S. pombe cells. Using this assay, we find that the 53BP1 orthologue and Rev7 specifically repress long-range resection through the RecQ helicase-dependent pathway, thereby preventing hyper-resection. These results suggest that ‘rewiring’ of BRCA1-deficient cells to employ an Exo1-independent hyper-resection pathway is a driver of PARPi resistance.Poly(ADP ribose) polymerase inhibitors (PARPi) target cancer cells deficient in homology-directed repair of DNA double-strand breaks (DSBs). In preclinical models, PARPi resistance is tied to altered nucleolytic processing (resection) at the 5’ ends of a DSB. For example, loss of either 53BP1 or Rev7/MAD2L2/FANCV derepresses resection to drive PARPi resistance, although the mechanisms are poorly understood. Long-range resection can be catalyzed by two machineries: the exonuclease Exo1, or the combination of a RecQ helicase and Dna2. Here, we develop a single-cell microscopy assay that allows the distinct phases and machineries of resection to be interrogated simultaneously in living  Healthy cells can typically repair damage to their DNA with high accuracy, keeping their genetic code intact. In contrast, cancer cells often lose this ability. Inaccurate repair leads to more frequent DNA mutations, which can make a tumor more aggressive. However, DNA repair-deficient tumors can be targeted with cancer therapies, such as PARP inhibitors, which kill cells that do not have working DNA repair mechanisms. PARP inhibitors show great promise clinically, but unfortunately some tumor cells can become resistant to these treatments over time. Recent work has shown that resistance to PARP inhibitors is often caused by further alternations to DNA repair machineries.Being able to visualize DNA repair in living cells is crucial to understanding this process and to find ways to improve cancer treatments. Previous studies have used repetitive DNA sequences called Lac operators (LacO) to visualize the dynamic behavior of DNA in live cells. Leland et al. have now adapted this system to watch individual DNA repair events in living yeast cells under the microscope. Their experiments reveal that when cells lose a single protein called Rev7, an early phase of DNA repair becomes hyperactive. Leland et al. traced the cause of this hyperactivity to an enzyme in the RecQ helicase family.A RecQ helicase becoming hyperactive in cells lacking Rev7 could explain how some cancer cells become resistant to PARP inhibitor treatments. This information could help fine-tune future approaches to treating cancer. For example, using an inhibitor of RecQ helicase alongside PARP inhibitors may help block this type of resistance from developing in the first place. As well as potentially paving the way for better cancer treatments, this method of visualization could improve scientists’ understanding of the basic processes of DNA repair. Brca1–/–p53–/– cells to become resistant to PARPi. In human cells, Rev7 appears to act in concert with another inhibitor of resection, 53BP1 – approximately twice as fast as Exo1-dependent long-range resection. As BRCA1 activity has been tied to Exo1-dependent long-range resection  when the repair machinery is primed for HR , as Rad52 loads at DSBs by exchanging with RPA on nascent ,resected ssDNA ends . Using qS. pombe .S. pombe (4 kb/hr) or Southern blot in S. cerevisiae (4.4 kb/hr)  . As this4 kb/hr) .exo1∆ cells. We also note a strong inhibition of resection (comparable to ctp1∆ cells) as close as 300 nts from the DSB in cells lacking Exo1 as detected by qPCR  and its orthologue Rad9 (in budding yeast) repress resection initiation ; in buddrev7∆ cells than in WT cells  -Dna2 pathway, or both . Interesependent . In star of Rqh1 . Consist pathway . When co(p=0.16) . Howeverres Rqh1 .crb2∆ or rev7∆ cells in which the RecQ helicase is derepressed are capable of resection rates in excess of 20 kb/hr, indicating that the RecQ helicases are more capable of driving hyper-resection of DSBs than the Exo1-dependent pathway. The strong inhibition of RecQ helicase-dependent resection by Crb2 and Rev7 also explains why Rqh1 is not a major player in WT S. pombe resection, since Rqh1 can be derepressed to such a large extent (by loss of Crb2 or Rev7) that Exo1 becomes dispensable for long-range resection in crb2∆exo1∆ and rev7∆exo1∆ cells  using a modified two-step integration procedure that first creates a site-specific DSB to increase targeting efficiency of linearized plasmid pSR10_ura4_10.3kb , 3.2 kb distal to the LacO insertion. The total distance between the HO cut site and the beginning of the 10.3 kb LacO array is 3.57 kb. As the LacO array can contract during the process of transformation, integrants were screened by HincII digest followed by southern blot  was transformed into S. pombe, which were then plated onto EMM-leu+thi-ura plates . After 4–5 days of growth at 30°C, 40–100 individual colonies were combined to obtain a reproducible plasmid copy number across the population. Cre-mediate HO gene exchange at the endogenous Urg1 locus (bleMX6urg1::RMCE) was induced by overnight culture in EMM-thi-ura+ade+NPG media : reduce photobleaching in microscopy experiments, prepared fresh). The following day, site-specific DSBs were induced in log-phase cultures by the addition of 0.50 mg/mL uracil. This induction strategy resulted in ~15% of cells making a DSB within ~2 hr (We used the uracil-responsive cut site . We perfin ~2 hr .Initiation of resection was assessed using a previously described qPCR assay where ssDNA produced by resection causes protection from ApoI digestion . ApoI cuAll images were acquired on a DeltaVision widefield microscope (Applied Precision/GE) using a 1.2 NA 100x objective (Olympus), solid-state illumination, and an Evolve 512 EMCCD camera (Photometrics). Slides were prepared ~20 min after adding 0.50 mg/ml uracil to log-phase cultures to induce HO endonuclease expression and DSB formation. Cells were mounted on 1.2% agar pads  and sealed with VALAP (1:1:1 vaseline:lanolin:paraffin). Image acquisition began between 40 and 80 min after uracil addition. Imaging parameters for all resection assay data acquisition were as follows. Transmitted light: 35% transmittance, 0.015 s exposure; mCherry: 32% power, 0.08 s exposure; GFP: 10% power, 0.05 s exposure. At each time point (every 10 min for 5–7 hr), 25 Z-sections were acquired at 0.26 μm spacing. Identical imaging parameters were used to image a strain expressing endogenously tagged Sad1-mCherry (Sad1 forms a single focus at the spindle pole body that contains between 450 and 1030 molecules) and relative mCherry foci intensities were used to determine that ~30 molecules of Rad52-mCherry are required to detect a visible focus with these imaging parameters .For the LacO resection assay, every cell cycle was tracked and quantified individually, including timing of nuclear division, cellular division, Rad52-mCherry focus formation, and LacO/LacI-GFP focus disappearance. Only on-target Rad52 foci  were considered, since many DSB events occur throughout the genome spontaneously, especially during S-phase. The number of cells and events used to generate the plots in all Figures is included as For focus intensity plots e.g. , quantifhttps://github.com/lelandbr/Leland_King_2018_eLife_Rev7_EndResection (https://github.com/elifesciences-publications/Leland_King_2018_eLife_Rev7_EndResection).Raw data were processed, visualized, and analyzed using R, in particular packages dplyr, ggplot2, and broom. Raw data, raw analysis for all individual cells included in plots, complete code, and other supporting materials are publically available on GitHub 600 and a Coulter Principle cell counter (Orflow Moxi Z). Cultures were diluted as needed to ensure identical numbers of cells were spotted for each genotype, starting with ~4×106 cell/mL and going down by sixfold dilutions. Plates were prepared using standard procedures  There are no graphs showing the quality of the kinetic data. The authors need to show graphs of intensity vs time, for each assay, with fitting statistics.2) It's a bit surprising that the array of 256 copies of lac repressor protein doesn't affect the measured rate. The authors need to show overlaid graphs of intensity vs time for the arrays; the PCR data for the arrays with lac repressor; and PCR data for the DNA arrays without lac repressors.S. cerevisiae.3) In the experiments with tagged RAD52, I don't understand why the foci persist. I would have expected RAD51 to replace the RAD52. Although the intensity does decrease (maybe just from photobleaching), it seems to be slower than the observations from the Rothstein lab on Rad52 in As stated above, the results are interesting and informative. However, the results rely entirely on the validity of the assay. In the current version, the authors don't present enough analysis and comparisons of the kinetic data to convincingly establish the assay. Presumably, they have the data; they need to provide it.Reviewer #2:S. pombe. DSB formation/early resection was detected by the appearance of Rad52-mCherry foci that co-localize with the lacO array marked with LacI-GFP. Resection of the entire lacO array results in loss of the GFP signal while the mCherry signal remains. Consistent with a previous study using population based qPCR to measure resection , the authors report that most resection is due to Exo1 activity and not Rqh1. Two years ago, several groups reported that Rev7 acts with 53BP1 to inhibit resection in mammalian cells. Here, the authors show that Rev7 and Crb2/53BP1 have a conserved role in preventing long-range resection in S. pombe. Furthermore, they show that Rev7 and Crb2 specifically block resection by Rqh1.DNA end resection is essential for homologous recombination, but excessive end resection can be detrimental to genome integrity. Long-range resection is catalyzed by either Exo1 or by a RecQ family helicase in collaboration with Dna2. The relative contribution of these two mechanisms and how they are regulated is not well understood. Here, the authors use a cytological approach to study end resection in single cells. An HO cut site was inserted 3.4-kb from a 10.3-kb lacO array located on Ch 2 of Overall, the authors demonstrate that live cell imaging can be used to study end resection in single cells, and their studies show considerable cell-to-cell variation in the initiation of resection, a feature missed by population-based DNA analysis. However, I'm not convinced that the microscopy-based single-cell assay is the best way to monitor resection. The generated data imply that resection speed can be measured accurately but that relies on some assumptions. In subsection “Image analysis” the authors write "The time between the first frame with an on-target Rad52-mCherry focus and the first frame with complete disappearance of the LacO/LacI-GFP focus is the duration of resection through 3.57 kb (between the HO cut site and the start of the LacO repeats) plus the full 10.3 kb LacO array". Is resection through the complete LacO array necessary for disappearance of the GFP focus? I could imagine that the GFP focus disappears before the complete LacO array is degraded. What is the minimum number of detectable LacO repeats? The authors could integrate LacO arrays of different lengths and check what the detection limit is. Or they should at least confirm the timing of the GFP focus disappearance with the qPCR assay .S. pombe and the efficiency of cutting is better than HO) would involve considerable more effort and time.Also, to my eye the Rad52 focus appearance and