Ive attached the instructions on writing the journal article critique. Ive also attached the article of choice and I need a minimum of 4 slides powerpoint presentation covering the article. all of the instructions are provided. Please follow the instructions precisely. No plagiarism. cite everything!
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Guidelines for Journal Article Critiques
The primary goal of the critique is to enable the reader to know the important results and
methods of the paper without having to read it himself. The secondary goal is to evaluate the
paper; this evaluation could include: the appropriateness of experiments/controls, how well the
data were interpreted, what additional experiments would be good, why you agree/disagree with
the conclusions, alternative explanations for their results, etc (one critique will not contain all
these elements). The critique is not a rewording of the abstract. It does not have to include every
detail/experiment in the paper. It should be written in paragraph form and not broken into
subsections, either formally or practically (i.e. do NOT have a paragraph that discusses methods
and a paragraph that discusses results; instead describe an experiment, give the result, and tell
how the authors interpreted it. Then move on to another experiment). The guidelines for grading
the critiques follow:
•
•
•
•
•
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The complete reference for the paper, as on a cited literature page, is at the top of the first
page.
The significant results and conclusions are clearly explained (without having to read the
paper)
The methods used to obtain the above results are explained (without having to read the
paper)
Correct spelling, grammar, paragraph structure, etc.
It is no longer than 2 pages (double spaced)
There is a significant, thoughtful evaluation of the paper including whether or not you
think it is a sound paper (simply saying it is good and offering no criticism is not
sufficient). This is worth ~10% of the grade
In addition to each student turning in a critique, each group will turn in the PowerPoint
presentation you use to present your article. The PowerPoint presentation must be at least 4
slides long and include at least two of the figures from the paper (you can get the pictures from
the MBOC website). The intention is that you could read your critique aloud while showing the
figures on PowerPoint that you describe in your critique.
90% of your presentation grade will be based on the following criteria:
• Following the above guidelines and matching the order/content of your critique
• The effective labeling of the figures so they are understandable in the context of your
critique.
• The efficient use of text (not copying the entire figure legend to accomplish the previous
goal)
• The effective use of text on any slides that do not have a figure on them. Too much text
on a slide is boring because your presentation becomes a glorified teleprompter.
• I generally dislike excessive use of animations and transitions; however they are
sometimes necessary and thus, you need to use at least one of each of them in this
presentation (animations and transitions are not the same thing, if you need help, ask
me).
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, foll …
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