Figure S1. Examples of the terminal phenotypes of UV-irradiated cells. Cells that failed to divide during the 12-24 h
period of time-lapse imaging were assigned terminal phenotypes. The number of cells with each phenotype are plotted
above for the 1st, 2nd, and 3rd cycles after UV irradiation (out of 300 irradiated cells, 600 daughter cells, 1200
granddaugther cells). For the rad51∆ experiments, only cells that divided at < 17 µM at the first cleavage are included in
the analysis. The smaller sample sizes for the rad51∆ analysis are indicated under the plot.
Cells that turned black, abruptly lost size, and exhibited static cytoplasm were assigned a “dead cell” phenotype:
d = dead cell
ds = died with a division septum in place
dc = died immediately upon cleavage
Cells that did not grow in length for >1 h were assigned the following phenotypes:
ng = no growth after birth
sg = stopped growing
nc = no cleavage (formed a division septum, stopped growing, and never underwent cleavage)
Cells that stop growing in length for >1 h rarely resume growth and division, even over extended observation periods.
So most cells with “ng”, “sg”, and “nc” phenotypes are not viable in the sense that they do not form colonies, although
they often maintain a somewhat normal appearance (normal cell width and dynamic cytoplasm indicative of metabolic
activity). Cells that were growing slowly when the field became too crowded for observation were scored as “na”
(terminal phenotype not available).
Figure S2. The DNA repair defects of rad51∆ and mre11∆ cells are partially suppressed by spontaneous checkpoint delays. Data
obtained from the time-lapse movies described in Fig. 1 are analyzed further. (A,B) The percentages of cells that underwent cleavage are
shown as a function of time for WT cells and HDR mutants. The results are shown for all 300 cells in the initial population. HDR mutants
divide more slowly than WT cells. As with other DDT mutants, a UV dose of 5 J/m2 does not alter the kinetics of cell cycle progression for
the first cycle after irradiation in HDR mutants. (C-F) The moving average of cell viability (3rd cleavage success) and cell length at the
second cleavage are plotted as a function of the duration of the first cell cycle. A 15 min window average was calculated at 1 min
increments. The UV-induced loss of viability and checkpoint-mediated length increase are both decreased in cells that undergo
spontaneous delays during the first cycle (e.g. > 2 s.d. above the mean for wild-type cells, or > 2.5 h cycle times). The likely explanation for
this phenomenon is that cell undergoing spontaneous delays have more time for excision repair prior to S phase, so a reduced number of
UV lesions are carried into S phase and fewer abnormal structures that require HDR for repair are formed during DNA replication. (G,H)
Selecting rad51∆ and mre11∆ cells that are less than 17 µM in length at the first cleavage yields a population of cells that proliferate with
the kinetics of WT cells, allowing us to ask the question: what is the phenotype of rad51∆ and mre11∆ cells if we eliminate the
suppressive effects of spontaneous checkpoint delays? Because this subpopulation of HDR mutant cells cycles like a WT population, we
can approximate cell cycle stages at the time of irradiation using the first cleavage time as with WT cells and other DDT mutants.