LacI-GFP focus disappearance are not easily identified (based on the microscopy images shown). The authors should mark the frames they define as "Rad52 focus appearance" and "LacI-GFP focus disappearance". In the Materials and methods section, they don't say how they define these events. Do they do it manually or using some image analysis software? What is the threshold? More details are necessary here to judge the accuracy of the method. Also, the restriction of the assay to 5 hours by photobleaching seems to be a considerable drawback, which limits the number of "usable" cell trajectories . Perhaps by changing the imaging period and number of z-stacks they could extend the time window for monitoring resection. Other approaches, such as using more photostable fluorescent proteins , LEDs as light sources, or switching to an enzyme that cuts more efficiently than HO  considers the HO cut fraction.Rad52 foci are used as a read out for DSB formation and initiation of resection. Can these two steps be separated, for example, by measuring DSB formation by qPCR using primers flanking the HO cut site on the same samples used for the ApoI protection assay, or possibly using Mre11-mCherry instead of Rad52? It appears from Figure 1B (upper panel) that resection through most of the lacO array is required for a strong Rad52 signal.S. pombe cells are in G2 most of the time, if one sister chromatid was cut and engaged in repair with the uncut sister it could result in an underestimation of resection. Were cells with a transient Rad52 focus detected? The exo1 example in Figure 1 appears to have a Rad52 focus that appears early and then goes away. Does elimination of Rad51 change the number of cells resecting or rate of resection? At late times, when the mCherry signal is very bright, are both sister chromatids cut and resected?In theory, the HO-induced DSB is unrepairable, but because the HO cutting efficiency is quite low, and Langerak et al., used a qPCR assay to measure end resection and reported no defect in resection initiation (35 nt from HO cut site) in the exo1 mutant or exo1 rqh1 double mutant. Is the failure to detect Rad52 foci in most exo1 cells because resection tracts are <90 nt or because the amount of ssDNA required to support a Rad52 focus is much longer than 90 nt? Given that the Rad52 single is quite weak until the lacO array disappears, I think the authors might be under-estimating the amount of ssDNA to visualize a Rad52 focus. Does the exo1 mutant show normal DSB formation (measured with primers flanking the HOcs) and resection to the ApoI site located 168 nt from HOcs? Similarly, is DSB formation normal in the rev7 mutant and can early resection be detected by the qPCR assay?Why does rqh1 suppress the early resection defect of rev7? An odd result that is not discussed in the text.While the overall findings on the role of Rev7 and Crb2 repressing the Rqh1 resection pathway are certainly of interest to the field, the data analysis needs to be improved.Reviewer #3:DNA end resection is a process that initiates recombination-based DNA double strand break repair. As resection also generally inhibits non-homologous end-joining, regulation of DNA end resection is an important process. The human 53BP1 protein, though its various effectors, has been found to be an inhibitor of DNA end resection, although mechanistic insights are lacking. These processes appear to be at least partially conserved in low eukaryotes.S. pombe as a model system, where they developed an assay allowing the monitoring of resection in live cells . The assay is based on the disappearance of GFP-LacI and appearance of Rad52-mCherry signal next to HO-endonuclease induced DSB. This is an interesting method that will be useful for high throughput microscopy-based screenings. However, the method has certain limitations in contrast to established southern blotting, PFGE or RTPCR-based methods:The authors are using a a) The assay measures Rad52 accumulation, which is a step after resection. Rad52 is a mediator that loads Rhp51 on RPA-coated resected DNA. Using RT-PCR based assay, the authors established that there is a correlation between both processes (resection and RAD52 loading). However, this was only done in wt background, and it is possible RAD52 loading might differ in the mutants analyzed, and Rad52 might be loaded with different kinetics dependent on the resection pathway. The authors calculate resection rates making the assumption that there is no difference.b) The assay measures resection of DNA bound by GFP-LacI, a non-physiologic binder. Therefore, the resection proteins must displace LacI for resection to occur. Under physiologic conditions, DNA near DSBs will likely be chromatinized and subsequently remodeled allowing resection. This is especially a concern when analyzing the role of chromatin binders such as 53BP1/Crb2. Also, the individual resection pathways may be affect by LacI binder to a different degree, which complicates interpretation.The most interesting finding is that Rev7 and 53BP1/Crb2 appear to repress long-range resection dependent on Rqh1 : In the absence of Rev7 and Crb2, long-range resection is accelerated, which is independent of Exo1 and depends on Rqh1.1) This observation should be verified using a previously established assay, given the concerns listed above. Related to this point, it remains a formal possibility that the action of Rqh1 is not specific to resection, but to strip LacI, allowing resection through another process. Performing resection with the same mutants in an established setup (with no LacI) will address this concern as well.The authors then go on to demonstrate differential effects of Rev7 and Crb2 on early resection steps, based on timing of Rad52 foci appearance upon break induction . I am concerned about these results: using Rad52 (a protein with a function downstream of resection) does not appear to be correct as a marker of "early" resection.2) The results should be analyzed in an established assay where the readout is clearly early resection. Also, to make claims on early resection, the analysis should include mutants deficient in short-range resection (e.g. mre11) and mutants in long-range resection, where both pathways have been inactivated (e.g. exo1 rqh1).3) In Figure 2, the authors demonstrate that rev7 mutants have accelerated long-range resection, which is dramatically decreased when rqh1 is additionally mutated (rev7 rqh1), supporting the hypothesis that resection in rev7 mutants is Rqh1 dependent. In contrast however, the in Figure 3, the "early" resection is increased when rqh1 is mutated in rev7 background, a completely opposite effect. This is very confusing. The authors comment that "early" resection is a combination of Mre11 and Exo1/Rqh1 dependent processes. This dichotomy reinforces my concerns about the robustness of the experimental setup.In summary, the manuscript presents an interesting assay and interesting pieces of data, but it seems rather preliminary at this point.[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Kevin Struhl as the Senior Editor.Thank you for submitting your article "Rev7 and 53BP1/Crb2 prevent RecQ helicase-dependent hyper-resection of DNA double-strand breaks" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.The biological findings of this manuscript are interesting, particularly the role of Rev7 in suppressing resection by the Rqh1 pathway, but there are significant concerns over the reliability of the assays used. The authors place equal importance on the fluorescence-based resection presented; however, the reliability of the assay is questionable and the weak point of this work. The conceptual idea of the assay is good, but there are reservations about its implementation and use to quantify resection speed. The authors have, for the most part, established the utility of their assay for the analysis of long-range resection, although they still need to provide a graphical example of their quantification. They discovered interesting roles of Rev7 and Crb2 in Rqh1-dependent resection. As both a methods contribution and a contribution to the recombination field, these findings could justify publication of a revised manuscript.However, the manuscript then goes on to dissect the roles of these proteins in proximal recombination. Here the work is both incomplete and internally inconsistent. It would appear that the data in Figure 4A and Figure 4C are inconsistent with one another: 4A show a reduction in "Cell cycles with Rad52 foci" for rev7delta, whereas 4C shows no change, within error, for rev7delta at 300 bps .Also, the conclusion that of Rev7 promotes early resection is based entirely on how quickly a Rad52 focus forms after break induction, and how many cells have visible Rad52 foci. It was suggested that the authors use a more direct measurement of break induction by qPCR using primers flanking the HO cut site. This has not been done and is a significant concern. If one compares the qPCR data for resection 168-bp from the HO cut site shown in Figure 1D and Figure 1—figure supplement 3B there are vast differences – 3% resection at 120 min in one figure compared with 15% resection at 120 min in the other. If there is this much variability between populations of cells then it could explain why the rev7 mutant looks different to wild type, and why rqh1 appears to suppress the early resection defect of rev7. How many independent inductions were performed for image analysis? The authors need a reliable method to assess DSB formation independent of Rad52 focus formation before drawing conclusions about a role for Rev7 in promoting early resection.Furthermore, In Figure 1D, the authors use a qPCR assay to detect formation of ssDNA 168 and 14,253 bp from the HO cut site. From this assay, very few (2%) cells exhibit resection to the end of the lacO array 360 min after HO induction. This would appear to contradict the microscopy assay, which shows resection through the array takes ~150 minutes. Also, data should really be from biological replicas, not technical replicates of qPCR.Consequently, whether or not Rev7 has a role in proximal resection is not clear from the authors' data.There was agreement that manuscript needs to be revised:1) It is essential that the confusing data on short range resection are clarified or removed from the manuscript. If the proximal resection data are retained, then the comments raised above need to be addressed. In addition, in the review of the prior version of this manuscript, reviewer #3 commented on this part in the original submission: "Also, to make claims on early resection, the analysis should include mutants deficient in short-range resection (e.g. mre11) and mutants in long-range resection, where both pathways have been inactivated (e.g. exo1 rqh1)." The authors did not address this previous request for clarification of this unexpected finding in this revised manuscript, using the analyses described for proximal resection .S. pombe, it is well-described in S. cerevisiae (Rad9). The second major finding is the effect of Rev7 on long-range resection: this was only shown in humans, but not in the microbial eukaryotes. Although significant, the impact of the current manuscript is diminished without the proximal resection data; consequently, the author would need to make the contributions of their sound work much clearer in an expanded Discussion section of their work in relation to the existing literature .It is unclear whether the authors can make these revision within the timeframe given. If not, then these