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MBoC | BRIEF REPORT
DNA damage triggers increased mobility
of chromosomes in G1-phase cells
Michael J. Smitha, Eric E. Bryantb, Fraulin J. Josepha, and Rodney Rothsteina,*
a
Department of Genetics and Development, Columbia University Irving Medical Center, New York, NY 10032;
Department of Biological Sciences, Columbia University, New York, NY 10027
b
ABSTRACT During S phase in Saccharomyces cerevisiae, chromosomal loci become mobile in
response to DNA double-strand breaks both at the break site (local mobility) and throughout
the nucleus (global mobility). Increased nuclear exploration is regulated by the recombination
machinery and the DNA damage checkpoint and is likely an important aspect of homology
search. While mobility in response to DNA damage has been studied extensively in S phase,
the response in interphase has not, and the question of whether homologous recombination
proceeds to completion in G1 phase remains controversial. Here, we find that global mobility
is triggered in G1 phase. As in S phase, global mobility in G1 phase is controlled by the DNA
damage checkpoint and the Rad51 recombinase. Interestingly, despite the restriction of
Rad52 mediator foci to S phase, Rad51 foci form at high levels in G1 phase. Together, these
observations indicate that the recombination and checkpoint machineries promote global
mobility in G1 phase, supporting the notion that recombination can occur in interphase
diploids.
Monitoring Editor
Kerry S. Bloom
University of North Carolina
Received: Aug 26, 2019
Accepted: Aug 30, 2019
INTRODUCTION
After DNA damage, cells must pursue timely repair to preserve the
integrity of their genomes. Developmental factors, signaling milieu,
cell type, and the characteristics of the lesion play a role in the repair
systems employed. One of the critical determinants in repair pathway choice is progression through the cell cycle, which introduces
complex challenges to nuclear organization and DNA metabolism
(Mathiasen and Lisby, 2014; Hustedt and Durocher, 2016). The two
main repair strategies used to resolve double-strand breaks (DSBs)
are ligation via nonhomologous end joining (NHEJ) and homologous recombination (HR). During NHEJ in Saccharomyces cerevisiae, DSB ends are first bound by the Ku70/Ku80 complex before
This article was published online ahead of print in MBoC in Press (http://www
.molbiolcell.org/cgi/doi/10.1091/mbc.E19-08-0469) on September 4, 2019.
The authors declare no competing financial interests.
*Address correspondence to: Rodney Rothstein (rothstein@columbia.edu).
Abbreviations used: CFP, cyan fluorescent protein; DIC, differential interference
contrast; DSB, double-strand break; GFP, green fluorescent protein; HR, homologous recombination; MSD, mean-square displacement; NHEJ, nonhomologous end joining; RFP, red fluorescent protein; ssDNA, single-stranded DNA;
WT, wild type; YFP, yellow fluorescent protein.
© 2019 Smith et al. This article is distributed by The American Society for Cell
Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported
Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
“ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of
the Cell®” are registered trademarks of The American Society for Cell Biology.
2620 | M. J. Smith et al.
ligation is catalyzed by Dnl4, Lif1, and Nej1 (Palmbos et al., 2005).
HR, however, requires a homologous template elsewhere in the
genome, for example, either the sister chromatid in S phase or the
homologue in a diploid. The commitment to HR is thought to occur
following resection of the 5′ ends of the DSB (Mathiasen and Lisby,
2014). The MRX complex (Mre11, Rad50, and Xrs2) is critical for
initiating initial resection, while Sgs1, Exo1, and Dna2 are responsible for more extensive resection (Mathiasen and Lisby, 2014).
Following single-stranded DNA (ssDNA) generation, replication
protein A (RPA) is recruited to the 3′ ends and catalyzes ATR/Mec1
checkpoint signaling (Zou and Elledge, 2003), the recruitment of the
Rad52 recombination mediator, and the mitotic recombinase Rad51
(Sung et al., 2003; Lisby et al., 2004). Rad51 filaments then search
the genome for homology and catalyze strand invasion and repair
(Qi et al., 2015).
