Human CST suppresses origin licensing and promotes AND-1/Ctf4 chromatin association

This study suggests a novel role of the telomere-associated human CST complex in suppressing origin licensing, through interactions with MCM, while promoting the recruitment of AND-1 and DNA polymerase alpha for replisome formation.

In this paper, Wang et al report ed two dist inct roles of the CTC1-STN1-TEN1 (CST) complex in general DNA replicat ion process. CST has similar propert ies wit h RPA and funct ions mainly at telomeres. The aut hors previously showed that CST plays a role in rest art of st alled replicat ion forks under DNA replicat ion st ress. Here, the aut hors found that reduct ion of cellular STN1 caused increase in binding of minichromosome maint enance (MCM) prot eins to chromat in using immunofluorescence microscopy. Consist ent ly, overexpression of CST result s in decrease of chromat in-bound MCM. They demonst rat ed that STN1 int eract s wit h MCM4 and MCM6 and that CST caused reduct ion in the int eract ion of CDT1 wit h MCM. These result s suggest that CST suppresses pre-RC format ion in some regions where CST localizes. Int erest ingly, CST also int eract s wit h AND-1/Ct f4 that is import ant for recruit ment of DNA polymerase alpha/primase for replisome format ion and init iat ion of Okazaki fragment synt hesis. shSTN1 caused significant reduct ion of chromat in-bound AND-1 and DNA Pol alpha, showing that CST enhances format ion/ maint enance of replisome, possibly in some regions of chromosome. These findings imply novel roles of CST in unpert urbed cell cycle in addit ion to the role in rest art of st alled replicat ion forks under the replicat ion st ress. However, I have two concerns on the consequence and significance of the st udy. CST plays a role in suppressing pre-RC format ion in early st age of init iat ion, whereas it enhances chromat in binding of AND-1 and DNA Pol alpha increasing replisome. I am a lit tle confused what is the overall consequence of CST in DNA replicat ion. Unfort unat ely, the aut hors did not provide sufficient int erpret at ion regarding the relat ions bet ween these apparent ly opposit e roles of CST in DNA replicat ion. The second concern is the aut hors did not conduct the analyses on specific regions of chromosomes, despit e assuming that CST plays in specific regions including G4s and GC-rich regions. It is quit e int erest ing to det ermine the regions where CST affect s MCM-loading AND-1/Pol alpha loading, respect ively, and whet her promot ion of AND-1/DNA Pol alpha binding occurs in the same region where pre-RC is suppressed. It is also int erest ing where CST localizes in G1 and S phases, respect ively. Lack of evidence and discussion on these point s made this work uncert ain and less at tract ive.
Minor comment s 1. Fig.1B and ot hers. Aut hors should specify the colors for merged image, magent a/MCM7, blue/DAPI and green/EdU. 2. Fig.1F. Dot dist ribut ion of WT (mean 47.8) differs subst ant ially from that in Fig.3C shNT (mean 36.4). Can the aut hors present a dat a set in which cont rols are consist ent wit h each ot her. 3. Fig.1F and Fig. EV1D. When CST-OE decreased severely chromat in-bound MCM7 and MCM6, did it affect DNA replicat ion and cell cycle progression? 4. Fig.2C. Aut hors need to add rat ionale and just ificat ion for the dot line at MCM6 int ensit y value 2 and for "high int ensit y MCM6". What happens if int ensit y above the background similarly to Fig.2B is compared for MCM6? 5. Fig.4B. Expression of CST severely impaired the CDT1-MCM int eract ion. I wonder if DNA replicat ion is delayed under the condit ions. Fig.6C. To just ify the comparison, the fold change of IB: H3 should be present ed. Alt ernat ively the values should be normalized using IB: H3. 6. Several references including Bhat tacharjee et al, Higa et al, are incomplet e.

