Originally published as Genetics Published Articles Ahead of Print on June 18, 2006.
Genetics, Vol. 173, 1951-1968, August 2006, Copyright © 2006
doi:10.1534/genetics.106.057794
The RAD6/BRE1 Histone Modification Pathway in Saccharomyces Confers Radiation Resistance Through a RAD51-Dependent Process That Is Independent of RAD18
John C. Game*,1,
Marsha S. Williamson*,
Tatiana Spicakova
and
J. Martin Brown
* Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 and
Department of Radiation Oncology, Stanford University School of Medicine, Stanford, California 94305
1 Corresponding author: Donner Laboratory, Room 326, Lawrence Berkeley National Laboratory, 1 Cyclotron Rd., Berkeley, CA 94720.
E-mail: jcgame{at}lbl.gov
Manuscript received March 2, 2006.
Accepted for publication June 3, 2006.
ABSTRACT
We examine ionizing radiation (IR) sensitivity and epistasis relationships of several Saccharomyces mutants affecting post-translational modifications of histones H2B and H3. Mutants bre1
, lge1
, and rtf1
, defective in histone H2B lysine 123 ubiquitination, show IR sensitivity equivalent to that of the dot1
mutant that we reported on earlier, consistent with published findings that Dot1p requires H2B K123 ubiquitination to fully methylate histone H3 K79. This implicates progressive K79 methylation rather than mono-methylation in IR resistance. The set2
mutant, defective in H3 K36 methylation, shows mild IR sensitivity whereas mutants that abolish H3 K4 methylation resemble wild type. The dot1
, bre1
, and lge1
mutants show epistasis for IR sensitivity. The paf1
mutant, also reportedly defective in H2B K123 ubiquitination, confers no sensitivity. The rad6
, rad51null, rad50
, and rad9
mutations are epistatic to bre1
and dot1
, but rad18
and rad5
show additivity with bre1
, dot1
, and each other. The bre1
rad18
double mutant resembles rad6
in sensitivity; thus the role of Rad6p in ubiquitinating H2B accounts for its extra sensitivity compared to rad18
. We conclude that IR resistance conferred by BRE1 and DOT1 is mediated through homologous recombinational repair, not postreplication repair, and confirm findings of a G1 checkpoint role for the RAD6/BRE1/DOT1 pathway.
RECENT research in eukaryotes has demonstrated a much greater role than was initially perceived for histone modifications in basic cellular processes, including transcription, gene silencing, control of carcinogenesis, and responses to DNA damage. As part of this, we reported that Saccharomyces strains deleted for any of several genes involved in histone modifications are substantially more sensitive than wild type to the lethal effects of ionizing radiation (IR) (GAME et al. 2005). The mutants included strains deleted for the DOT1 gene, which encodes the methylase that acts on the lysine 79 residue (K79) of the histone H3 protein (FENG et al. 2002; VAN LEEUWEN et al. 2002), as well as histone H3 mutants in which wild-type Dot1p cannot act because its target lysine is replaced with another amino acid. These findings complemented information from other laboratories that implicates histone H3 lysine 79 methylation in controlling the DNA damage checkpoint induced by ultraviolet radiation and other agents in yeast (GIANNATTASIO et al. 2005; WYSOCKI et al. 2005) and in damage recognition by the checkpoint protein 53BP1 in mammalian cells (HUYEN et al. 2004).
Substantial information is available indicating that the DOT1-mediated methylation of H3 K79 is dependent on the prior modification of histone H2B involving ubiquitination of lysine 123 in Saccharomyces (BRIGGS et al. 2002; NG et al. 2002a) or lysine 120 in mammals (KIM et al. 2005). Recently, it was shown that H3 K79 trimethylation and some di-methylation is dependent on H2B K123 ubiquitination, whereas mono-methylation of K79 still occurs fully even in mutants that fail to modify H2B K123 (SHAHBAZIAN et al. 2005). The Rad6 ubiquitin conjugase and the Bre1 ubiquitin ligase together ubiquitinate H2B K123 (ROBZYK et al. 2000; HWANG et al. 2003; WOOD et al. 2003a). In addition, the LGE1 gene product has been found to complex with Bre1 protein and is required for its function (HWANG et al. 2003), and mutants involving some members of the RNA polymerase II-associated PAF1 complex, specifically deletions of the RTF1 and PAF1 genes, have also been reported to abolish H2B K123 ubiquitination (NG et al. 2003a; WOOD et al. 2003b). Most recently, the Bur1/Bur2 cyclin-dependent protein kinase has also been implicated in H2B K123 ubiquitination through its role in activating the Rad6 protein by phosphorylation (WOOD et al. 2005).
Given this information, and to better understand the role of the RAD6 gene in different DNA repair pathways, we chose to study the X-ray sensitivity of additional Saccharomyces histone modification mutants, including those with reported defects in H2B K123 ubiquitination and H3 K79 methylation and those involved in methylation elsewhere on histone H3. In addition, we constructed double-, triple-, and multiple-mutant strains involving H2B K123 ubiquitination and H3 K79 methylation mutations combined with each other and with key mutations in previously known DNA repair pathways. We assessed IR sensitivity in these strains to determine epistasis relationships for this phenotype both within the proposed BRE1/DOT1-mediated histone modification pathway and between this pathway and others to identify the probable IR repair processes involved.