The differences in the repair of DSBs in G1 and S and in haploid
and diploid cells have been well studied. It has long been appreciated that diploid cells are more resistant to DSBs, which may be a
result of the presence of a homologous template throughout the
cell cycle (Friis and Roman, 1968; Heude and Fabre, 1993). This
difference extends to the G1 phase of the cell cycle, where evidence
indicates that G1 diploids are competent for HR and gene conversion (Luchnik et al., 1977; Esposito, 1978; Fabre, 1978; Lee and
Petes, 2010). The ability of both haploid and diploid cells to repair
DSBs depends on the characteristics of the break itself. So-called
“dirty” DSBs that require end processing are resected and prepared
Molecular Biology of the Cell
for HR, while “clean” breaks (formed by endonuclease cutting) are
predominantly repaired by NHEJ in haploids (Barlow et al., 2008). In
diploid cells, NHEJ is blocked by the a1/α2 repression of NEJ1
expression (Kegel et al., 2001), suggesting that even clean-break
repair events in G1 phase must occur by HR. However, other reports
indicate that HR requires S-phase CDK1 activation (Aylon et al.,
2004; Ira et al., 2004). In addition, the recruitment of Rad52 to repair
centers is cell cycle restricted to S phase in haploid cells (Lisby et al.,
2004; Barlow et al., 2008). Thus, it is unclear how recombination is
coordinated in the G1 phase.
Proper repair via HR requires the coordination of many enzymatic
and cell biological steps. One aspect of this process that has remained poorly understood is the search for homologous sequence
in the crowded nucleus following DSB formation (reviewed in Smith
and Rothstein, 2017). This search is especially critical in G1-phase
diploids, which are limited to interhomologous repair. Time-lapse
imaging studies have provided the most insight into this question
on a cell biological level. Yeast chromosomal loci are confined to a
small volume during S phase (Mine-Hattab and Rothstein, 2012) and
to a slightly larger volume during G1 phase (Dion et al., 2013;
Lawrimore et al., 2017). The motion regime of yeast chromosomes
is essentially subdiffusive (Mine-Hattab et al., 2017), but can be
approximated at longer timescales as undergoing Brownian
diffusion (Marshall et al., 1997). Following the induction of a sitespecific DSB in S-phase cells, loci proximal to the break expand their
explored volume 10-fold, in a process known as local mobility. Interestingly, undamaged loci throughout the nucleus also become more
mobile, although to a lesser extent, in a process known as global
mobility. These increases in explored volume may underlie the
homology search process, allowing highly mobile sequences close
to the break to move throughout the nucleus to seek homology,
aided in the search by the nucleus-wide increased motion permitted
by global mobility (Mine-Hattab and Rothstein, 2013).
The mechanisms of these mobility responses have not been definitively identified, although the regulatory underpinnings are becoming clearer. The DNA damage checkpoint activated by Mec1 is
critical for both global and local mobility, while the recombination
machinery itself, particularly Rad51, Rad52, and Rad54, likely
regulates the ability of the checkpoint to trigger increased mobility
(Dion et al., 2012; Mine-Hattab and Rothstein, 2012; Smith et al.,
2018). Downstream of checkpoint activation, a diverse array of
factors have been implicated in the mobility response, including
microtubules (Strecker et al., 2016; Lawrimore et al., 2017), actin
(Spichal et al., 2016), and chromatin remodelers (Hauer et al., 2017).
Importantly, increased chromosomal mobility after DNA damage
seems to be remarkably well conserved, and has been observed in
human and insect cells, with regulation similar to yeast (Dimitrova
et al., 2008; Chiolo et al., 2011; Lottersberger et al., 2015).
Most studies of chromosomal mobility have been performed in
S-phase cells, but the response to DNA damage in the G1 phase is
less clear. Recent work has indicated that G1-phase haploid cells
treated with phleomycin are able to undergo a global mobility response, but the response in diploids, where a repair template is available, has not been examined. To gain insight into G1-phase repair
dynamics, we explored whether G1-phase diploid cells undergo
global mobility. We find that, compared with S-phase cells, G1-phase
diploid cells have an elevated baseline mobility that undergoes a
further increase following irradiation, demonstrating that G1-phase
diploid cells also induce global mobility. This increase in mobility is
regulated similarly as in S-phase cells and is dependent on the DNA
damage checkpoint and the recombinase Rad51, consistent with the
idea that homology search can occur in the G1 phase of the cell
Volume 30 October 1, 2019
cycle. Surprisingly, despite a strong defect in Rad52 recruitment, we
find that Rad51 is recruited to sites of DNA damage in G1 phase,
further supporting the notion of interphase recombination. Thus, our
results demonstrate that global increased DNA mobility is part of the
response to DSBs in interphase diploid cells and that checkpoint and
recombination factors regulate this process.