Referee #3 Review
This submission by Wang et al explores the effect s of manipulat ing the CST complex in human cells on two different aspect s of DNA replicat ion, origin licensing and the recruit ment of a Pol alpha and replicat ion checkpoint recruit er, AND-1. Manipulat ing the levels of CST has downst ream effect s on MCM chromat in associat ion that are consist ent wit h CST as a negat ive regulat or of MCM loading. The experiment s are well-cont rolled and rigorous, and the dat a are generally of very high qualit y. The IF and FACS analysis of MCM loading are admirably quant it at ive. In addit ion, the effect of CST overproduct ion on the co-immunoprecipit at ion bet ween (overproduced) Cdt 1 and MCM is st riking. I have two major concerns however that preclude my support for publicat ion in this journal in it s present form: A) There isn't quit e enough subst ance to the finding that CST inhibit s MCM loading to const it ut e a major advance.
1. What is the biological import ance of ~3-fold higher/lower MCM loading in G1? As report ed by ot hers, modest changes in MCM loading in G1 don't appear as overt proliferat ion defect s, but they might creat e some new sensit ivit ies. Is there a cellular phenot ype that can connect these CST manipulat ions on a physiological paramet er? For example, does G1 lengt h change? Are cells more or less sensit ive to genet ic pert urbat ions to licensing (e.g. geminin product ion, part ial deplet ion of a licensing prot ein)? Changes in numbers of act ive forks? the aut hors should choose how best to demonst rat e a downst ream consequence of the MCM loading difference. 2. The overexpression and deplet ion experiment s analyze cells that have had long-t erm changes -shRNA or tet -induced st able expression (for how long?). Can they aut hors rule out the possibilit y that the MCM loading changes are indirect effect s of cell cycle dist ribut ion, a secondary effect of S phase pert urbat ions on subsequent G1 phases, et c? 3. The genet ic rescue experiment s in Figure 2 are not part icularly convincing. In part icular, the red and grey signals in Figure 2E look the same whereas red should resemble the blue cont rols if STN is complement ing well. (If this assay were working bet ter, it could be the foundat ion to test CST mut ant s.) In addit ion, the gat ing of "high" MCM cells is an art ificial designat ion that magnifies the phenot ype quant ified in 2D; there should be some just ificat ion for where this cut off is placed, and it would be even bet ter to not impose a binary classificat ion on dat a that are largely cont inuous. Finally, this figure -while impressive in it s technical sophist icat ion -is just one biological replicat e. 4. The aut hors should demonst rat e int eract ion of CST wit h MCM and int erference wit h Cdt 1-MCM association without the need for the very high expression typically associated with plasmid transfections. Is Cdt1-MCM binding enhanced by CST depletion? Analyzing endogenous proteins will also eliminate the need to normalize for differences in total MCM expression. 5. Is the CST effect on origin licensing separable from its well-known effect at replication forks? For example, is ssDNA binding required for the effect on MCM loading? There is very little molecular dissection of this new phenomenon that could put it in context of overall CST function. The text emphasizes the similarity to RPA, but ssDNA is not thought to be relevant for MCM loading. B) This study is essentially two smaller projects presented together rather than one complete study. The majority of the novelty and the bulk of the stronger data are the potential role of CST in origin licensing as an addition to it's more well-known role at replication forks. The very modest effects of CST on And-1 in Figure 6 and the CST chromatin binding in S phase in Figure 5 are too thin and underdeveloped to warrant inclusion, and they distract from the principal finding. Minor: a) The claim that CST "directly" binds MCM is a minor overstatement. One would need to test the complexes in isolation since it is formally possible that an evolutionarily-conserved bridging protein mediates the binding. b) The authors should report how many cells were analyzed in the dot plots and how many independent biological replicates were performed (co-IPs, FACS, etc.). c) The interpretation that HU has no effect on binding doesn't necessarily rule out a role for replication stress in chromatin recruitment in Figures 5 and 6. Cells generate endogenous replication stress every S phase. Given that, the S phase chromatin association of CST in Figure 5 could have nothing to do with MCM loading and everything to do with its replication fork function. d) The yeast spotting assays are spliced images; it would be better to not need that extra manipulation to present the data. e) The two-hybrid reporters encode genes for adenine and histidine biosynthesis, not GAL4 itself (page 8, top). f) The general protein stain Ponceau S is mis-spelled on several figures. Thank you for transferring your manuscript entitled "Human CST suppresses origin licensing and promotes AND-1/Ctf4 chromatin association" to Life Science Alliance. The manuscript was assessed by expert reviewers at another journal before, and those reviewer reports were transferred to us with your permission.