data on proximal resection would need to be removed, and the conclusions of the manuscript refocused. One major finding is the effect of Crb2 on long-range resection: while this is new in 2) The authors use appearance of a Rad52-mCherry focus to identify onset of resection and disappearance of a lacO/LacI-GFP focus to identify resection past a site some 14 kb away from the DSB site. The problem is that both signals are rather fuzzy in many of the image series shown. This is especially true for the Rad52-mCherry focus . The decision if a signal is judged as a focus or not is crucial, as it is the basis to calculate resection speed. This decision is made manually. Although the authors try to "equalize" the error by randomizing the image series prior to analysis, it seems to be a quite ambiguous approach of questionable reliability. What is their criterion to judge if a focus is present or not? Do they compare with background control strains? Why don't the authors use software to quantify the fluorescence signals and generate intensity trajectories? There are several non-commercial image analysis software packages available dedicated to exactly this purpose. Based on thresholds defined by appropriate control strains, appearance and disappearance of the signals could then be identified in a more controlled and rational way. A minimal requirement is that the author provides an x-y graph of foci fluorescent intensity vs time for each their examples of time-lapse video data in the manuscript.If the authors submit another revised version, then the decision to accept or reject will be final, and no subsequent revisions will be considered. [Editors’ note: the author responses to the first round of peer review follow.]Essential revisions:Reviewer #1:This is an interesting paper with potentially important conclusions. The results depend entirely on the assay, which seems to be reliable, but some questions remain:We are pleased that the reviewer found our study interesting and have endeavored to support our conclusions with additional experimental data as well as some clarifications.1) There are no graphs showing the quality of the kinetic data. The authors need to show graphs of intensity vs time, for each assay, with fitting statistics.We agree that if the resection rates were derived by measuring the kinetic loss of GFPLacI intensity this would be a critical point. Indeed, this is an aspect of this approach that can be exploited. However, for the data reported in this manuscript, we found that the most robust approach to determining the median long-range resection rate is an endpoint measurement . Thus, we derive a rate from the resection of the entire LacO array rather than from the rate of GFP-LacI loss. This being said, as we show in 2) It's a bit surprising that the array of 256 copies of lac repressor protein doesn't affect the measured rate. The authors need to show overlaid graphs of intensity vs time for the arrays; the PCR data for the arrays with lac repressor; and PCR data for the DNA arrays without lac repressors.To further verify the (perhaps surprising) result that the LacO array does not strongly influence the measured resection rates, we carried out qPCR analysis of resection within a population of WT cells on both sides of the induced DSB. Although we cannot use qPCR to assess resection within the LacO array because of its repetitive nature, we did assess the extent of resection on the far side of the LacO array (~14 kb from the DSB site) and compared this to resection ~14 kb on the other (non-LacO containing) side of the DSB. We find that the rates on both sides of the induced DSB appear to be highly similar . Thus, although it may be surprising, we have not found any indication that the LacO array alters the process of resection. Further, our extensive analysis by qPCR in the revised manuscript is entirely consistent with the interpretations made from the LacO-based system we have developed.3) In the experiments with tagged RAD52, I don't understand why the foci persist. I would have expected RAD51 to replace the RAD52. Although the intensity does decrease (maybe just from photobleaching), it seems to be slower than the observations from the Rothstein lab on Rad52 in S. cerevisiae.To further verify the (perhaps surprising) result that the LacO array does not strongly influence the measured resection rates, we carried out qPCR analysis of resection within a population of WT cells on both sides of the induced DSB. Although we cannot use qPCR to assess resection within the LacO array because of its repetitive nature, we did assess the extent of resection on the far side of the LacO array (~14 kb from the DSB site) and compared this to resection ~14 kb on the other (non-LacO containing) side of the DSB. We find that the rates on both sides of the induced DSB appear to be highly similar . Thus, although it may be surprising, we have not found any indication that the LacO array alters the process of resection. Further, our extensive analysis by qPCR in the revised manuscript is entirely consistent with the interpretations made from the LacO-based system we have developed.As stated above, the results are interesting and informative. However, the results rely entirely on the validity of the assay. In the current version, the authors don't present enough analysis and comparisons of the kinetic data to convincingly establish the assay. Presumably, they have the data; they need to provide it.Indeed, as suggested by the reviewer, we are pleased to be able to provide additional support for the interpretation for the vast majority of our assertions in the initial submission. In particular, we have carried out extensive qPCR analyses that support the fundamental soundness of the assay design and implementation . However, as we argued in our initial submission, this single cell assay can reveal attributes of individual events that are poorly captured in the population-based qPCR data, particularly for cells lacking Rev7, in which slow initial steps of resection initiation and faster long-range resection are conflated in a population-based approach . Indeed, we believe this is one of the reasons that this assay can be a powerful resource to complement existing population-based assays. We are confident that the inclusion of these additional data will provide the necessary validation to highlight the utility of this experimental approach and the resulting insights into DSB end resection.Reviewer #2:DNA end resection is essential for homologous recombination, but excessive end resection can be detrimental to genome integrity. Long-range resection is catalyzed by either Exo1 or by a RecQ family helicase in collaboration with Dna2. The relative contribution of these two mechanisms and how they are regulated is not well understood. Here, the authors use a cytological approach to study end resection in single cells. An HO cut site was inserted 3.4-kb from a 10.3-kb lacO array located on Ch 2 of S. pombe. DSB formation/early resection was detected by the appearance of Rad52-mCherry foci that co-localize with the lacO array marked with LacI-GFP. Resection of the entire lacO array results in loss of the GFP signal while the mCherry signal remains. Consistent with a previous study using population based qPCR to measure resection , the authors report that most resection is due to Exo1 activity and not Rqh1. Two years ago, several groups reported that Rev7 acts with 53BP1 to inhibit resection in mammalian cells. Here, the authors show that Rev7 and Crb2/53BP1 have a conserved role in preventing long-range resection in S. pombe. Furthermore, they show that Rev7 and Crb2 specifically block resection by Rqh1.Overall, the authors demonstrate that live cell imaging can be used to study end resection in single cells, and their studies show considerable cell-to-cell variation in the initiation of resection, a feature missed by population-based DNA analysis. However, I'm not convinced that the microscopy-based single-cell assay is the best way to monitor resection. The generated data imply that resection speed can be measured accurately but that relies on some assumptions.First, we appreciate the reviewer’s determination that this single-cell LacO resection assay can be used to measure resection, including determination of cell-to-cell variability. We would like to emphasize that we expect that this assay is complementary to existing approaches (and is in no way intended to replace such methods). Moreover, we show that this assay has unique strengths  and future applications . Admittedly, as with any technique, there are aspects that may be better addressed with orthogonal methods. We appreciate the concerns that the reviewer has about the various parameters necessary for the assay to work robustly, and we have worked to challenge the “assumptions” with additional data, specifically with extensive qPCRbased analysis .In subsection “Image analysis” the authors write "The time between the first frame with an on-target Rad52-mCherry focus and the first frame with complete disappearance of the LacO/LacI-GFP focus is the duration of resection through 3.57 kb (between the HO cut site and the start of the LacO repeats) plus the full 10.3 kb LacO array". Is resection through the complete LacO array necessary for disappearance of the GFP focus? I could imagine that the GFP focus disappears before the complete LacO array is degraded. What is the minimum number of detectable LacO repeats? The authors could integrate LacO arrays of different lengths and check what the detection limit is. Or they should at least confirm the timing of the GFP focus disappearance with the qPCR assay .Indeed, we had carried out such analysis to determine our sensitivity to both Rad52 loading  and “full” resection of the LacO array. This was carried out as suggested by the Reviewer through analysis of LacO arrays of decreasing lengths . Based on these analyses, we expect our determination of the time at which resection initiates to be slightly AFTER the true time of resection initiation (by ~300 bps) and we expect our determination of the time at which resection through the LacO array is complete to be slightly BEFORE the true time of complete LacO resection (by <500 bps). This is based on the following observations: (1) New qPCR analysis of cells lacking Exo1 that demonstrates that the extent of resection required to visualize Rad52-mCherry at the induced DSB is ~300-500 bp . (2) Our ability to robustly detect a 1kb LacO array signal above background under our imaging conditions  – from this further analysis, we conservatively estimate that the LacO/GFP-lacI array can be visualized until <500 bps of the array is retained. In the context of 13.9 kb of total resection distance (from HOcs to the end of the LacO array), we argue that <800 bps will only slightly influence the determined rate (<5% difference). However, as reviewer 2 correctly notes, the fact that we are detecting the initiation of resection slightly later than the true initiation time, and we are detecting the completion of resection slightly earlier than the true completion time, means that we are slightly underestimating the total duration of resection and thus slightly overestimating the rate of resection. This actually could partially explain the reason our measured rate for WT cells (median = 7.6 kb/hr) is slightly higher than previous published qPCR-based approaches, although we would predict an over-estimate maximally of 0.4 kb/hr. Indeed, the extent to which this could influence our interpretation of the