RESULTS AND DISCUSSION
Increased chromosomal mobility after DNA damage occurs
in G1-phase cells
To gain insight into G1-phase repair dynamics, we made use of a
previously described system (Mine-Hattab and Rothstein, 2012). We
imaged cells containing a multiple tandem array of the bacterial
tetO sequence bound by red fluorescent protein (RFP)-tagged TetR.
To correct for the motion of the cell or the movement of the nucleus,
we also tagged a structural component of the spindle pole body,
Spc110, with yellow fluorescent protein (YFP). As the SPB is embedded in the nuclear wall and largely immobile (Berger et al., 2008),
we corrected positional measurements of the tetO array, taken
every 10 s for 30–70 time points, by the position of the SPB. Using
these positional measurements, we calculated a metric known as
mean-square displacement (MSD), which models how displacement
lengths change over given time intervals (Heun et al., 2001). Previous work has shown that yeast chromosomes undergo confined
Brownian diffusion within a small volume at this timescale and thus
display plateaued MSD curves (Marshall et al., 1997). The radius of
that confined volume (Rc) can be calculated based on the height of
the plateau. The URA3 locus in particular is confined to a volume
with an Rc of ∼450 nm in S-phase cells (Mine-Hattab and Rothstein,
2012; Smith et al., 2018).
To analyze the mobility of the URA3 locus in G1-phase cells, we
restricted our analysis to unbudded cells with an undivided spindle
pole body. We find that G1-phase diploids, like haploids (Heun
et al., 2001; Lawrimore et al., 2017), exhibit a higher baseline Rc
(Figure 1A, Rc = 570 ± 70 nm) than S-phase cells, possibly due to
differences in cohesin loading between G1 phase and S phase
(Dion et al., 2013). To examine the mobility of URA3 in an HR-
specific context, we used ionizing radiation to create “dirty”
(Barlow et al., 2008), which are preferentially repaired by HR in
haploid cells. Breaks formed in this way in G1-phase cells show
markers of resection, such as ssDNA formation (through the appearance of RPA foci) and Mec1-dependent checkpoint activation
(through the formation of Ddc1 foci), indicating the engagement
of the HR pathway (Table 1). We therefore detected damaged G1phase cells via these Ddc1–cyan fluorescent protein (CFP) foci
(Lisby et al., 2004; Barlow et al., 2008), and measured the mobility
of the URA3 locus. Following DSB formation, G1-phase diploid
cells undergo an additional increase in Rc (Figure 1A, Rc = 730 ±
100 nm, p value compared with undamaged = 0.02), indicating
that global mobility also occurs during G1 phase. This increase in
Rc corresponds to a two- to threefold increase in nuclear volume
explored.
Genotype
0 Gy
RFA1-YFP
30%
Cells
40 Gy
Cells
59
82%
45
DDC1-CFP
7.0%
143
56%
108
DDC1-CFP+20 mM caffeine
9.2%
109
46%
97
108
54%
97
DDC1-CFP rad51∆
10%
TABLE 1: Percent of G1 cells with DNA damage foci.
Global mobility occurs in G1
| 2621
a DSB. An expansion in nuclear volume
following damage could contribute to an
expansion in the volume that loci explore.
Recent work has shed light on a possible
link between the DNA damage response
and nuclear plasticity (Kumar et al., 2014;
Kidiyoor et al., 2016); thus, we wanted to
investigate whether global mobility is related to changes in nuclear volume. To
address this question, we tagged Nic96, a
component of the nuclear pore complex,
with green fluorescent protein (GFP) and
used it to estimate nuclear volumes in
G1-phase diploid cells before and after
irradiation. As depicted in Figure 3A, we
calculated volumes by assuming a spherical
FIGURE 1: Global mobility occurs in G1-phase diploids and is regulated by the DNA damage
nucleus and measuring the inner diameter
checkpoint. (A) Undamaged (blue) G1-phase diploids show mobility that is slightly elevated
of the Nic96 ring. When we applied this
compared with S-phase cells (Mine-Hattab and Rothstein, 2012; Smith et al., 2018). After
method to undamaged cells (Figure 3B), we
irradiation (red) there is a further increase in exploration (Wilcoxon rank-sum test p value
= 0.02). (B) Caffeine treatment blocks global mobility in G1-phase cells, with irradiated cells (red)
found that our median volume calculations
showing no difference in mobility compared with undamaged cells (blue) (Wilcoxon rank-sum
were only slightly larger than the mean
test p value = 0.8).
values reported for haploid nuclei (Winey
et al., 1997; Jorgensen et al., 2007). ImporRecent evidence has demonstrated that the DNA damage
tantly, we observed no change in median nuclear volume following
checkpoint is necessary and sufficient for global mobility in both
irradiation (0 Gy = 2.7 µm3, 40 Gy = 2.6 µm3, unpaired t test p value
diploid and haploid cells during S phase (Seeber et al., 2013; Smith
= 0.87), indicating that global mobility is not mediated by gross
et al., 2018). Moreover, damaged G1-phase haploid cells exhibit a
changes in nuclear morphology.