With the exception of paf1 deletion strains, we found increased sensitivity to X-rays in all the mutants that we tested that are reported to affect histone H3 K79 methylation. We also found that set2 mutants, which fail to methylate histone H3 lysine 36, show mild X-ray sensitivity, whereas mutants that abolish histone H3 lysine 4 methylation retain wild-type resistance to X-rays. We obtained evidence that genes required for histone H3 K79 methylation predominantly fall into a single RAD6-dependent IR epistasis group that falls outside the well-known family of recovery processes mediated by RAD6/RAD18-dependent postreplication/translesion synthesis mechanisms. Instead, these histone modification genes appear to function in a process that facilitates RAD51-dependent homologous recombinational repair (HRR), although they are not completely required for such repair since significant RAD51-dependent IR resistance remains in dot1
, bre1
and related mutants. We show, in agreement with evidence from others, that some aspects of the DNA damage response cell-cycle checkpoints are abrogated in mutants unable to methylate histone H3 K79, and discuss this as a possible cause of their IR sensitivity.
MATERIALS AND METHODS
Yeast strains:
As described earlier (
GAME et al. 2005), our starting strains
were from the library of

4700 individual haploid deletion strains
in the

mating type (background strain BY4742) created by an
International Consortium and obtained from Research Genetics,
Huntsville, Alabama (now Invitrogen Life Technologies). The
genotype of strain BY4742 and the construction of the deletion
strains have been described (
BRACHMANN et al. 1998;
WINZELER et al. 1999).
Information is also available at the Saccharomyces Genome Deletion
Project website at
http://www-sequence.stanford.edu/group/yeast_deletion_project/deletions3.html.
We also used our background-isogenic strains MW5067-1C and g1201-4C,
described earlier (
GAME et al. 2005), as a wild type for survival
curves and a wild-type
MATa parent for initial crosses with
MAT
mutants from the deletion strain library, respectively.
For crosses involving
rad51, we primarily used a
rad51::URA3 disruption
null allele (originally obtained from Vladimir Larionov)
that we had backcrossed eight times into the library background
to give expected unlinked nonisogenicity <1%. This enabled
us to use the
URA3 marker in place of
KanMX4 to quickly distinguish
rad51 from other mutants in crosses. In the text, we refer to
the
rad51::URA3 allele as
rad51null and the
rad51::KanMX4 replacement
allele from the library as
rad51
. Strains containing either
of these
rad51 alleles show equivalent survival curves, as shown
in
Figure 7.
Genetic methods and media:
Genetic methods including tetrad dissection were as described
(
SHERMAN et al. 1982). Cultures were routinely incubated at
30°. Rich media (YPD) and supplemented minimal media were
prepared as described (
SHERMAN et al. 1982). To induce meiosis,
we incubated cultures for 4 or more days, usually at 30°,
on solid Fogel's sporulation medium. This contains 9.65 g potassium
acetate, 1 g glucose, 2.5 g yeast extract (Difco), and 2% agar
per liter. To score geneticin (GEN) resistance, hygromycin B
(HYG) resistance, or nourseothricin (NAT) resistance, we used
YPD plates separately supplemented with geneticin (Sigma, St.
Louis), hygromycin B (Research Products International), or nourseothricin
(Werner BioAgents) added from filter-sterilized solution shortly
before pouring plates to give a final concentration of 150 µg/ml
(GEN), 300 µg/ml (HYG), or 100 µg/ml (NAT), respectively.
Transformations:
To facilitate scoring multiple deletion mutations in crosses,
for several relevant genes we replaced the
KanMX4 cassette that
was used to create the original deletion library with cassettes
containing
LEU2 (obtained from James A. Brown, Stanford University)
or a hygromycin B (
HYG) or nourseothricin resistance (
NAT) gene
(obtained from Beth Rockmill, Yale University), using described
cassettes (
GOLDSTEIN et al. 1999) and a standard transformation
procedure (
ITO et al. 1983). To restore
BRE1 to
bre1
mutant
strains, we transformed with a
CEN URA3 plasmid containing
BRE1,
obtained from James A. Brown, using a lithium acetate procedure
(
GIETZ et al. 1995).
Determining X-ray sensitivity:
As described earlier (
GAME et al. 2005), for X-ray exposures
we used a Machlett OEG 60 X-ray tube with a beryllium window
and a Spellman power supply operated at 30 kV and 15 mA to deliver
a dose rate of 1.3 Gy (130 rad)/sec of "soft" X-rays. To determine
initially whether a mutant strain was likely to exhibit IR sensitivity,
we essentially followed the spot-testing procedure described
previously (
GAME et al. 2005). To quantify the degree of sensitivity,
we obtained X-ray survival curves using log-phase cells from
overnight liquid YPD cultures, freshly sonicated to reduce any
clumpiness, as described in the same article. Colonies were
counted after incubation for 46 days at 30°. We obtained
survival curves for at least two separate strains for most of
the single-, double-, or multiple-mutant genotypes that we present
here, and in many cases additional survival assays (not shown)
over part or all of the dose range served to confirm our findings.