Figure S3. Elimination of Rad51 has a dramatically different effect on the duration of the
checkpoint responses to UV than elimination of Mre11. Moving averages of length at the 2nd
cleavage (a measure of the checkpoint response) and cell viability are plotted as described in figure
3. To compare the phenotype of HDR-defective cells to WT cells, it was necessary to control for the
spontaneously occurring checkpoint delays and cell death in these strains. Only cells that were < 17
µM in length at the first cleavage were analyzed as described in figure S2 to eliminate spontaneous
delays that occurred during the first cycle. To control for spontaneous checkpoint delays during the
second cycle in the top panel, the average length of a mock irradiated population at the 2nd cleavage
was subtracted from the data for cells exposed to 5 J/m 2. To control for spontaneous cell death in the
bottom panel, viabilities are expressed relative to the mean viability of a mock irradiated control. In
contrast to rad51∆ cells, mre11∆ cells have normal checkpoint delays, indicating that Mre11 is not
required for the repair of daughter-strand gaps at UV lesions or to generate the checkpoint signal that
delays cleavage in WT cells. Because mre11∆ cells completely lack the capacity to repair DSBs,81
this result also indicates that the duration of the UV-induced checkpoint response in WT cells is
unrelated to the presence of UV-induced DSBs. Surprisingly, cells lacking Mre11 showed reduced
survival after exposure to 5 J/m2 of UV, suggesting that HDR may be required to repair UV-induced
DSBs in some cells. Thus, DSBs may form after 5 J/m 2 in WT cells but fail to influence the duration of
the checkpoint response, perhaps because they are rare relative to daughter-strand gaps and are
typically completely repaired prior to the completion of PRR. This interpretation requires that the
DSBs formed in mre11∆ cells do not elicit a checkpoint response because they are too few in number
or because Mre11 is required for DSB-induced checkpoint activation (fission yeast mre11∆ cells
exhibit checkpoint delays in response to ionizing radiation,82 but this response could potentially be the
result of base damage or single-strand breaks rather than DSBs). Alternatively, Mre11 might repair
structures whose production is delayed for some reason until the refractory period of the 2 nd cycle or
some point during the 3rd cycle.
Figure S4. Cell-to-cell variability in the duration of the S. pombe cell cycle is mostly due to variability in the
duration of G2 phase. A strain expressing GFP fused to alpha-tubulin was imaged at 2 min intervals over the course of 6
hours. The times of birth, formation of the mitotic spindle (determined as described in detail in figure S8), and cleavage
were recorded while manually inspecting ~200 cells. (A) Duration of the cell cycle (cleavage to cleavage interval) is highly
correlated with the duration of G2 phase (defined as the interval between birth and spindle assembly) in individual cells.
The linear correlation coefficient is 0.975. (B) Duration of the cell cycle is not correlated with the remaining interval
between spindle assembly and the subsequent cleavage (linear correlation coefficient -0.044). (C) The means and
standard deviations are shown for the overall cell cycle times and the durations of the G2 phase and post-G2 phase
periods. Standard deviation in the duration of G2 phase is slightly greater than that of the overall cycle time because there
is more error involved with scoring the time of spindle assembly than the time of cell cleavage (cycle time calculations do
not require scoring spindle assembly). The standard deviation in the post-G2 phase duration is not much larger than
measurement error. Calculation of G2 and post-G2 periods involves identical measurement errors, so the variability in
these parameters can be compared more directly. Such a comparison gives a conservative estimate of the variability in
the cell cycle attributable to G2 phase  80%  with the actual value likely to be significantly higher.
Figure S5. Elimination of all excision repair prolongs the second
cycle after UV irradiation. Cleavage kinetics are plotted for three cell
cycles after exposure of wild-type cells and excision repair defective cells
to 5 J/m2 of UV.
Figure S6. Comparison of wild-type cells and DDT mutants exposed to a UV dose of 5 J/m2. Populations of 300
cells were exposed to 5 J/m2 and imaged by time-lapse microscopy. In the case of rad51∆ cells, only those cells that
underwent 1st cleavage at < 17 µM in length are plotted as described in figure S2. The checkpoint response and cell
viability are plotted as a function of progress through the cell cycle as described in Fig. 3. Because the phenotype of
mutagenic TLS mutants is not much larger than experimental error at 5 J/m 2, a dose of 10 J/m2 is shown in the main text.
When cells reach ~35 µM in length, increasing the UV dose not increase cell length (data not shown). Likewise, when
cells are at 10-20% viability, increasing the UV dose does not substantially decrease viability in our assay. Because the
checkpoint delay and loss of viability approach these stationary levels in rad51∆ cells exposed to 5 J/m2 (e.g. the
relevant phenomena are not entirely in the dynamic range of our assay), a UV dose of 2 J/m 2 was shown for rad51∆ cells
in the main text.
Figure S7. Rad51 and Pol function in separate pathways. The cleavage kinetics of cells lacking
Rad51 are compared to those of cells lacking both Rad51 and Pol activity. All 300 irradiated cells and
their progeny are included in the analysis. Elimination of Pol activity from rad51∆ cells increased UVinduced cleavage delay and reduced viability (3rd cleavage success after 5 J/m2: rad51∆ = 31%,
rad51∆eso1-cs = 11%). Thus, Pol carries out a function that is separable from HDR.
