Can you write a journal article critique?

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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: • • • • • • 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, 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.). REFERENCES Aylon Y, Liefshitz B, Kupiec M (2004). The CDK regulates repair of doublestrand breaks by homologous recombination during the cell cycle. EMBO J 23, 4868–4875. Barlow JH, Lisby M, Rothstein R (2008). Differential regulation of the cellular response to DNA double-strand breaks in G1. Mol Cell 30, 73–85. Barlow JH, Rothstein R (2009). Rad52 recruitment is DNA replication independent and regulated by Cdc28 and the Mec1 kinase. EMBO J 28, 1121–1130. Berger AB, Cabal GG, Fabre E, Duong T, Buc H, Nehrbass U, Olivo-Marin JC, Gadal O, Zimmer C (2008). High-resolution statistical mapping reveals gene territories in live yeast. Nat Methods 5, 1031–1037. Brunborg G, Resnick MA, Williamson DH (1980). 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Running head: ARTICLE CRITIQUES

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Journal Article Critiques
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ARTICLE CRITIQUES

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DNA damage triggers increased the mobility of chromosomes in G1-phase cells
This journal article perfectly-investigated the mobility of chromosomes in G1-phase of
diploid cells. The article provides solutions to questions concerning the movement of
chromosomal loci in G1-phase after DNA damage. The study utilized the results obtained in the
related projects to make conclusions. The conclusion captured the objective of the experiment
and the data was adequately interpreted. The experiment investigated the relationship between
chromosomal loci mobility in G1-phase of Saccharomyces cerevisiae and the breakage of DNA
double-strand at one site (local mobility) and breakage of the double-strand in the whole nucleus
(Smith, Bryant, Joseph, & Rothstein, 2019).
20mM of the working solution was made by reducing the concentration of 100mM of
stock solution. The dilution was then illuminated in the presence of caffeine and used to treat
cells for 30 minutes. Cells grown overnight were diluted and cultured in new media at 23oC for
60 minutes...


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