For the most part, we find good agreement in X-ray sensitivity
between different spore clones with the same genotype in the
same genetic background. We prefer to present individual survival
curves instead of averaging measurements at each dose from separate
curves, both because dose points within a curve are related
by serial dilutions and because their statistical robustness
will vary from curve to curve on the basis of colony counts
as well as on the accuracy of the unirradiated control. This
means that error bars calculated for a mean value based on separate
curves can be misleading (see
GAME et al. 2005). In addition,
despite our isogenic genetic background, we prefer to obtain
confirmatory survival curves using separate spore clones rather
than repeating the same strain, as a better control for modifier
mutations that might arise. Clearly, taking average values for
separate strains would obscure any variability that we hope
to expose.
Ultraviolet radiation treatments:
Log-phase cells were prepared for UV survival as for X-ray curves.
They were irradiated on YPD plates using a shielded apparatus
containing five General Electric G8T5 tubes giving most of their
radiation at 254 nm. Plates were incubated in the dark and colonies
counted as for X-ray curves.
Cell cycle checkpoint studies:
Standard methods (
DAY et al. 2004) were adapted as follows:
To study the IR-induced G
1 checkpoint, cells were arrested at
G
1 using

-factor (Zymo Research). One microliter of 10 mM

-factor
was added to 2 ml of log-phase cells shaken in liquid YPD at
OD

0.2 at 30°. After 1.5 hr, a second microliter was added
and synchrony was assessed microscopically after another 1.5
hr. The cultures were split: one-half was irradiated using a
137Cs source (Mark 1 model 3 from J. L. Shepherd, San Francisco;
dose rate 28.4 Gy/min) and one-half was mock treated. After
irradiation, cells were released from the block and 0.25-ml
aliquots were fixed in 70% ethanol at 15-min intervals. Fixed
cells were spun down and washed with 1 ml 0.05 M sodium citrate.
Cell pellets were mixed with 0.5 ml 0.05 M sodium citrate containing
0.25 mg/ml RNase A and incubated at 50° for 1 hr. After
addition of propidium iodide (16 µg/ml final concentration),
samples were incubated at room temperature for 30 min, briefly
resonicated, and analyzed by flow cytometry (
NASH et al. 1988)
with a FACSCalibur machine.
To study the IR-induced G2 checkpoint, nocodazole (Sigma) was added (15 µg/ml final concentration) to midlog-phase cultures (OD
0.2) shaking in liquid YPD and cells were incubated for 2.5 hr at 30° to achieve >90% large buds. Cultures were split and then mock treated or irradiated with a 137Cs source (see above), nocodazole was removed by resuspending in sterile water, and cells were then resuspended in fresh YPD and shaken at 30°. Aliquots of 250 µl were fixed in 70% ethanol at 30-min intervals, spun down, resuspended in PBS (120 mM sodium chloride; 2.7 mM potassium chloride; pH 7.3 with 10 mM phosphate buffer), and incubated with DAPI (1 µg/ml final concentration) (WILLIAMSON and FENNELL 1979) at room temperature for 20 min. Percentages of uninucleate and binucleate cells were assessed by fluorescence microscopy.
RESULTS
IR survival of mutants separately blocked in histone H3 K4, K36, and K79 methylation:
In addition to histone H3 K79, two other histone H3 lysine residues,
K4 and K36, are known to be methylated in both Saccharomyces
and higher eukaryotes (
ROGUEV et al. 2001;
STRAHL et al. 2002;
LEE and SKALNIK 2005). We studied mutants blocked in each of
these methylations to determine if they too played a role in
IR resistance, as is the case for H3 K79 methylation. H3 K4
methylation resembles that of H3 K79 in being dependent on prior
ubiquitination of histone H2B K123 for di- and trimethylation
of the lysine residue (
DOVER et al. 2002;
SUN and ALLIS 2002;
SHAHBAZIAN et al. 2005). Methylation of H3 K4 is carried out
by the SET1 protein complex, also known as COMPASS, which is
thought to include at least eight component proteins (
MILLER et al. 2001;
ROGUEV et al. 2001;
KROGAN et al. 2002;
SCHNEIDER et al. 2005).
Information is available concerning the IR sensitivity ranking
for homozygous diploid deletion mutants involving six of the
COMPASS-encoding genes relative to the rest of the mutants in
a pooled deletion library after a single dose (200 Gy) of IR
(
BROWN et al. 2006). These mutants are deleted for
BRE2,
SDC1,
SHG1,
SPP1,
SWD1, and
SWD3, respectively. This assay involves
microarray hybridization to assess the relative prevalence of
molecular markers for each mutant relative to the whole pool
(
BIRRELL et al. 2001;
GAME et al. 2003;
BROWN et al. 2006).
While the assay is less rigorous than survival curves, none
of the six COMPASS-component mutants tested in this way came
within the top 20% of mutants ranked in the pool for IR sensitivity
(
BROWN et al. 2006), collectively arguing strongly against a
significant role for the COMPASS complex in ensuring diploid
survival after IR. Additional observations based on qualitative
assays of replica plates with patches of haploid cultures of
the same mutants also showed no evidence of sensitivity. To
confirm lack of sensitivity, we assayed survival
vs. dose for
one of these mutants, the
MAT
haploid deleted for the
SWD1 gene,
and found sensitivity equivalent to that of wild type (
Figure 1).