Rad9-dependent checkpoint arrest (Siede et al., 1993). To examine
whether or not G1-phase global mobility in diploids is regulated
Rad51 forms foci in G1-phase cells without concomitant
by the checkpoint, we treated cells with the PI3K-like kinase inhibiformation of Rad52 foci
tor caffeine (Gentner and Werner, 1975; Hall-Jackson et al., 1999;
Previous evidence in haploid cells has suggested that Rad52 activity
Heffernan et al., 2002) in the presence and absence of damage to
is restricted to S phase and that Rad52 foci do not form on G1block checkpoint activation. Interestingly, caffeine treatment did not
phase DSBs until Cdc28 activity allows cells to become competent
affect Ddc1 focus recruitment (Table 1). However, as in S-phase cells,
for HR (Barlow et al., 2008). Because we observed Rad51-depenglobal mobility was blocked in damaged cells subjected to caffeine
dent global mobility in G1-phase diploids, we were curious whether
treatment (Figure 1B, undamaged: Rc = 580 ± 80 nm, damaged: 570
Rad52 foci form in G1-phase diploids and whether they recruit
± 40 nm, p value = 0.8), indicating that the regulatory mechanisms
Rad51. To answer this question, we examined the appearance of
of mobility present in S phase are preserved in G1 phase.
Rad51 and Rad52 foci in G1- and S-phase diploid cells before and
after irradiation. Singly tagged (YFP-RAD51/RAD51 or RAD52-CFP/
G1-phase global mobility requires the recombinase RAD51
RAD52; Figure 4B, black points) and doubly tagged (YFP-RAD51/
In S-phase cells, global mobility is controlled by a regulatory circuit
RAD51 RAD52-CFP/RAD52; Figure 4B, red points) strains were
established by the recombination machinery and the DNA damage
used. The doubly tagged cells were used to show that neither
checkpoint (Smith et al., 2018). The recruitment of Rad51 to resected DNA stimulates global mobility signaling alongside the DNA
damage checkpoint. To test whether these regulatory systems are
also present in G1 phase, we examined rad51∆ cells. As shown in
Table 1, rad51∆ did not affect recruitment of the Ddc1 checkpoint
protein. When assaying cells for global mobility, we noted a slight
increase in the baseline Rc of rad51∆ G1-phase cells compared with
wild type (WT) cells (Figure 2, Rc = 670 ± 40 nm, p value compared
with undamaged WT = 0.06). This increase is consistent with earlier
reports that RAD51 deletion in S phase leads to elevated baseline
mobility (Dion et al., 2013; Lawrimore et al., 2017). However, following irradiation, there was no further change in mobility (Figure 2, Rc
= 640 ± 50 nm, p value compared with undamaged rad51∆ = 0.7),
indicating that Rad51, as in S-phase cells, is required for the global
mobility response.
Global mobility in G1-phase diploids is not a consequence
of changes in nuclear volume
A simple explanation for changes in the volume explored during
global mobility is that the nucleus changes in size or shape following
2622 | M. J. Smith et al.
FIGURE 2: rad51∆ cells display no global mobility response. Both
undamaged (blue) and damaged (red) cells exhibit similar radii of
confinement (Wilcoxon rank-sum test p value = 0.7).
Molecular Biology of the Cell
data indicate that, despite the relative scarcity of Rad52 foci, Rad51 is able to access
damaged sites in G1 phase.
