have generally higher levels of NHEJ
than the yeast systems.
NHEJ is required for radiation resistance during G1
Previous studies in yeasts showed that survival follow-
ing DSB induction depended entirely on HR, whereas
NHEJ mutants were insensitive to DSBs (Siede et al.
1996; Manolis et al. 2001). Our observation that NHEJ
levels increase during G1 prompted us to test whether
NHEJ becomes important for survival of DSBs during G1
arrest. Using wild-type cells and mutants in the HR
(−N) cultures were treated with varying doses of ?-radiation. NHEJ mutants are only sensitive during the G1 arrest induced by nitrogen
starvation. (A) Serial dilution assay. (B) Quantitative survival analysis. (C) Cells were exposed to 100 Gy of ? radiation and allowed to recover
for varying amounts of time. G1-arrested wild-type (wt) cells undergo DSB repair faster than NHEJ mutants, whereas NHEJ is dispensable for
repair during log growth. (D) Quantitation of the data in C. Ethidium bromide signals corresponding to broken DNA were normalized to the
signal for intact chromosome II for each lane. The value obtained at time 0 was designated as 1.00.
NHEJ is required for survival and repair of damage induced by ?-radiation during G1. (A,B) Logarithmic (Log) and nitrogen starved
Ferreira and Cooper
2252GENES & DEVELOPMENT
(rad22−and rhp51−) and NHEJ (pku70−and lig4−) path-
ways, we analyzed the viability of logarithmic and nitro-
gen-starved cultures exposed to varying doses of ?-irra-
diation (Fig. 4A,B). Whereas logarithmically growing HR
mutants were extremely sensitive to ?-irradiation, NHEJ
mutants exhibited viabilities comparable to that of wild-
type cells (Fig. 4A). During nitrogen starvation, however,
NHEJ mutants were 10-fold more sensitive to ?-irradia-
tion than wild-type cells, denoting a role for NHEJ in
survival of radiation during G1 (Fig. 4A,B). Interestingly,
wild-type cells lose viability more severely during G1
than during logarithmic growth (Fig. 4B), perhaps indi-
cating that elevated use of potentially inaccurate NHEJ
during G1 confers some lethality. HR genes are also im-
portant for survival of ?-irradiation during nitrogen star-
vation (Fig. 4A,B), although dependence on HR is less
severe in nitrogen-starved than logarithmic cultures (Fig.
4B). This could reflect HR that occurs following release
from nitrogen starvation, the ∼30% of cells that do not
arrest in G1 upon nitrogen starvation, or HR-related pro-
cesses that occur during G1 arrest.
NHEJ is required for efficient DSB repair in G1
As assessment of survival can only address the success of
DSB repair and not when it was undertaken, we directly
monitored the recovery of whole chromosomes follow-
ing breakage by ionizing radiation. G1-arrested cells
were exposed to 100 Gy of ionizing radiation to induce
DSBs, then kept in G1 and analyzed by PFGE to monitor
DSB repair as a function of time. Whereas whole chro-
mosomes from untreated cells are visible as three intact
bands (Fig. 4C), the irradiated cells accumulate degraded
DNA appearing as a smear of higher-mobility DNA frag-
ments. Remarkably, G1-arrested wild-type cells not only
sustain less DNA damage, but also recover chromosome
integrity faster than pku70−and lig4−mutants (Fig.
4C,D). This result demonstrates that repair occurs while
the cells are blocked in G1, and that efficient repair re-
quires the NHEJ pathway. pku70−and lig4−mutants
only started to regain intact chromosomes upon pro-
longed recovery periods, consistent with the moderate
decline of viability of NHEJ mutants. In IR-treated loga-
rithmic cultures, recovery rates were independent of
pku70−and lig4−(Fig. 4D), reinforcing the idea that NHEJ
is a prominent mode of DSB repair during G1 exclu-
As manipulating Cdc2 kinase activity
can alter the relative levels of NHEJ
and HR, we infer that Cdc2 is the ul-
timate determinant of the choice of
repair pathway, with low levels of
Cdc2 activity dictating
NHEJ and high levels directing HR.
Indeed, replication studies have es-
tablished that levels of Cdc2 kinase
activity distinguish cells that have or
have not accomplished genome dupli-
cation. This same kinase may direct
DSBs to error-free repair pathways
when sister chromatids are present.