data (0.4 kb/hr) is minor compared to the variability within the population, or any of the median rates that we find to be statistically significant between genotypes. Given this, rather than making assumptions, we felt that it was more transparent to use the complete distance from HOcs to the end of the LacO array in our calculations, as this is the only distance that it is possible to know with complete certainty. Lastly, as suggested by the reviewer, we have also now acquired significant qPCR-based data that supports the conclusions from this live cell assay (see below).Also, to my eye the Rad52 focus appearance and LacI-GFP focus disappearance are not easily identified (based on the microscopy images shown). The authors should mark the frames they define as "Rad52 focus appearance" and "LacI-GFP focus disappearance".As requested, we now indicate both Rad52-mCherry loading and GFP-lacI focus disappearance for all kymographs, as well as the resection duration and rate. Further, we now present many more examples of the population of cells, each indicating the frame that we score for “Rad52 focus appearance” in Figure 1—figure supplement 2.In the Materials and methods section, they don't say how they define these events. Do they do it manually or using some image analysis software? What is the threshold? More details are necessary here to judge the accuracy of the method.We now include additional details on our image analysis pipeline, which uses a combination of manual and automated routines. We would emphasize that, although aspects are carried out manually, the hundreds of individual 5D image fields from all genotypes were all pooled together, randomly sorted, and then presented to the person scoring DSB events with all identifying file name information removed. This method of blinding across ~900 image fields distributes any human error evenly across the 11 genotypes analyzed.Also, the restriction of the assay to 5 hours by photobleaching seems to be a considerable drawback, which limits the number of "usable" cell trajectories . Perhaps by changing the imaging period and number of z-stacks they could extend the time window for monitoring resection. Other approaches, such as using more photostable fluorescent proteins , LEDs as light sources, or switching to an enzyme that cuts more efficiently than HO (I-PpoI has been successfully used in S. pombe and the efficiency of cutting is better than HO) would involve considerable more effort and time.We invested substantial effort and time into exploring numerous induction systems, site-specific cut sites/endonuclease pairs and imaging conditions that could produce results superior to those described in this manuscript, as suggested by the reviewer. Based on this exhaustive exploration, the system described here presented the most robust and reproducible performance. Although it is likely that further improvements can still be made to this system, we would argue that our ability to characterize a wide array of genetic backgrounds to reveal new insights into regulation of DSB end resection in fission yeast highlights that the assay is of sufficient throughput. Unfortunately, it is not possible to reduce the number of z-slices without losing the ability to accurately detect sub-diffraction foci in Z or avoid loss of foci above/below the z-stack.The authors stress that they use a single cell assay. But it is not clear what the advantage of single cell data is in their work. In the end they are comparing population medians, which could also be generated with bulk experiments.rev7Δ cells, this is absolutely critical. As we show, fewer cells successfully initiate resection the absence of Rev7 . However, once initiation occurs, long-range resection occurs much more rapidly than in WT cells . We would predict that these two opposite effects would be very challenging to convincingly (and certainly quantitatively) assess using a population-based resection assay . Indeed, our qPCR analysis, presented in Figure 4C, makes this point, with distinct effects 300 bp and 3 kb from the HO cut site. However, given only this information: a quite subtle early resection delay in the population (300 bp from the HO cut site) and a gain of resection 3 kb from the HO cut site in the population, it would be challenging to fully interpret this data based on qPCR alone. Thus, we think this example (unlikely to be the only case) illustrates how it will be revealing to compare results using these two approaches in future studies.To the contrary, we would strongly argue that the data presented here illustrate precisely why such a single cell assay provides a valuable complement to population assays. Perhaps it was not clear that the long-range resection rates are only determined from individual cells that have successfully initiated resection. For the Further insights can be concluded for other genetic backgrounds, for example we can discern that loss of Crb2 does not affect resection initiation in otherwise WT backgrounds, while it increases long-range resection rates . More generally, as no DSB induction system is entirely efficient, even in budding yeast, this approach allows us to decouple early events in DSB processing from long-range resection without weighing heavily on estimates of DSB induction. Lastly, we would argue that going forward it is clear that having a cell biological assay will open up the door for analysis not possible with qPCR approaches such as DSB mobility and subnuclear compartmentalization.The low DSB formation efficiency might be a motivation to use a single cell assay to restrict the analysis to the few cells with a DSB. However, bulk experiments generally take the cutting efficiency into account, e.g. the qPCR-based assay described by Zierhut and Diffley, (2008) considers the HO cut fraction.While this is true, because this is population approach it does not allow individual cells to be monitored for the rate of long-range resection independently from delays in resection initiation. In the assay described here, even if there is a strong reduction in resection initiation (say 25% of WT), we can still monitor the rate of long-range resection in that 25% of cells; again, this cannot be assessed by qPCR. Indeed, cells lacking Rev7 are a good example of how these two phases can be analyzed distinctly.Rad52 foci are used as a read out for DSB formation and initiation of resection. Can these two steps be separated, for example, by measuring DSB formation by qPCR using primers flanking the HO cut site on the same samples used for the ApoI protection assay, or possibly using Mre11-mCherry instead of Rad52? It appears from Figure 1B (upper panel) that resection through most of the lacO array is required for a strong Rad52 signal.In the revised manuscript, we include our further characterization for the extent of resection required to load sufficient Rad52-mCherry to be robustly visualized in the assay. This was achieved, as suggested by the Reviewer, through comparison of live cell imaging and ApoI protection/qPCR, which can be found in Figure 1—figure supplement 3. Based on this, we now estimate that ~300 bps of resection are required.In theory, the HO-induced DSB is unrepairable, but because the HO cutting efficiency is quite low, and S. pombe cells are in G2 most of the time, if one sister chromatid was cut and engaged in repair with the uncut sister it could result in an underestimation of resection. Were cells with a transient Rad52 focus detected? The exo1 example in Figure 1 appears to have a Rad52 focus that appears early and then goes away. Does elimination of Rad51 change the number of cells resecting or rate of resection? At late times, when the mCherry signal is very bright, are both sister chromatids cut and resected?crb2Δ cells, which proceed into mitosis due to the checkpoint defect associated with this allele. We found that in the vast majority of cases (21/27), both daughter cells inherited a fully resected site-specific DSB  in which Rad52 associates with the LacO array and is then lost, followed by repair; in this case we would expect the cells to avoid checkpoint arrest, and proceed into mitosis. In the example the Reviewer points out, cells lacking Exo1 , any cell that recruits Rad52 fails to resect, but remains checkpoint arrested, suggesting failed repair rather than repair of a single DSB using the sister as a template. This interpretation is further supported by the observation that loss of Crb2 in this background fully recovers Rad52 loading . To further address the frequency at which both sisters are cut, we analyzed Rad52 foci upon cell division in  DSB see . In the Langerak et al., used a qPCR assay to measure end resection and reported no defect in resection initiation (35 nt from HO cut site) in the exo1 mutant or exo1 rqh1 double mutant. Is the failure to detect Rad52 foci in most exo1 cells because resection tracts are <90 nt or because the amount of ssDNA required to support a Rad52 focus is much longer than 90 nt? Given that the Rad52 single is quite weak until the lacO array disappears, I think the authors might be under-estimating the amount of ssDNA to visualize a Rad52 focus. Does the exo1 mutant show normal DSB formation (measured with primers flanking the HOcs) and resection to the ApoI site located 168 nt from HOcs? Similarly, is DSB formation normal in the rev7 mutant and can early resection be detected by the qPCR assay?As stated in the manuscript, we experimentally estimate that at least 30 copies of Rad52 must be loaded onto the resected DNA to be visualized. Unfortunately, the number of Rad52 copies that are expected to remain associated with the remodeled nucleoprotein filament has not been clearly established experimentally. We initially set lower bounds as 90 nts. To provide further insight, we carried out qPCR experiments on cells lacking Exo1 and Ctp1 using the approach described in Langerak et al., as suggested by the Reviewer, but with many more primer sets. All genetic backgrounds (including cells lacking Exo1 or Ctp1) have similar levels of resection 168 bps from the HO cut site (not shown). Most useful is the finding that we do see a substantial resection defect in cells lacking Exo1 using primers 300 bps from the HO cut site, almost to the extent seen in cells lacking Ctp1 . Moreover, we see no resection 3 kb from the DSB at 90’ post-induction. Taken together we estimate that cells lacking Exo1 have a block between ~150 bps and ~300 bps of resection based on these population data. Importantly, we do visualize transient Rad52 loading in cells lacking Exo1 , consistent with an upper bounds of ~300 nts of resected DNA being sufficient to load enough Rad52-mCherry copies to be visualized. However, we have given a broader range for the extent of resection necessary to observe robust Rad52-mCherry recruitment as between 150-300 bps in the revised manuscript. The effect of loss of Rev7 by qPCR is now included in Figure 4C. Again, consistent with our initial interpretation, there is a slight defect in resection 300 bps from the HO cut site, but a gain in resection 3 kb away. This orthogonal approach therefore supports our hypothesis that Rev7 promotes early steps in resection but has a gain in long-range resection rate (once this early phase is overcome).Why does rqh1 suppress the early resection defect of rev7? An odd result that is not discussed in the text.We do not yet have a molecular understanding of how loss of Rqh1 is able to suppress the early loss of Rad52-mCherry loading in cells lacking Rev7. We do acknowledge this in