Implications for the control of HR
in G1 cells
While it has long been appreciated that
DSBs can be repaired in G1-phase diploid
cells, the regulation of DSB repair in interphase is not well understood, and the similarities and differences from S phase remain
to be delineated. We show here that the
mechanisms of one facet of HR, increased
chromosomal mobility, are preserved in G1phase diploids, and the regulation of chromosome mobility is similar to that observed
in S phase. Additionally, we find that, deFIGURE 3: Nuclear volume does not change following irradiation of G1-phase diploid cells.
spite a cell cycle restriction of Rad52 foci to
(A) A schematic of the method used to estimate nuclear volumes. The inner diameter of the
S, Rad51 foci frequently form, demonstratNic96 ring is used to calculate a spherical volume (see Materials and Methods). In the case of
ing that recombination proteins can be
ellipsoid nuclei, the longest available diameter is used. Scale bar: 0.6 microns. (B) Scatter plot of
loaded in G1 phase and suggesting that the
calculated nuclear volumes from undamaged (left, median = 2.7 µm3, N = 114 cells) and
irradiated (right, median = 2.6 µm3, N = 129 cells) (unpaired t test p value = 0.87). Box plots
mobility processes we observe are a prodrepresent median and interquartile range.
uct of HR. We have previously shown that
the recruitment of recombination factors to
tagged protein affects localization of the other. As previously
sites of damage is critical for mobility, and these observations are
reported, we observe fewer Rad52 foci in G1-phase cells after 40 Gy
consistent with those findings (Mine-Hattab and Rothstein, 2012;
(Figure 4, A and B). However, Rad51 foci form at high levels in damSmith et al., 2018). Therefore, we propose that DSBs formed in G1
aged diploid G1-phase and S-phase cells (Figure 4, A and B). These
phase are resected to yield ssDNA overhangs that catalyze the recruitment of checkpoint and recombination
complexes. The interactions between these
two complexes drive increases in chromosomal mobility to promote HR.
This model raises several interesting
questions. First, does the complete HR
reaction occur in G1-phase cells? Supporting this view, we observe the loading of
Rad51 as well as the induction of global mobility, a possible prerequisite for homology
search. On the other hand, the recombination machinery may be loaded in G1 phase,
yet remain inactive until S phase begins
and Cdk1 activity increases, as observed in
haploid cells (Barlow et al., 2008). It is also
possible that global mobility is induced
alongside checkpoint activation but that Sphase entry is required to stabilize Rad51
presynaptic filaments to drive local mobility
and repair. We favor the hypothesis that
repair reactions are proceeding to completion in G1 phase based both on previous
reports of interphase gene conversion and
repair (Luchnik et al., 1977; Esposito, 1978;
Fabre, 1978; Brunborg et al., 1980; Kadyk
and Hartwell, 1992; Lee and Petes, 2010)
and our observations of Rad51 loading and
FIGURE 4: Rad51 forms repair foci in G1 phase. (A) Representative images of G1- and S-phase
increased chromosomal mobility (Figures 1
cells depicting YFP-Rad51 and Rad52-CFP foci each tagged in the same strain. White arrowhead
and 4). However, as Lawrimore and colindicates a Rad52 focus colocalizing with a Rad51 focus. Scale bar: 2 µm. (B) Measurements of
Rad52 (left) or Rad51 (right) focus formation in G1- and S-phase cells, with and without treatment leagues also observed global mobility in
haploid G1-phase cells following treatment
with 40 Gy of gamma radiation. Black points represent the percent foci for each tagged protein
with the radiomimetic drug phleomycin, as
in independent experiments. Red points represent an independent experiment in which Rad52
well as after endonuclease cutting, it is also
and Rad51 are both tagged in the same cells. Error bars represent 1 SEM for each group of
experiments.
possible that the signaling reactions and
Volume 30 October 1, 2019
Global mobility occurs in G1
| 2623
downstream mobility evoked by DSB formation are not directly
coupled to the completion of interhomologous recombination, as
these cells lack a viable repair template (Lawrimore et al., 2017). Still,
it seems unlikely that diploid cells would delay repair until S phase,
given that the homologue is always available as a template, and the
alternative repair strategy, NHEJ, is down-regulated (Kegel et al.,
2001).
Second, why is Rad52 focus formation restricted to S? The finding that Rad51 foci form in G1 phase despite low levels of Rad52
foci is, at first, incongruous with the notion that Rad52 foci are required for Rad51 recruitment. However, it is possible that Rad52 is
still functional in G1 phase but fails to form a focus, that is, there
are not enough molecules to be visualized. In this way, Rad52
could be stimulating Rad51 filament formation and providing regulatory input into the control of global mobility despite a lower
level of expression or of binding to resected ssDNA. Alternative
mediators such as Rad55, Rad57, and Rad59 are unlikely to play a
role in Rad51 focus formation in G1 phase, because Rad51 foci
require Rad52 irrespective of the cell cycle (Smith et al., 2018).
Thus, we suggest that Rad52 remains functional in G1-phase diploids and promotes the formation of the HR machinery.