To test for a role for H3 K36 methylation in IR resistance,
we studied the
set2
mutant, since Set2p is responsible for methylating
this residue (
STRAHL et al. 2002). We observed that the
set2
MAT
haploid strain showed mild X-ray sensitivity in survival
curves, which was reproducible in
set2
spore clones derived
from a backcross of this strain to wild type (
Figure 1). A clear
segregation for sensitivity in this cross was difficult to observe
on replica plates, although segregation for a borderline X-ray-sensitive
phenotype was apparent after 1540 Gy of X-rays. A homozygous
set2
/set2
diploid strain constructed from our spore segregants
also showed a small increase in sensitivity compared to a wild-type
diploid (not shown). In addition, the
set2 diploid from the
deletion library pool showed a ranking of 105 for relative growth
after IR treatment (
BROWN et al. 2006), consistent with mild
sensitivity. We conclude that methylation of histone H3 K36
plays at least a minor role in resistance to radiation.
Mutants defective in histone H2 K123 ubiquitination are X-ray sensitive:
We reported earlier (
GAME et al. 2005) that either yeast strains
deleted for the
DOT1 gene, whose product methylates the histone
H3 K79 residue (
NG et al. 2002b), or yeast strains in which
the H3 K79 residue is altered to another amino acid, showed
sharply increased X-ray sensitivity compared to wild type. At
the same time, work from other laboratories showed that methylation
of histone H3 K79 as well as H3 K4 is dependent on prior ubiquitination
of histone H2B at residue K123 (
BRIGGS et al. 2002;
NG et al. 2002a).
The H2B K123 ubiquitination reaction has been shown to result
from the combined action of the
RAD6-encoded ubiquitin conjugase
and the
BRE1-encoded ubiquitin ligase (
ROBZYK et al. 2000;
HWANG et al. 2003).
IR sensitivity in
rad6 mutants was first reported in 1968 (
COX and PARRY 1968)
and is well known (
GAME and MORTIMER 1974;
LAWRENCE 1994) but
has been thought previously to result from the interaction of
Rad6p with Rad18p and their joint role in the ubiquitination
of proliferating cell nuclear antigen (PCNA) (
HOEGE et al. 2002).
The
dot1
mutant's IR sensitivity implied that the role of
RAD6 in H2B K123 ubiquitination might also contribute to the sensitivity
conferred by
rad6
, and we anticipated that
bre1
itself should
confer X-ray sensitivity comparable to that conferred by
dot1
but less than that conferred by
rad6
. In addition, deletion
mutations involving the
LGE1,
RTF1, and
PAF1 genes have also
each been reported to abolish H2B K123 ubiquitination (
HWANG et al. 2003;
NG et al. 2003a;
WOOD et al. 2003b) and hence might also be
expected to confer IR sensitivity. While Lge1p directly interacts
with Bre1p, both Rtf1p and Paf1p are members of the separate
Paf1/RNA polymerase II complex and may have additional effects
on other histone modifications (
MUELLER and JAEHNING 2002;
KROGAN et al. 2003).
We therefore tested X-ray sensitivity in
bre1
,
lge1
,
rtf1
, and
paf1
deletion strains.
Initial plate tests indicated that MAT
haploid strains carrying any of bre1
, lge1
, or rtf1
showed clearly increased X-ray sensitivity compared to wild type, whereas the paf1
mutant showed at most marginal sensitivity. The bre1
, lge1
, and rtf1
diploids from the deletion library pool show rankings of 36, 51, and 32, respectively, for relative growth after IR treatment (BROWN et al. 2006), consistent with sensitivity and in the same range as the dot1
mutant (rank 42). The paf1
mutant, which has a slow-growth phenotype (SHI et al. 1996), is not ranked in the pool assay. As done with other mutants from the library (GAME et al. 2005), we backcrossed each of the mutant MAT
haploid strains to a wild-type strain (g1201-4C) carrying the same genetic background as the deletion library to confirm haploidy and to test whether the X-ray-sensitive phenotype cosegregates with the geneticin-resistance phenotype marking the known deletion mutation. Results are shown in Table 1, where it can be seen that a cosegregation for both phenotypes occurs in crosses of bre1
, lge1
, and rtf1
, confirming that the deletion itself is responsible for conferring the X-ray sensitivity. For paf1
, no segregation for X-ray sensitivity was apparent, but a convincing cosegregation was observed for the geneticin-resistance marker and a slow-growth phenotype conferred by the original mutant (see SHI et al. 1996).
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TABLE 1 Meiotic spore viability and cosegregation data for four deletion-mutant heterozygous diploids
|
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Next, to quantify sensitivity, we performed X-ray survival curves
for at least two haploid strains carrying each of the deletion
mutations, using the original strains from the deletion library
and one or more spore clones derived from the crosses to wild
type. Results are shown in
Figures 2 and
3, where an additional
dot1
mutant survival curve is shown for comparison. It can be
seen that the
bre1
,
lge1
, and
rtf1
mutations confer sensitivity
comparable to that seen in
dot1
strains, consistent with a repair
defect that in each case arises from abolition of the Dot1p-mediated
histone H3 K79 methylation. In the case of
BRE1, we further
confirmed that the deletion itself conferred the IR sensitivity
by transforming a
bre1
strain with a plasmid containing the
BRE1 gene and finding that this restored wild-type resistance
(
Figure 2). Surprisingly, however, the
paf1 deletion mutant
shows a wild-type response to IR, in contrast to the other three
mutants (
Figure 3). We considered that the lack of sensitivity
of
paf1
might arise from a secondary mutation in the mutant
strain acting as a suppressor or modifier, but rejected this
as unlikely when we found a uniform lack of sensitivity in
paf1
spore clones segregating from a cross with wild type, as judged
from irradiated replica plates. A survival curve of one of these
paf1
spore clones shown with the original mutant in
Figure 3 resembles wild type, and a curve from another spore clone (not
shown) was equivalent. We note that Paf1p has multiple functions
in addition to facilitating H2 K123 ubiquitination (
CHANG et al. 1999;
KROGAN et al. 2003;
MUELLER et al. 2004;
SHELDON et al. 2005),
and it seems possible that the slow-growth phenotype of
paf1
counteracts the expected IR sensitivity, as discussed later.