the revised text, but further insight will require additional experimentation, which is ongoing.While the overall findings on the role of Rev7 and Crb2 repressing the Rqh1 resection pathway are certainly of interest to the field, the data analysis needs to be improved.We appreciate that the reviewer found the findings revealed by this work to be of interest and have endeavored to address the remaining concerns regarding image analysis and validation using orthogonal assays in this revision.Reviewer #3:DNA end resection is a process that initiates recombination-based DNA double strand break repair. As resection also generally inhibits non-homologous end-joining, regulation of DNA end resection is an important process. The human 53BP1 protein, though its various effectors, has been found to be an inhibitor of DNA end resection, although mechanistic insights are lacking. These processes appear to be at least partially conserved in low eukaryotes.The authors are using a S. pombe as a model system, where they developed an assay allowing the monitoring of resection in live cells . The assay is based on the disappearance of GFP-LacI and appearance of Rad52-mCherry signal next to HO-endonuclease induced DSB. This is an interesting method that will be useful for high throughput microscopy-based screenings. However, the method has certain limitations in contrast to established southern blotting, PFGE or RTPCR-based methods:We appreciate the reviewer’s assessment of the value of a microscopy-based resection assay. While we agree that the assay has some weaknesses compared to previously developed population-based assays, we would argue that qPCR and Southern blot approaches also have limitations in contrast to the microscopy-based method developed here . In the larger scope, we expect that both assays will provide complementary insights into the control of DSB end resection.a) The assay measures Rad52 accumulation, which is a step after resection. Rad52 is a mediator that loads Rhp51 on RPA-coated resected DNA. Using RT-PCR based assay, the authors established that there is a correlation between both processes (resection and RAD52 loading). However, this was only done in wt background, and it is possible RAD52 loading might differ in the mutants analyzed, and Rad52 might be loaded with different kinetics dependent on the resection pathway. The authors calculate resection rates making the assumption that there is no difference.exo1Δ, rev7Δ, ctp1Δ) using the qPCR based method . Our findings support the notion that the extent of Rad52-mCherry loading is coincident with resection of at least 150-300 bps of DNA flanking the DSB .In the revised manuscript, we include substantial additional support for our conclusion by analyzing additional genetic backgrounds (b) The assay measures resection of DNA bound by GFP-LacI, a non-physiologic binder. Therefore, the resection proteins must displace LacI for resection to occur. Under physiologic conditions, DNA near DSBs will likely be chromatinized and subsequently remodeled allowing resection. This is especially a concern when analyzing the role of chromatin binders such as 53BP1/Crb2. Also, the individual resection pathways may be affect by LacI binder to a different degree, which complicates interpretation.We agree that in principle the LacI binding could alter how resection proceeds through the array. In addition to the experimental evidence presented in the initial manuscript supporting the validity of resection through the array, we now also include qPCR analysis on the other (non-LacO containing) side of the HO nuclease cut site; this analysis supports the conclusion that the LacO array does not strongly influence resection . This concern has also been explored by more broadly including analysis of resection using the orthogonal, qPCR based assay .The most interesting finding is that Rev7 and 53BP1/Crb2 appear to repress long-range resection dependent on Rqh1 : In the absence of Rev7 and Crb2, long-range resection is accelerated, which is independent of Exo1 and depends on Rqh1.1) This observation should be verified using a previously established assay, given the concerns listed above. Related to this point, it remains a formal possibility that the action of Rqh1 is not specific to resection, but to strip LacI, allowing resection through another process. Performing resection with the same mutants in an established setup (with no LacI) will address this concern as well.In the revised manuscript, in addition to the validation by the qPCR assay that the LacO array does not alter resection , we provide further evidence that cells lacking Rev7 show more rapid long-range resection using the qPCR approach . These data strongly support that resection, rather than some other activity (such as loss of LacI) is responsible for the progressive loss of GFP-LacI signal.The authors then go on to demonstrate differential effects of Rev7 and Crb2 on early resection steps, based on timing of Rad52 foci appearance upon break induction . I am concerned about these results: using Rad52 (a protein with a function downstream of resection) does not appear to be correct as a marker of "early" resection.2) The results should be analyzed in an established assay where the readout is clearly early resection. Also, to make claims on early resection, the analysis should include mutants deficient in short-range resection (e.g. mre11) and mutants in long-range resection, where both pathways have been inactivated (e.g. exo1 rqh1).The revised manuscript includes substantial new data to address how to place Rad52 loading in the context of resection progression. We now leverage cells lacking Exo1 to obtain a more precise estimate of the extent of resection required to load Rad52. By comparing qPCR analysis  and our image analysis , we now estimate that ~200-300 bp of resection is necessary to visualize Rad52-mCherry loading at the DSB. Moreover, we compare cells lacking Exo1 with those lacking CtIP/Ctp1 (a “true” resection initiation factor); these two genetic backgrounds have very similar resection defects 300 bp from the HO cut site. We would therefore argue that while Exo1 is not required for “resection initiation”, it is required for the early phase of long-range resection (defined here as the earliest phase in which Exo1 is required).3) In Figure 2, the authors demonstrate that rev7 mutants have accelerated long-range resection, which is dramatically decreased when rqh1 is additionally mutated (rev7 rqh1), supporting the hypothesis that resection in rev7 mutants is Rqh1 dependent. In contrast however, the in Figure 3, the "early" resection is increased when rqh1 is mutated in rev7 background, a completely opposite effect. This is very confusing. The authors comment that "early" resection is a combination of Mre11 and Exo1/Rqh1 dependent processes. This dichotomy reinforces my concerns about the robustness of the experimental setup.As stated in our response to reviewer 2, it is true that at present we do not fully know how to interpret this result, which will require further experimentation  to dissect. However, with regards to the general robustness of the assay, we believe that the revised manuscript provides substantial new support for the experimental setup and the validity of the interpretations made from the data.In summary, the manuscript presents an interesting assay and interesting pieces of data, but it seems rather preliminary at this point.We have endeavored to convince the reviewer that, with the addition of supportive data from orthogonal assays and further validation, the manuscript is now sufficiently developed to support publication.[Editors' note: the author responses to the re-review follow.]The biological findings of this manuscript are interesting, particularly the role of Rev7 in suppressing resection by the Rqh1 pathway, but there are significant concerns over the reliability of the assays used. The authors place equal importance on the fluorescence-based resection presented; however, the reliability of the assay is questionable and the weak point of this work. The conceptual idea of the assay is good, but there are reservations about its implementation and use to quantify resection speed. The authors have, for the most part, established the utility of their assay for the analysis of long-range resection, although they still need to provide a graphical example of their quantification.We are gratified that the biological findings of the manuscript were found to be of interest and that there was enthusiasm for the utility of our assay. The remaining point, the need for graphical examples of the quantification, has been addressed in the revised manuscript – please see Figure 1D,E, Figure 1—figure supplement 2, Figure 2B, and Figure 2—figure supplement 1.They discovered interesting roles of Rev7 and Crb2 in Rqh1-dependent resection. As both a methods contribution and a contribution to the recombination field, these findings could justify publication of a revised manuscript.We appreciate that the insights into the mechanisms by which Rev7 and Crb2 influence long-range resection (in an Rqh1-dependent and Exo-independent manner) were found to be of interest, therefore warranting further consideration of our manuscript.However, the manuscript then goes on to dissect the roles of these proteins in proximal recombination. Here the work is both incomplete and internally inconsistent. It would appear that the data in Figure 4A and Figure 4C are inconsistent with one another: 4A show a reduction in "Cell cycles with Rad52 foci" for rev7delta, whereas 4C shows no change, within error, for rev7delta at 300 bps .We acknowledge the concerns of the reviewers with respect to the measurement and interpretation of data corresponding to proximal resection. As we have not obtained additional data to clarify these points, we have removed this section of the manuscript . This revision is therefore focused solely on the long-range resection assay and the novel finding that Rev7 acts as an inhibitor of RecQ helicase-mediated resection.Also, the conclusion that of Rev7 promotes early resection is based entirely on how quickly a Rad52 focus forms after break induction, and how many cells have visible Rad52 foci. It was suggested that the authors use a more direct measurement of break induction by qPCR using primers flanking the HO cut site. This has not been done and is a significant concern.Although we have removed the data and text related to proximal resection, the revised manuscript includes qPCR analysis of the efficiency at which the site-specific DSB is induced in the population. The addition of this data supports the assertion that resection of the site-specific DSB takes place in the vast majority of fission yeast in S/G2 with a DSB, as the fraction of cells that (1) show loss of the qPCR product across the HO cut site , (2) undergo resection 168 bps from the HO cut site , and (3) ultimately load Rad52-mCherry  are similar (~15-20%).If one compares the qPCR data for resection 168-bp from the HO cut site shown in Figure 1D and Figure 1—figure supplement 3B there are vast differences – 3% resection at 120 min in one figure compared with 15% resection at 120 min in the other. If there is this much variability between populations of cells then it could explain why the rev7 mutant looks different to wild type, and why rqh1 appears to suppress the early resection defect of rev7. How many independent inductions were performed for image analysis?In this revision, we include population data (qPCR) to test the reproducibility