The effect of the cell cycle on DSB repair has been long appreciated, but many of the precise details still remain to be resolved. Do
broken chromosomes in G1 phase undergo local as well as global
mobility? The recruitment of Rad51 to sites of damage suggests
that local mobility and homology search are occurring. What are the
differences in mobility and repair between G1-phase haploids and
diploids? Heterozygosity of the mating-type locus or the presence
of a sister homologue may have broad effects on repair pathway
choice and attendant phenomena such as increased mobility. How
are recombination proteins loaded to different types of breaks in G1
phase, and how does that loading differ from S phase? The reduced
recruitment of a variety of repair factors in G1 phase (Barlow et al.,
2008) indicates that the cell may take a separate approach to DSBs
that form in interphase. In any case, the results presented here show
that global increased chromosomal mobility following DSB formation is a facet of the DNA repair response that is present in both G1
and S phases and suggest that the mechanisms of homology search
are preserved.
were resuspended at higher density from overnight cultures before
being placed upon a 1.4% agarose slab for visualization. Images
were acquired on a Leica DM5500B upright microscope using a
1.46 numerical aperture 100× magnification Plan Apochromat lens
illuminated with a 100-W mercury arc lamp (Leica Microsystems).
High-efficiency filter cubes were used for fluorophore imaging
(Chroma 41028, Chroma 31044v2, and Chroma 41002C, for YFP,
CFP, and RFP, respectively), and images were captured with a
Hamamatsu Orca AG cooled digital CCD (charged-coupled device).
All microscopy was performed at 23°C. Analysis of image data was
performed with Volocity software (Perkin-Elmer). For mobility
experiments, we captured 15 z-stacks spaced by 300 nm every 10 s
for 70 time points. Exposure times were as follows: differential interference contrast (DIC) (30 ms), YFP (100 ms), RFP (100 ms), and CFP
(2s for Ddc1-CFP). DIC and CFP images were taken once before
time-lapse imaging began. For Rad51 and Rad52 focus experiments, we captured 21 z-stacks spaced as for mobility experiments.
DIC exposure time was 30 ms, and the YFP and CFP exposure time
was 800 ms.
MATERIALS AND METHODS
Strains
ACKNOWLEDGMENTS
All strains are RAD5+ derivatives of W303 (Thomas and Rothstein,
1989; Zhao et al., 1998). Strains were created as listed in Supplemental Table S1 (Jiang et al., 1996; Ryan et al., 2003; Lisby et al.,
2004; Mine-Hattab and Rothstein, 2012; Reid et al., 2016).
Caffeine treatment
Caffeine treatment was performed as described in Barlow and
Rothstein (2009). Cells were treated for 30 min with 20 mM caffeine,
which was diluted from a freshly prepared 100 mM stock, and irradiated in the presence of caffeine.
γ-Irradiation
Overnight cultures of strains were diluted slightly in fresh medium
and allowed to grow for 1 h at 23°C. Aliquots were exposed to
radiation using a Nordion 220 60Co irradiator and were then prepared for imaging.
Microscopy
Microscopy was performed as described previously (Lisby et al.,
2004; Mine-Hattab and Rothstein, 2012; Smith et al., 2018). Cells
2624 | M. J. Smith et al.
Nuclear volume calculations
Multiple z-stacks were obtained as for mobility experiments. The
section with the largest diameter was selected, and three innerdiameter measurements were made and averaged for the GFPNic96 ring. When the nucleus was ellipsoid, the longest available
diameter was measured. These diameters were used to calculate
spherical volumes and are likely to be overestimates. Statistical
comparisons between irradiated and undamaged cells were made
using an unpaired t test.
Data analysis and statistics
Analysis and statistics were performed as previously described
(Smith et al., 2018). We calculated mean MSD plots from the population of cells in each experiment as well as values for each individual
cell. These individual values were used to calculate ±SEM values for
each experiment and Wilcoxon rank-sum tests (Mann and Whitney,
1947) to evaluate significance. A table of all results and test values
can be found in Supplemental Table S2. Analyses were performed in
R (R Core Team, 2016). All code is available upon request.
We thank the entire Rothstein lab for experimental feedback,
suggestions, and assistance with an early version of the article. This
work was supported by National Institutes of Health grants P30
CA013696 for use of the HICCC shared radiation resource, T32
GM007088 (M.J.S. and F.J.J.), T32 GM008798 (E.E.B.), T32
CA009503 (E.E.B. and F.J.J.), TL1TR001875 (E.E.B.), R35 GM118180S1 (to F.J.J.), and R35 GM118180 (R.R.).
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