Mutants defective in histone H2 K123 ubiquitination or H3 K79 methylation interact epistatically for IR sensitivity:
If IR sensitivity in mutants defective in H2B K123 ubiquitination
arises from their downstream effects on H3 K79 methylation,
then combining
bre1
and
lge1
with each other or with
dot1
should
add no additional sensitivity. We constructed strains with each
of the double-mutant genotypes involving these three genes,
as well as triple-mutant strains.
Figures 46

show that
all three genes interact epistatically. The data are compelling
for
bre1
lge1
(
Figure 4),
bre1
dot1
(
Figure 5), and the
bre1
dot1
lge1
triple-mutant strain (
Figure 6). For
dot1
lge1
strains,
we observed some scatter among strains of equivalent genotype,
with two strains showing possibly increased sensitivity and
a third falling closer to the single mutants (
Figure 6).
Both rad6
and rad51
are epistatic to dot1
:
Most previously characterized mutants that show substantial
X-ray sensitivity in Saccharomyces either are defective in HRR,
mediated by
RAD51,
RAD52, and related genes, or are defective
in one or more aspects of postreplication repair/translesion
synthesis that are dependent on the
RAD6 and
RAD18 genes.
Mutants in the latter group, including rad6
and rad18
, confer additional sensitivity in double-mutant combinations with rad51
(MCKEE and LAWRENCE 1980; GAME 2000; see Figure 7), supporting the view that these repair processes are essentially separate. However, rad6
mutants show substantially greater X-ray sensitivity than rad18
mutants (see Figure 8), although rad6
and rad18
mutants are equally defective in ubiquitination of PCNA, which is a prerequisite for the subsequent steps of postreplication repair/translesion synthesis (HOEGE et al. 2002; STELTER and ULRICH 2003; HARACSKA et al. 2004). This suggests an additional role for RAD6 in mediating IR resistance outside the PCNA ubiquitination pathway. Further support for a separate role for RAD6 in DNA transactions may come from the fact that rad6 mutants are completely defective in meiotic division and fail to commit to meiotic recombination (GAME et al. 1980), whereas rad18 mutants show little if any meiotic phenotype (GAME and MORTIMER 1974; DOWLING et al. 1985).
Given the X-ray sensitivity of
bre1
, we anticipated that this
additional role for
RAD6 could be mediated by its involvement
in H3 K79 methylation through its function with
BRE1 in H2B
K123 ubiquitination, and this in turn could involve the
RAD51-dependent
HRR pathway. We therefore constructed double mutants involving
dot1
with
rad6
and with
rad51
.
Figure 7 shows that
dot1
confers
no additional X-ray sensitivity when combined with either
rad6
or
rad51
. However, we and others have observed that
rad6
single
mutants tend to vary in radiation sensitivity and to quickly
pick up modifier mutations, especially in the
SRS2 gene (
SCHIESTL et al. 1990).
To address possible variation here, we performed seven survival
assays involving six
rad6
strains. In comparing double mutants
with r
ad6
, we show either the curve with the median IR sensitivity
(
Figures 8,
9,
12, and
17) or a
rad6
strain from the same cross
as the double mutant to which we compare it (
Figure 7). Also,
we show both the most sensitive and the least sensitive of the
six strains in
Figure 10. The
dot1
rad6
double mutant (g1238-2B,
Figure 7) has a sensitivity equivalent to the most related
rad6
single mutant (g1238-7B,
Figure 7) and very similar to that
of the median
rad6
strain (MW5094-8A, shown on the same scale
in
Figure 17). Hence, we conclude that
DOT1 mediates a pathway
of radiation resistance that requires the
RAD6 gene but also
facilitates HRR, thus demonstrating a role for
RAD6 in enabling
effective HRR.
The bre1
, lge1
, and dot1
mutations add extra IR sensitivity when combined with the rad18
mutation:
Given that the HRR mutation
rad51
is epistatic to
dot1
, we expected
that the latter mutation would confer increased sensitivity
in double-mutant combinations with
rad18
, since
RAD18 is known
to act in postreplication repair (PRR) and itself interacts
additively with mutants in HRR (
MCKEE and LAWRENCE 1980;
GAME 2000).
Figures 8 and
9 show that there is a strong, rather similar
increase in sensitivity in each of the double mutants that we
constructed involving
rad18
with
bre1
,
lge1
, or
dot1
compared
to the component single mutants. This both confirms that histone
H3 K79 methylation is not involved in PRR and supports the functional
separation of PRR from the HRR pathway.