of DSB induction; these data  demonstrate that HO nuclease has ~20% efficiency from multiple biological replicates. As detailed in Supplementary file 2 for all genotypes, the data for the image analysis is from 6 independent inductions (WT) and 5 independent inductions (rev7Δ). More generally, there is substantial evidence that the differences between resection rates that we measure in different genotypes are highly reproducible.The authors need a reliable method to assess DSB formation independent of Rad52 focus formation before drawing conclusions about a role for Rev7 in promoting early resection.Again, although we have removed the data/interpretation related to a role for Rev7 in proximal resection, we do now include qPCR analysis across the HO cut site that supports the conclusion that Rev7 does not influence the efficiency of DSB induction .Furthermore, In Figure 1D, the authors use a qPCR assay to detect formation of ssDNA 168 and 14,253 bp from the HO cut site. From this assay, very few (2%) cells exhibit resection to the end of the lacO array 360 min after HO induction. This would appear to contradict the microscopy assay, which shows resection through the array takes ~150 minutes.We understand that this appears contradictory at first glance, but importantly the time courses for the qPCR (population experiment) and the measurement of the time to resect the array (in single cells) cannot be directly compared. The “time 0” for the qPCR experiments is the addition of uracil to the media. Thus, there is a substantial lag  for cells to express the HO nuclease, reach S phase , and initiate resection. For the single cell assay we isolate measuring the rate of long-range resection by setting the “time 0” to the point at which Rad52-mCherry has visibly loaded. Thus, on the surface this apparent “contradiction” instead reflects that the qPCR data is influenced by many rates of sequential steps . Indeed, this comparison highlights yet another the advantage of the assay described in the manuscript, which specifically interrogates long-range resection without these other confounding steps contributing to the measured rate. We have also edited the text to clarify this point.Also, data should really be from biological replicas, not technical replicates of qPCR.We agree, and these data include multiple biological replicates .Consequently, whether or not Rev7 has a role in proximal resection is not clear from the authors' data.Again, we have removed this aspect of the manuscript.There was agreement that manuscript needs to be revised:1) It is essential that the confusing data on short range resection are clarified or removed from the manuscript. If the proximal resection data are retained, then the comments raised above need to be addressed. In addition, in the review of the prior version of this manuscript, reviewer #3 commented on this part in the original submission: "Also, to make claims on early resection, the analysis should include mutants deficient in short-range resection (e.g. mre11) and mutants in long-range resection, where both pathways have been inactivated (e.g. exo1 rqh1)." The authors did not address this previous request for clarification of this unexpected finding in this revised manuscript, using the analyses described for proximal resection .It is unclear whether the authors can make these revisions within the timeframe given. If not, then these data on proximal resection would need to be removed, and the conclusions of the manuscript refocused. One major finding is the effect of Crb2 on long-range resection: while this is new in S. pombe, it is well-described in S. cerevisiae (Rad9). The second major finding is the effect of Rev7 on long-range resection: this was only shown in humans, but not in the microbial eukaryotes. Although significant, the impact of the current manuscript is diminished without the proximal resection data; consequently, the author would need to make the contributions of their sound work much clearer in an expanded Discussion section of their work in relation to the existing literature .We agree with points of the reviewers with respect to the proximal resection data and have removed this from the manuscript. Further, we acknowledge (and now discuss in greater detail in the discussion) that an influence of Rad9 on long-range resection in budding yeast has been described previously. Further, data argue that this effect could involve Sgs1, the Rqh1 orthologue. However, we would argue that the following conclusions coming from this study are unique and impactful:1) An entirely novel (and indeed the key) aspect of our study is that Rev7 acts to inhibit long-range resection through the RecQ helicase (Rqh1) pathway. Although it is true that Rev7 was suggested to inhibit resection in mammalian cells, these measurements were largely indirect, and the phase of resection influenced by Rev7 was not addressed.2) We show that the influence of Crb2 on long-range resection is also through repression of Rqh1. This is consistent with budding yeast studies but is clearly shown to occur through a specific effect on long-range resection in our study.We have articulated these points more clearly in the revised Discussion section.2) The authors use appearance of a Rad52-mCherry focus to identify onset of resection and disappearance of a lacO/LacI-GFP focus to identify resection past a site some 14 kb away from the DSB site. The problem is that both signals are rather fuzzy in many of the image series shown. This is especially true for the Rad52-mCherry focus . The decision if a signal is judged as a focus or not is crucial, as it is the basis to calculate resection speed. This decision is made manually. Although the authors try to "equalize" the error by randomizing the image series prior to analysis, it seems to be a quite ambiguous approach of questionable reliability. What is their criterion to judge if a focus is present or not? Do they compare with background control strains? Why don't the authors use software to quantify the fluorescence signals and generate intensity trajectories? There are several non-commercial image analysis software packages available dedicated to exactly this purpose. Based on thresholds defined by appropriate control strains, appearance and disappearance of the signals could then be identified in a more controlled and rational way. A minimal requirement is that the author provides an x-y graph of foci fluorescent intensity vs time for each their examples of time-lapse video data in the manuscript.We evaluated the utility of several image analysis software . Indeed, we are experts at developing quantitative image analysis routines , but all were inadequate due to the uniquely stringent criteria that we placed on identifying a Rad52-mCherry focus (described in the Methods), which goes well beyond simply an intensity measurement. Nonetheless, to satisfy the reviewer’s request, we include data to address the issue of “background control strains” now in Figure 1—figure supplement 3D-G. As you can see, there is little to no chance that background fluorescence or “noise” could reproducibly contribute to a focus assignment. Moreover, as stated in the manuscript, the frequency of identifying a Rad52-mCherry focus that meets our criteria  and found that this can essentially never be observed if HO nuclease is not expressed ”). We do recognize that it is imperative to that we can robustly identify these events, and ideally to provide a quantitative framework for how we do so. To that end, as requested, we include fluorescent intensity versus time plots in the revised manuscript. Depicting the data in this manner makes it qualitatively quite clear, for example, that cells lacking Rev7 resect the array much more quickly than WT strains.


Osteoarthritis (OA) is a leading cause of disability in the United States. Although no disease-modifying therapies exist, patients with knee OA who increase walking may reduce risk of functional limitations.The objective of the study is to evaluate the impact of a mobile app (OA GO) plus wearable activity monitor/pedometer (Jawbone UP 24) used for 90 days on the mobility of patients with knee OA treated with hylan G-F 20.2 were excluded. All patients received a single 6-mL injection of hylan G-F 20 and wore the Jawbone monitor. The patients were then randomized 1:1 to Jawbone and OA GO  with visible feedback (unblinded) or Jawbone only  with no visible feedback (blinded). The primary endpoint was mean change from baseline in steps per day at day 90 between Groups A and B.Patients with knee OA aged 30 to 80 years who were eligible to receive hylan G-F 20 and were familiar with smartphone technology were enrolled in this randomized, multicenter, open-label study. Patients who had a body mass index above 35 kg/mP=.03) and the mean percentage change  from baseline in favor of Group A over Group B. There was a greater reduction in pain from baseline during the 6-minute walk test in Group A versus Group B. . Most patients (65.4%) and surveys of physicians (67.3%) reported they would be likely or very likely to use/recommend the devices. Patient Activity Measure-13 scores improved from baseline (LS mean change for Groups A and B: 5.0 vs 6.9), with no significant differences between groups. The occurrence of adverse events was similar in the 2 groups.Baseline characteristics were similar between groups. There were significant differences between the increases in least squares (LS) mean number of steps per day (1199 vs 467, Use of a novel smartphone app in conjunction with a wearable activity monitor provided additional improvement on mobility parameters such as steps per day and pain with walking in the 6-minute walk test in patients with knee OA who were treated with hylan G-F 20. Results also highlight the amenability of patients and physicians to using mobile health technology in the treatment of OA and suggest further study is warranted. Osteoarthritis (OA) is a leading cause of disability in the United States . From 20To date, there is no disease-modifying therapy available for OA . OptimalWalking has been shown to significantly reduce symptoms and the risk of functional limitations due to knee OA ,20. In oAdoption of mobile health apps has the potential to improve patient outcomes. Mobile health apps have been shown to be a useful tool in weight loss programs ,22. In aThe use of mobile apps in health care may be one such strategy to increase physical activity and enhance OA management. However, there are currently no published data on the use of mobile apps for the management of knee OA.The objective of this study was to examine the impact on mobility in knee OA patients who were treated with hylan G-F 20 and standard of care follow-up plus a mobile smartphone app (OA GO) and a wearable activity monitor (Jawbone UP 24) versus hylan G-F 20 and standard of care follow-up only over 90 days. We hypothesized that patients using the OA GO app and a wearable activity monitor would experience a positive impact on their mobility compared with those not using the app.Consecutive patients with knee OA whom the physician investigator decided to treat with one 6-mL injection of hylan G-F 20 (in accordance with the US label) who consented and qualified were enrolled in this open-label, multicenter, randomized, parallel-group study. Study sites and physicians were recruited using a detailed questionnaire and site qualification visit. Patients were recruited from the selected private community-based practices and research-only practice sites. All eligible