Figure S8. Scoring cell cycle landmarks. Asynchronous cell population were imaged at 2 min intervals using
confocal microscopy (A-C) or widefield microscopy (D) under illumination conditions that did not effect the duration of
the cell cycle. (A) The time of mitotic spindle assembly was determined using a strain expressing GFP fused to alphatubulin. When cells transit from G2 phase into mitosis, the interphase microtubules that run the length of the cell
abruptly disappear and a bright dot appears at the middle of the cell, indicating that mitotic spindle assembly has
begun.83 (B,C) The time of nuclear separation and spindle disassembly were determined using a strain expressing
Rad11-mYFP, which allowed visualization of nuclei. The time of nuclear separation was defined as the first frame in
which the nuclei are fully separated. After nuclei are pushed to the cell poles during mitosis, the mitotic spindle
disassembles and the nuclei abruptly reverse their migration and move back towards the middle of the cell.84 The time
of spindle disassembly was defined as the first frame in which this reversed migration was evident. (D) The time of
septation was defined as the first frame in which a septum was clearly visible. This landmark was the most difficult to
score because septum formation is not a discrete event when cells are imaged at 2 min intervals but occurs slowly
over time. For this reason, the cell cycle illustrations in this paper use the point of maximal checkpoint response to UV
as a more accurate estimate of the beginning of S phase.
For figure 2C (main text), the percent of cells at a given stage were calculated using the following formulas:
G2 =
prophase – anaphase B =
anaphase B =
G1 =
S=
100 minus % past mitotic spindle assembly
% past mitotic spindle assembly minus % past nuclear separation
% past nuclear separation minus % past spindle disassembly
% past spindle disassembly minus % past septation
% past septation
Table S1. S. pombe strains used in this study
Strain
Genotype
AJC-G9
AJC-E57
AJC-F17
AJC-D12
AJC-D15
AJC-N24
AJC-L69
AJC-J29
AJC-D69
AJC-H23
AJC-G64
AJC-G6
AJC-F7
AJC-F53
AJC-G67
AJC-F56
AJC-E64
AJC-F4
smt0 leu1-32
smt0 rad8::ura4+ leu1-32 ura4-D18
smt0 rhp18::ura4+ leu1-32 ura4-D18
smt0 rad32MRE11::ura4+ leu1-32 ura4-D18
smt0 rad51::ura4+ leu1-32 ura4-D18
smt0 lys1::Pnmt1-GFP-ATB2 leu1-32
smt0 rad11-mYFP-kanMX leu1-32
smt0 hht1-mRFP-KanMX leu1-32
smt0 chk1::ura4+ leu1-32 ura4-D18
smt0 rhp14::kanMX uve1::leu2+ leu1-32
smt0 eso1::eso1-D147N-kanMX leu1-32
smt0 rad8::ura4 rhp18::ura4+ leu1-32 ura4-D18
smt0 rad8::ura4+ eso1::eso1-D147N-kanMX leu1-32 ura4-D18
smt0 rev1::ura4+ leu1-32 ura4-D18
smt0 rev3::kanMX leu1-32
smt0 rev1::ura4+ rev3::kanMX leu1-32 ura4-D18
smt0 rad51::ura4+ rev3::kanMX leu1-32 ura4-D18
smt0 rad51::ura4+ eso1::eso1-D147N-kanMX leu1-32 ura4-D18
Table S2. Mutant alleles used in this study
Allele (Source/Reference)
smt0 85
rad51::ura4+ 86
rad32MRE11::ura4+ 81
rev3::kanMX 87
rev1::ura4+ 88
eso1::eso1-D147N 88
rad8::ura4+ 89
rad11-mYFP (this study)
rhp18::ura4+ 90
lys1::Pnmt1-GFP-ATB2 91
hht1-mRFP-kanMX 92
rhp14::kanMX 93
uve1::leu2+ 94
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