Figure 10 shows that
the
dot1
bre1
rad18
and
dot1
lge1
rad18
triple mutants as well
as a quadruple mutant involving
bre1
,
lge1
, and
dot1 with
rad18
fall within the range of these doubles, further confirming epistasis
of
bre1
,
lge1
, and
dot1
. It is noteworthy that these strains,
and specifically the
bre1
rad18
double mutant (
Figure 8), which
is defective in ubiquitination of two separate repair-involved
targets of the
RAD6 ubiquitin ligase, resemble the
rad6
single
mutant in sensitivity. The median curve in
Figure 8 as well
as the
rad6
curves in
Figure 10 confirm that the additional
sensitivity of the
rad6
single mutant compared to the
rad18
mutant can be accounted for by the role of
RAD6 in the
BRE1-mediated
histone ubiquitination step. However, as noted below, we also
tested the N-end rule protein ubiquitination activity of
RAD6 for a possible effect on IR resistance by studying
ubr1
mutant
strains.
A role in IR repair for the RAD6-dependent UBR1 ubiquitin ligase:
The
UBR1 gene encodes the ubiquitin ligase that interacts with
Rad6p in its major role in poly-ubiquitinating proteins targeted
for degradation according to the N-end rule (
DOHMEN et al. 1991).
This pathway is not specific to DNA repair, but
ubr1
mutants
have been found to affect chromosome stability, probably through
an indirect effect on sister-chromatid cohesion by affecting
the degradation pathway for cohesin (
RAO et al. 2001). The
ubr1
diploid from the deletion library pool showed a ranking of 38
for relative growth after IR treatment (
BROWN et al. 2006),
consistent with IR sensitivity, and we found a mildly increased
sensitivity in
ubr1
haploid survival curves, as shown in
Figure 11.
A mild sensitivity on plate tests appeared to cosegregate with
the
ubr1
allele in crosses (not shown). To determine if the
sensitivity is manifested through an effect on the
RAD18 or
BRE1 pathways or perhaps neither of these, we made double and
triple mutants involving
ubr1
,
rad18
, and
bre1
. We found little
or no increased sensitivity in
bre1
ubr1
double mutants (
Figure 11),
but a significant increase in
rad18
ubr1
doubles (
Figure 12).
This enhancement of
rad18
sensitivity is consistent with a role
for
UBR1 in HRR, as might be expected from the reported effects
of
ubr1
on chromosome stability and cohesin degradation (
RAO et al. 2001).
Given the mild sensitivity of
ubr1
, it is less compelling that
BRE1 is really epistatic to
UBR1. However, the
rad18
bre1
ubr1
triple mutants shown in
Figure 12 resemble the
rad18
bre1
double
mutant as well as the
rad6
single mutant. We expect this triple-mutant
genotype to mimic
rad6
since it should lack all three known
ubiquitination activities that
RAD6 mediates, but a potential
contribution to IR sensitivity from
ubr1
in the triple mutant
might be difficult to discern in the context of the high sensitivity
of the
rad18
bre1
double mutant, which already resembles
rad6
(see above).
The rad5
mutation is additive for IR sensitivity with bre1
and with rad18
:
The Rad5 protein acts downstream from Rad18p in the ubiquitination
steps of PCNA and thereby plays a major role in postreplication
repair (
HOEGE et al. 2002;
TORRES-RAMOS et al. 2002). However,
while
rad5
and
rad18
interact epistatically with respect to
UV sensitivity (
JOHNSON et al. 1992; this study; data not shown),
we observed an additive response for IR sensitivity (
Figure 14),
in agreement with other reports (
FRIEDL et al. 2001;
CHEN et al. 2005).
In addition,
CHEN et al. (2005) presented data showing that
RAD5 has another function that contributes to IR resistance
independently of its PCNA-modifying role and is probably related
to an
MRE11/RAD50/XRS2-mediated repair activity. To study
RAD5 in relation to the
BRE1/DOT1 pathway, we constructed
rad5
bre1
and
rad5
dot1
double mutants.
Figures 13 and
14 show that the
rad5
mutation adds sensitivity to
bre1
and
dot1
as well as to
rad18
. When taken with data for
rad18
combined with
bre1
and
dot1
(
Figures 8 and
9), this implies that
RAD5,
RAD18, and
BRE1/DOT1 mediate three at least partly independent IR resistance mechanisms.
Surprisingly, the
dot1
rad5
rad18
triple shows only slight sensitivity
beyond each of the component double mutants (
Figure 14). It
is difficult to assess the significance of this, but it seems
less sensitive than would be expected from double-mutant data.
Roles for Rad5p in more than one IR repair pathway might account
for this, as discussed later.
IR epistasis and colony-size effects of rad50
with bre1
and dot1
:
We constructed double and triple mutants involving
bre1
,
dot1
,
and
rad50
to test whether the Mre11/Rad50/Xrs2 complex (MRX)
is involved in repair affected by histone H3 K79 methylation.