patients were able to read and understand English and provided informed consent prior to starting the study. The protocol complied with the 18th World Health Congress  recommendations and applicable amendments and with any applicable country-specific laws, regulations, and guidelines. The protocol was approved by relevant ethics committees and/or institutional review boards. After working with the US Food and Drug Administration (FDA) to initiate registration of the trial, the sponsor deemed the study not eligible to be posted on ClinicalTrials.gov considering that clinical studies using devices whereby the primary outcome measure relates solely to feasibility and not to health outcomes are excluded from registration. The FDA concurred there was not a requirement to register the trial at that time.To be included in the study, all patients must have had unilateral knee OA and have been suitable for treatment with hylan G-F 20 based on the decision of the physician investigator. Study sites were requested to report whether or not a knee examination was performed or x-ray was taken but the results of these examinations were not captured.Patients were excluded if they were aged younger than 30 years or older than 80 years, were unfamiliar with smartphones, or had baseline pain greater than 9 on the 11-point numeric pain rating scale  in the target-for-treatment knee while walking on a flat surface. Patients with bilateral disease were excluded with the exception of patients who were treated in only one knee and had contralateral knee pain less than 4 on NPRS while walking on a flat surface. Patients whose baseline daily step average was less than 500 or more than 8000 as assessed during the screening and run-in phases were not eligible. Also excluded were patients who had a body mass index (BMI) greater than 35 or life expectancy less than 12 months, were currently using a wearable activity monitor or analogous device, had planned surgery on any lower extremity joint or any significant medical condition that would interfere with study participation, were chronic narcotic users, or were pregnant or breastfeeding or likely to become pregnant. The protocol did not specifically exclude patients based on use of previous intra-articular injections .All eligible patients received hylan G-F 20. Participants wore a commercially available activity monitor (Jawbone UP 24) on their wrists and were instructed to remove the monitor only during the weekly charging times and in situations where the device would be submerged in water. Patients were randomized 1:1 (stratified by site) to Group A or Group B. The randomization scheme was generated by the study sponsor and stratified by site. Sealed envelopes, numbered in an ascending order for use, were provided to each site. The envelopes were opened according to ascending sequence to ensure proper randomization.Group A patients were provided with regular follow-up as per standard-of-care  plus an unblinded wearable activity monitor and a mobile app (OA GO) . The OA The primary endpoint was mean change from baseline to day 90 in mobility as measured by steps per day. Baseline steps per day was the average over at least 3 days, and last assessment was the average steps per day over the 7 days immediately preceding the last visit. Change was calculated as the difference between the 2 averages. Use of average steps per day avoided bias due to any one particularly good or bad day for the patient. The secondary endpoints were mean percentage change from baseline in steps per day at each assessment visit (average of a 7-day period), and at day 90, mean percentage change from baseline in the 6-minute walk test (distance and pain assessed by the NPRS), patient and physician satisfaction with treatment, percentage change in Patient Activation Measure (PAM)-13 questionnaire score , percentFor Group A, the satisfaction of patients and physicians was captured using survey questionnaires. The patient survey consisted of 7 questions: the first 6 were answered on a 7-point Likert scale related to the extent to which the patients experienced changes , and the seventh was a 4-point scale asking about their likelihood of using the device in the future . Valid questionnaires were those in which at least 4 of the first 6 questions were answered. A physician survey was similarly constructed with a final question, using the 4-point scale, which asked whether they would recommend the device in the future (not likely to very likely). Physicians answered the survey at day 90, before viewing the data, and must have answered at least 3 of the first 4 questions. All responses to survey questions were based on patient and physician observations and opinions. The survey questionnaires were developed for this study and were not externally validated.The PAM-13  was usedThe VAMS was used to assess mood as captured in 8 domains with raw scores transformed into T-scores with a mean of 50 and standard deviation of 10. The VAMS has been validated in both normal and neurologically impaired individuals as a brief measure of internal mood state ,33.The Consolidated Standards of Reporting Trials guidelines were followed . A samplAll efficacy endpoints were analyzed in the modified intent-to-treat population, which included all randomized patients who had both a baseline value and an on-treatment value for the primary endpoint on or after day 30. All efficacy endpoint data comparing change from baseline between groups were obtained from an analysis of covariance model with baseline mean steps per day as the covariate and treatment assignment and pooled site as class variables. They are presented as least squares (LS) means with 95% confidence intervals (CIs). Statistical analyses were based on initial verification of parametric model assumptions, and a rank analysis of covariance was then considered if conditions for a parametric model were not met. To allay concerns by the readers, nonparametric analyses are presented here. Patient and physician satisfaction surveys at last visit were summarized.Safety endpoints were analyzed for all patients provided with a wearable activity monitor. Adverse events (AEs) were presented as the number and percentage of patients experiencing an AE. Multiple occurrences of the same event in the same patient were counted only once. The denominator for computation of percentages is the safety population within each randomized group.All patients had a knee exam at baseline, and 119 of 211 (56.4%) had an x-ray of the knee. A total of 211 knee OA patients treated with hylan G-F 20 were randomized , and nearly all patients completed the 90-day observation period  . Almost P<.001). Improvement in mobility at day 90 was significantly greater in Group A than in Group B. LS mean change in number of steps per day was 1199 vs 467, a mean difference of 732 steps   .P=.03), at day 30 was 40.1% versus 9.0% , and at day 90 was 35.8% versus 11.5%   .P=.007) (P=.96)  . There w (P=.96) .A greater number of Group A patients  reported they would be likely or very likely to use the devices compared with patients  who reported that they would be somewhat likely or not at all likely to do so . A totalP=.99).PAM-13 scores improved from baseline to day 90 in both groups. The LS mean change from baseline was 5.0% in Group A versus 6.9% in Group B , which should give a more valid and reliable report of pain over time as it does not rely on patient recall of pain experienced. In contrast, visual analog scales  and the In this study, the results of the distance portion of the 6-minute walk test were not statistically different between groups. However, this assessment represents only a very brief snapshot in time; distance monitored continuously may be a more reliable measure of improvement in patient mobility. Despite these results not achieving statistical significance, those using the OA GO app ended the study with an average of more than 5500 steps per day, which approaches 6000 steps per day, a level that has been shown to cut the risk of developing functional limitation by half within 24 months .PAM-13 scores were also not statistically different between groups in this study. The observed lack of statistical difference between groups could be due to the high mean baseline activation state of patients in this study (71.2 [SD 13.2]), which may have resulted from the selection criteria  and may have been higher than for those excluded from the study. Indeed, the baseline activation score in this study was higher than scores for the general population in the US (61.9 [SD 21]) and populations with other chronic conditions , and acThe practical aspects of incorporating a mobile health app into treatment paradigms to improve patient mobility should be further investigated. This strategy requires access to a smartphone, which may be a financial barrier for some patients . PatientOverall, the results of this study provide evidence that in patients suffering from knee OA who received hylan G-F 20, additional improvement was achieved in this study, based on mobility parameters of steps per day and pain reduction, with use of a mobile app and wearable activity monitor.Reduced mobility in patients with knee OA can be a significant issue negatively affecting quality of life and experience of pain. Increasing patients’ motivation to walk and thereby increasing their mobility may reduce these negative effects. Use of a novel smartphone app in conjunction with a wearable activity monitor provided additional improvement on mobility parameters such as steps per day and pain with walking in the 6-minute walk test in patients with knee OA who were treated with hylan G-F 20 see . These r


To describe a case of complete remission of thrombotic microangiopathy after treatment with eculizumab in a patient with non-Shiga toxin-associated bacterial enteritis.Case report: Medical/surgical intensive care unit (ICU) of a university teaching hospital.A 62-year-old man presented to a local hospital with mucous and bloody stool persisting for 1 month and worsening abdominal pain for 2 weeks. He had thrombocytopenia and renal dysfunction and was admitted with a diagnosis of sepsis due to intraabdominal infection. However, he did not respond to antimicrobial therapy, and after 7 days he was transferred to the Chiba University Hospital ICU.Antimicrobial therapy was continued, and continuous hemodiafiltration was initiated on ICU day 3, but the patient's condition deteriorated and he became anuric. Plasma exchange (PE) was initiated on ICU day 11, but anuria and thrombocytopenia persisted. Intravenous eculizumab therapy was initiated on day 26 and resulted in quick recovery of urine output and platelet count and successful discontinuation of renal support.E coli in feces and anti-Shiga-toxin antibody in blood led to suspicion of atypical hemolytic uremic syndrome (aHUS). Genetic test identified a nonsynonymous mutation  in the membrane cofactor protein gene (MCP).The diagnosis of thrombotic microangiopathy was established by the presence of schistocytes on the peripheral blood smear on ICU day 9. A plasma sample collected prior to initiation of PE showed a disintegrin-like and metalloproteinase with thrombospondin type 1 motifs member 13 (ADAMTS13) activity level of >10% (25.1%). The absence of both Shiga-toxin producing Although the pathological significance is currently unknown, this mutation may have been the cause of adult-onset aHUS in our patient. In this case, eculizumab was successfully introduced and discontinued, and the patient remained relapse-free