These ideas complement a recent re-
port (Caspari et al. 2002) describing a
mutation in the B-type cyclin Cdc13 that reduces the
formation of Rhp51 foci and impairs a later step in HR in
response to DSBs during G2. It will be of great interest to
identify the targets of Cdc2 kinase that control the regu-
lation of DNA repair.
Materials and methods
Strains and medium
Strains are listed in Table 1. Cultures were grown at 32°C in standard
YES or EMM medium with or without NH4Cl and any required supple-
Gene disruption strains
Gene disruption was performed using the method of Bahler et al. (1998),
and verified by PCR using primers for the integrating constructs and
flanking genomic sequences.
Nitrogen starvation and FACS analysis
Cultures were grown to log phase (0.5–1 × 107cells/mL), washed exten-
sively with EMM-N, resuspended in EMM-N at a density of 1–5 × 106
cells/mL, and starved for 36–72 h. FACS was performed on ethanol-fixed
cells on a Becton Dickson FACScan.
Pulse-field gel electrophoresis, in-gel hybridization,
and Southern blotting
Pulsed-field gel electrophoresis was performed as previously described
(Ferreira and Cooper 2001). Telomere overhangs were analyzed as in
Tomita et al. (2003). Overhang signals were quantified using Molecular
Dynamics ImageQuant software.
The NHEJ plasmid assay was performed essentially as described (Boulton
and Jackson 1996). The plasmid pKan1 (Haering et al. 2000) was linear-
ized with KpnI and cells transformed in triplicate with 1 µg of linear or
circular pKan1. Cells were spread on YES containing 100 µg/mL G418
and colonies counted after 5 d at 32°C. The HR plasmid assay was similar
to that previously described (Keeney and Boeke 1994). Strains auxotro-
phic for leu1+were transformed in triplicate with 1 µg NdeI-linearized
pJK148 as above. To assess the uptake of DNA, an aliquot of competent
cells of each strain was transformed with 1 µg of pJK148-ars1 (same as
pJK148, but with the EcoRI fragment of ars1 filled in and cloned into the
SmaI site of pJK148). Transformants were selected on EMM lacking leu-
cine, and integration frequencies calculated by dividing the number of
pJK148 leu1+colonies by the number of pJK148-ars1 leu1+colonies. For
each such ratio, the average and standard deviation of at least three
replicates of each transformation is presented.
Radiation sensitivity and DSB repair assay
Cultures were either diluted in triplicate to yield 200 colonies per plate
or serially diluted 1:5 in 96-well plates and spotted on YES medium. The
cells were immediately exposed to ?-rays using an IBL 637 Irradiator
S. pombe strains used in this work
h−ade6-M210 leu1-32 ura4-D18
h−taz1?ura4+ade6-M210 leu1-32 ura4-D18
h−rum1?ura4+ade6-M216 leu1-32 ura4-D18
pku70?kanrhis3-D1 leu1-32 ura4-D18 ade6−
h−pku70?ura4+leu1-32 ura4-D18 ade6-M210
h+rad22?LEU2 his3-D1 leu1-32 ura4-D18
h−rad22?ura4+ade6-M210 leu1-32 ura4-D18
h+rhp51?ura4+ade6-704 leu1-32 ura4-D18
Cell cycle regulation of DSB repair
GENES & DEVELOPMENT2253
(137Cs source at a rate of 3.45 Gy/min). Plates were then incubated for Download full-text
3–5 d at 32°C. The results are presented as the percentage of survivors of
a given dose relative to the viabilities of the same strains unirradiated.
Each experiment was repeated three times.
DSB repair assay
Logarithmic and nitrogen-starved cultures were exposed to 100 Gy ?-ra-
diation in liquid medium. Samples were collected at various times and
processed for PFGE as described above. Whole chromosomes were sepa-
rated on a CHEF-DRIII apparatus (Bio-Rad) according to the manufactur-
er’s specifications. Gels were stained with ethidium bromide and digi-
tized using a UVP VisiDoc-IT system without oversaturation. Broken
DNA and intact chromosome signals were quantified using NIH Image
version 1.63 software.
We thank the members of the Cooper lab for discussion and J. Hayles and
F. Uhlmann for critically reading the manuscript. We thank the labora-
tories of T. Carr, T. Cech, S. Moreno, A. Pastink, and P. Nurse for sharing
strains and plasmids. This work was supported by the NIH, the Human
Frontiers Science Program, the Pew Scholars Program in the Biomedical
Sciences, and Cancer Research UK. M.G.F. is a recipient of a Cancer
Research UK postdoctoral fellowship.
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