Figure 15 shows that combining
bre1
,
dot1
, or both mutants with
rad50
adds no further sensitivity to
rad50
alone, as might be
expected from the role of MRX in HRR (
BRESSAN et al. 1999;
GAME 2000)
and our double-mutant data with
rad51
. As discussed later, there
is no support for a separate IR damage repair role involving
nonhomologous end-joining (NHEJ) from these data, since the
strains in
Figure 15 have survival curves equivalent to those
of the
rad51
mutant included for comparison. However, we did
observe a strong effect of the
rad50
bre1
double mutant and
the
rad50
bre1
dot1
triple-mutant genotypes on the colony size
of meiotic spore clones, which was sharply reduced compared
to that of other spore clones in the same cross. This implies
a slow-growth phenotype presumably caused by interaction of
rad50
with
bre1
and confirms similar findings from large-scale
random spore analysis (
TONG et al. 2004). Since this phenotype
was absent in our
rad50
dot1
double-mutant spore clones, it
is presumably not mediated by abrogation of H3 K79 methylation.
However, synthetic lethality has been reported (
TONG et al. 2004)
between
rad50
and two mutants for genes in the COMPASS complex,
swd3
and
bre2
, responsible for methylating the histone H3 K4
residue (
KROGAN et al. 2002). Since di- and trimethylation of
this residue depends on histone H2 K123 ubiquitination, it is
plausible that the slow-growth phenotype of
rad50
bre1
double
mutants also arises from the impact of
bre1
on H3 K4 methylation.
A role for the BRE1/DOT1 pathway in IR-damage-induced checkpoint control:
While this work was in progress, several reports suggested that
histone H2B K123 ubiquitination and histone H3 K79 methylation
are important for checkpoint arrest after DNA damage. It was
recently shown that the 53BP1 checkpoint protein in mammalian
cells recognizes and binds to methylated histone H3 K79 residues
and that the methylation is important for attracting 53BP1 to
double-strand-break (DSB) sites (
HUYEN et al. 2004). Saccharomyces
Rad9 protein, which has a central role in establishing checkpoint
delays after irradiation (
WEINERT and HARTWELL 1988,
1989;
SIEDE et al. 1993),
shares homologous domains with 53BP1, including a recently described
major domain very similar to the Tudor domain in 53BP1 that
interacts with methylated mammalian H3 K79 (
ALPHA-BAZIN et al. 2005).
Hence, the mammalian 53BP1 findings are strongly suggestive
of a role for H3 K79 methylation in
RAD9-mediated checkpoints
in yeast. In addition, others have shown directly that
rad6
,
bre1
, and
dot1
mutants reduced or abolished the checkpoint delay
seen in wild type after UV- and chemical DNA-damaging treatments
in G
1 and intra-S phase cells without affecting the G
2 checkpoint
(
GIANNATTASIO et al. 2005). These authors also showed that phosphorylation
of Rad9 protein was reduced or abolished in these mutants after
similar DNA-damaging treatments, leading in turn to defective
activation of Rad53 checkpoint protein. Most recently,
WYSOCKI et al. (2005) identified
dot1
in a screen for mutants that abrogate the G
1 damage checkpoint. Surprisingly, however, using more qualitative
tests, these authors found little evidence of IR sensitivity
in the
dot1
mutant. Genetic differences in the strain backgrounds
used probably account for these different results (see DISCUSSION).
To investigate whether the substantial IR sensitivity of
bre1
and
dot1
mutants in our strains is conferred through an effect
on checkpoint controls, and to extend the epistasis analysis,
we studied double mutants involving
rad9
as well as tested directly
for abrogation of checkpoints in
bre1
and
dot1
mutants using
IR.
Double-mutant analysis with rad9
:
Since
RAD9 has a well-established role in DNA-damage-induced
checkpoint controls (
WEINERT and HARTWELL 1988,
1989;
SIEDE et al. 1993),
we anticipated that if
bre1
confers sensitivity through abrogating
one or more
RAD9-dependent checkpoints, epistasis between
rad9
and
bre1
would be observed, whereas an additive response would
suggest a different mechanism. Mutations in the
RAD9 gene are
known to be IR sensitive (
COX and PARRY 1968;
GAME and MORTIMER 1974),
but surprisingly, we could find little published information
about the epistasis relationships of
rad9 in combination with
mutants in other
RAD genes. An exception is the
rad9
rad6
combination,
which has been reported to show additive sensitivity compared
to the single mutants (
SCHIESTL et al. 1989). It has also been
shown that activation of some Saccharomyces HRR proteins after
radiation is dependent on an intact
RAD9 gene (
BASHKIROV et al. 2000)
and that the ATM-mediated checkpoint is required for normal
function of the
RAD54 HRR gene in chicken DT40 cells (
MORRISON et al. 2000).
We tested the double-mutant
rad9
bre1
and the triple-mutant
rad9
bre1
dot1
in the deletion library background, where each
of the single mutants is sensitive, to determine whether
RAD9 affects the same pathway as
BRE1 and
DOT1, as might be expected
on the basis of results with mammalian cells (
HUYEN et al. 2004).