after 1 year of follow-up. The duration of eculizumab therapy for patients with aHUS should be determined on a case-by-case basis and possibly according to the causative genetic mutation, even though discontinuation of eculizumab therapy once initiated is not generally recommended. Two major variants of TMA are hemolytic uremic syndrome (HUS) caused by Shiga toxin and thrombotic thrombocytopenic purpura (TTP) associated with decreased activity of a disintegrin-like and metalloproteinase with thrombospondin type 1 motifs member 13 (ADAMTS13]). Other etiologies of TMA include infection, drugs, and malignancy. TMA associated with abnormalities in the complement pathway is specifically designated as atypical hemolytic uremic syndrome (aHUS). More recently, the clinical efficacy of eculizumab in the treatment of aHUS has been reported. Eculizumab is a monoclonal antibody that specifically binds the 5th component of the complement (C5) and suppresses excessive complement activation.aHUS has traditionally been treated by plasma exchange and infusion of fresh frozen plasma. However, these supportive therapies provide only transient remission and have been associated with poor long-term prognosis.We herein report complete remission of a refractory case of TMA upon initiation of intravenous infusion of eculizumab in a patient with bacterial enteritis. Findings in the case suggested that the patient had aHUS in association with an abnormality in the complement pathway derived from a recently reported genetic mutation.24/μL), thrombocytopenia (6.4 × 104/μL), and elevated blood urea nitrogen  and serum creatinine (2.56 mg/dL) levels. The patient was admitted with a suspicion of sepsis secondary to intraabdominal infection, and broad-spectrum antimicrobial therapy was initiated. However, his condition deteriorated, and after 7 days, he was transferred to the intensive care unit (ICU) at Chiba University Hospital.A 62-year-old man presented to his local hospital after 1 month of mucous and bloody stool and 2 weeks of worsening abdominal pain. He had leukocytosis , the C3 level was 85 mg/dL , and the C4 level was 23 mg/dL .On admission to the ICU, the 165.3 cm tall patient weighing 99.3 kg was fully conscious , but he had a tendency to somnolence. His blood pressure was 154/103 mm Hg, heart rate 115/minutes, respiratory rate 30/minutes, body temperature 37.9 °C, and SpOEnterococcus species and Corynebacterium striatum, a diagnosis of severe sepsis derived from bacterial enteritis was established and wide-spectrum antimicrobial therapy was initiated. Continuous hemodiafiltration was initiated on ICU day 3 but oliguria progressed to anuria, thrombocytopenia persisted, and laboratory values did not improve. Blood, urine, and spinal fluid cultures taken on admission to the ICU were all negative, which eliminated the possibility of additional foci of infection. Two polyps (one each in the sigmoid colon and the rectum) were discovered on lower gastrointestinal endoscopy, which were diagnosed at histopathology as early-stage nonhemorrhagic adenocarcinoma. The polyps did not appear to be the cause of the bloody stool. Additional laboratory evaluations were negative for other hematological disorders or connective tissue diseases. On ICU day 9, schistocytosis (0.5%) was noted on the peripheral blood smear, and a diagnosis of TMA was established. The Hct level also had dropped to 23.5% on the same day and the declining trend continued, therefore red blood cell transfusion was performed days later when he complained dyspnea. Plasma exchange (PE) therapy was initiated on ICU day 11. The patient had only a slight remission of thrombocytopenia after a total of 8 PE sessions (2.1 × 104/μL before the 1st PE and 5.9 × 104/μL after the 8th PE), but there was no notable clinical improvement and the anuria persisted  was initiated on ICU day 26. An increase in urine output was observed on the next day, and renal support was successfully discontinued on the same day. And also marked recovery of the platelet count was obtained in combination with the effectiveness of supportive therapy including PE. Biweekly eculizumab infusion was repeated 4 times and then discontinued.The patient's clinical course is summarized in the Fig. CFHR1/3 on chromosome 1q32 using a commercially available kit , and found that CFHR1/3 did not show gene deletions. However, moderate hemolysis was induced in concomitant hemolytic assays using sheep red blood cells (RBCs), and analysis for mutations potentially associated with aHUS identified a nonsynonymous mutation  in the gene encoding membrane cofactor protein (MCP). All screened genes are presented in the Table After approval by the institutional review boards of Chiba University, the University of Tokyo, and National Cerebral and Cardiovascular Center, an anticomplement factor H (CFH) antibody assay and an analysis of complement factor H-related (CFHR) genes were performed using a blood sample collected from the patient prior to initiation of PE. Also written informed consent was obtained from the patient before the assays. The anti-CFH antibody level was 6.16 AU/mL, which indicated negativity for anti-CFH antibody. We performed the multiplex ligation-dependent probe amplification (MLPA) analysis to screen the copy numbers of The patient was discharged from the ICU on day 37 and was discharged to home on day 58. He was followed up regularly as an outpatient and was receiving 40 mg/day of prednisolone. At approximately 1 year after discharge, the patient remained in stable condition without relapse or resumption of eculizumab therapy.3CFH was first reported in 1998 and since then additional genes that may be associated with aHUS have been identified. In our case, a patient with infectious enteritis developed TMA. We suspected aHUS after excluding HUS, TTP, and secondary TMA. As PCR studies were not performed for this case thorough his disease course and antibody screening for Shiga toxin-producing E coli was only performed at admission, it is undeniable that our examination was not enough to eliminate the possibility of Shiga toxin-producing E coli infection for this case. However, the responsiveness of PE and also the effectiveness of eculizumab administration were obvious enough to diagnose this case as complement mediated aHUS.aHUS is a peculiar type of TMA that develops in association with endothelial dysfunction and platelet surface activation induced by dysregulation and chronic activation of the alternative complement pathway. Familial HUS caused by abnormalities in the gene encoding  In the present case, the patient was negative for anti-CFH antibody and had no gene deletions in CFHR1 and CFHR3. However, moderate hemolysis in a hemolytic assay using sheep RBCs as an auxiliary diagnostic method suggested an abnormality in the alternative complement pathway,,18 and a nonsynonymous MCP mutation, Ala311Val, was detected. Mutations in MCP have previously been associated with aHUS. This patient has no family history of aHUS, suggesting that the mutation may not have been inherited. The minor allele frequency for this novel mutation has not been extensively compared with that in nonaffected individuals. Therefore, it is categorized as a “variant of uncertain significance.”Plasma C3, C4, complement factor B (CFB) levels, anti-CFH antibody titers, and measurement of MCP expression by leukocytes are recommended for the diagnosis of aHUS. Since C3b is capable of binding to and injuring pathogens as well as autologous cells, rapid cleavage of C3b protects autologous cells from injury by the complement system. Development of aHUS secondary to MCP mutation involves a loss of function of the MCP that leads to failed C3b inactivation and the resulting chronic activation of complement.MCP is a membrane-bound protein that serves as a cofactor of complement factor I (CFI) and facilitates CFI-mediated cleavage of C3b expressed on the same cell membrane.MCP have been reported in approximately 10% to 20% of patients with aHUS. The Ala311Val point mutation is found within the serine–threonine–proline-enriched domain of MCP. Up to 90% of patients with aHUS who have abnormalities in regions of MCP encoding parts of the transmembrane domain or any of the 4 complement control protein (CCP) domains recover after PE or supportive care treatment alone, and renal support is successfully terminated in most of these cases.,20 Although an amino acid substitution in MCP suggestive of an MCP mutation causing aHUS was discovered in our patient, he did not respond well to plasma exchange.Abnormalities in  and in the present case, the initiation of eculizumab induced a rapid increase in urine output, allowing successful withdrawal of renal support in combination with plasma therapy, etc. Criticism that the initiation of eculizumab just coincide with resolution of the disease may arise because the platelet count was gradually rising prior to start of diuretic phase. However, the eculizumab infusion must have at least triggered the occurrence because the period of anuria was fairly long even though we continued PE regularly and the diuretic phase came suddenly following administration of eculizumab. Progression to end-stage renal failure (ESRF) is a key determinant of survival in patients with aHUS, and systemic complications of aHUS are serious. In 2 small open-label prospective trials, up to 80% of patients with aHUS were able to discontinue renal support after initiation of eculizumab therapy. As approximately 30% (5 in 18) of the patients who missed eculizumab doses experienced severe subsequent complications of TMA, discontinuation of eculizumab was not favored. However, cases of aHUS associated with MCP abnormalities seem to have a more favorable prognosis. Accordingly, in the present case, we decided to discontinue the eculizumab treatment after 4 weekly doses, and there was no deterioration in renal function during 1 year of follow-up. This suggests a possibility that safe and successful discontinuation of eculizumab is expected in mild aHUS caused by MCP mutations. We understand the exact association of the Ala311Val mutation and pathogenesis of aHUS is still opaque. Actually, this case is adult-onset aHUS, therefore carriers of the mutation are suggested to pose potential risk to cause the disease by severe infection, that is, septic insult as this case had. As aHUS is an extremely rare disease, with an annual incidence as low as 2/1,000,000 in adults and 7/100,000 in children, continued accumulation of knowledge regarding patterns of disease onset and response to treatments under different genetic backgrounds will be essential for the development of future treatment strategies.,23Eculizumab has been effective in patients with aHUS irrespective of their response to PE therapy,4MCP. However, this genetic variant has not yet been proven to be disease causing and its frequency in the general population has not yet been fully studied. Therefore, further genetic testing using a larger patient cohort is needed. The patient had a complete remission of TMA after initiation of intravenous eculizumab therapy, which was sustained as of 1 year after discontinuation of treatment. The present case demonstrates that the duration of eculizumab therapy for patients with aHUS should be determined on a case-by-case basis and possibly according to the causative genetic mutation.We encountered a case of TMA in a patient with bacterial enteritis. The patient had an amino acid substitution in MCP, suggesting that the TMA was due to aHUS associated with a mutation in