We found that the rad9
single mutant is significantly more sensitive than the bre1
or dot1
strains, but that double or triple mutants involving rad9
with either or both of these two are no more X-ray sensitive than rad9
alone (Figure 16). This agrees with findings by WYSOCKI et al. (2005), who observed qualitatively that dot1
did not potentiate the sensitivity of rad9
in a different genetic background where there was little or no IR sensitivity of the dot1
single mutant. The epistasis implies either that the X-ray resistance mediated by BRE1 and DOT1 is dependent on a RAD9-mediated checkpoint or that the RAD9 checkpoint itself is partially dependent on an intact BRE1/DOT1-mediated H3 K79 methylation pathway. In the latter case, which is consistent with data from mammalian cells (HUYEN et al. 2004), RAD9 function is presumably only partly dependent on H3 K79 methylation, since substantial RAD9-dependent resistance remains in bre1
and dot1
single mutants (Figure 16).
On the basis of our observations that
rad51null and
rad9
are
each epistatic to
bre1
and
dot1
, we expected that
RAD9 itself
would fall into the
RAD51 epistasis group with respect to X-ray
sensitivity. This would also be consistent with reported additivity
between
rad9
and
rad6
(
SCHIESTL et al. 1989) since known mutants
in HRR such as
rad51null show increased sensitivity in combination
with
rad6
(
Figure 17; see also
MCKEE and LAWRENCE 1980;
GAME 2000).
Since we find no additivity between
bre1
and
rad9
,
increased
sensitivity contributed by
rad6
in the
rad6
rad9
double mutant
seems likely to arise from the
RAD18-dependent aspect of
RAD6 repair. To test this, we studied double mutants involving
rad9
with
rad18
,
rad51null, and
rad5
as well as retesting the
rad6
rad9
combination.
We found a sharp increase in sensitivity in rad9
rad18
(Figure 18), a slightly lesser increase in rad9
rad5
(Figure 18), and, at most, only a slight increase in the rad9
rad51null strains (Figure 17), compared to the single mutants in each case. Hence RAD18-mediated repair seems to be largely independent of the RAD9-mediated checkpoint, whereas RAD51-mediated HRR is heavily dependent on RAD9 function. As expected from these observations as well as previous work (SCHIESTL et al. 1989), we found that the rad6
rad9
double mutant also shows strongly increased IR sensitivity compared to the single mutants. In fact, this double mutant is equivalent to rad6
rad51null double mutants (Figure 17), again suggesting that RAD9 is required for most or all RAD51-mediated IR recovery. The equivalent sensitivity of the rad9
rad18
double mutant to rad9
rad6
and rad6
rad51null, despite the lower sensitivity of rad18
compared to rad6
, also implies that RAD9 is required for the part of RAD6-mediated resistance that is independent of RAD18 and that this, in turn, is dependent on RAD51. The rad9
rad6
rad51
triple mutants in Figure 17 are possibly slightly more sensitive than the rad6
rad51
double mutants; hence a minor additional role for RAD9 outside either PRR or HRR cannot be excluded. Interestingly, in double-mutant combinations both rad9
and rad18
also show increased sensitivity with rad5
, but the rad5
rad9
rad18
triple mutants in Figure 18 are no more sensitive than the rad9
rad18
double mutants, perhaps implying that RAD5 acts in more than one pathway, as discussed later.
The dot1
and bre1
mutants are defective in the G1 but not the G2 IR-induced cell cycle checkpoint:
Wild-type yeast cells irradiated in the G
2 phase of the cell
cycle become arrested before proceeding through cell division.
This arrest is dependent on the
RAD9 gene and is important for
subsequent cell survival:
rad9 mutants substantially fail to
arrest in G
2 after irradiation, and this is thought to be part
of the reason for their increased killing by IR compared to
wild type (
WEINERT and HARTWELL 1988). Using the
rad9
mutant
and wild type as controls, we tested haploid
dot1
and
bre1
mutants
for an effect on the IR-induced G
2 checkpoint by monitoring
their ability to progress into mitosis following a nocodazole-induced
accumulation in G
2.
Figure 19 shows that, as expected, without
irradiation, all four strains promptly enter nuclear division
when released from nocodazole. After 500 Gy of
137Cs gamma irradiation,
however, wild-type,
dot1
, and
bre1
cells remain arrested, with
little sign of division up to 90 min following irradiation.
In contrast,
rad9 shows significant escape from the G
2 checkpoint,
although at this dose it too shows delayed division compared
to the unirradiated control. Results are similar after 1000
Gy of radiation (not shown), and together these data indicate
that
dot1
and
bre1
mutations do not significantly abrogate the
IR-induced G
2 cell cycle checkpoint.
To assess radiation-induced G
1 arrest, we followed the cell
cycle progression of
dot1
and
bre1
mutants released from

-factor-induced
synchrony with and without 500 Gy of
137Cs gamma irradiation
and, as reported by others (
GIANNATTASIO et al. 2005;
WYSOCKI et al. 2005),
observed a significant effect of both mutants in abrogating
the arrest response seen in wild type (see
Figure 20). In fact,
both mutants are essentially equivalent in this phenotype to
the
rad9 strain. We used
rad9 as a positive control because
previous work (
SIEDE et al. 1993) has shown that
RAD9 is involved
in the G
1 checkpoint as well as in the G
2 checkpoint. The three
strains differ from wild type, where IR-induced arrest can clearly
be seen. Despite differences in IR sensitivity, our findings
of a G
1 but not a G
2 checkpoint defect in
dot1
or
bre1
mutants
in the deletion library background are thus consistent with
recent findings by others in the W303 background (
GIANNATTASIO et al. 2005;
WYSOCKI et al. 2005).