The reviewers at the other journal appreciated the quality of your work, but thought that the support for a biological significance of the results and for the mechanism of CST competing with CDT1 for MCM binding was not sufficient. Given the interest of the newly found CST gain-/loss-offunction effects particularly on replication licensing, these concerns do not preclude publication in Life Science Alliance, and we would like to invite you to provide a revised version for publication here. We would like to ask you to provide a full point-by-point response to the previously raised concerns and to address all minor/specific concerns of the referees. Certain controls (CTC1/TEN1 knockdown effect (reviewer #1, point 2)) should get added and the concern regarding indirect cell cycle-related effects (reviewer #3, point 2) should get addressed. Ideally (but failure to do so will not preclude publication), a second biological replicate for the assay in Fig. 2 should be provided (reviewer #3, point 3), and the effect of CST knockdown on CDT1-MCM interaction should get tested (reviewer #3, point 4).
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Response to Reviewers Comments
Referee #1: 2. Throughout the analyses, the authors only showed the effect of STN1 knockdown on the levels of MCM proteins. Have the authors ever examined the impact of the depletion of the other two proteins of the CST complex, CTC1, and TEN1?
We sincerely thank the reviewer for this suggestion, which has revealed STN1 as the major contributor of origin licensing supression. We have now performed this experiment following siRNA knockdown of CTC1, STN1 or TEN1 ( Figure S4). Our results for STN1 are reproduced with siRNA, providing further confirmation that STN1 knockdown leads to increased MCM. Interestingly, we find that neither CTC1 or TEN1 knockdown results in increased MCM chromatin association by IF or flow cytometry. These findings are consistent with our yeast-two-hybrid data demonstrating that STN1 and not CTC1 or TEN1 interact with MCM ( Figure 4).
We have added the following section "Depletion of CTC1 or TEN1 is not sufficient to increase chromatin-bound MCM" to describe these new results: "To demonstrate that this phenotype is not caused by long-term changes from stable knockdown, we next examined whether transient siRNA knockdown of STN1 also increased chromatin-bound MCM. We also determined whether CTC1 or TEN1 knockdown increased MCM chromatin association. Cells were treated with siRNA targeting CTC1, STN1 or TEN1 and MCM levels were then assessed in preextracted cells by IF and flow cytometry, as described above in Figure 1 and 2. With siRNA depletion of STN1, we observed a similar increase in MCM compared to stable knockdown. However, we were surprised to find that MCM levels were not increased following CTC1 or TEN1 knockdown ( Figure S4). These results suggest that CTC1 or TEN1 depletion are not sufficient to increase MCM levels and STN1 is the critical component of CST required to alter MCM chromatin association (see additional details below)." To address these results in the context of the inhibition of the MCM-CDT1 interaction, we also added the following text to the section "CST disrupts the interaction between MCM and CDT1": "However, we do note that depletion of CTC1 or TEN1 did not increase origin licensing ( Figure S4). This could be due to incomplete knockdown of CTC1 or TEN1. We propose that blockage of CDT1 occurs through binding of CST to MCM, which prevents/obstructs stable binding of CDT1 ( Figure 4C). Disruption of the MCM-CDT1 interaction would directly affect origin licensing (i.e. MCM loading) by preventing MCM recruitment, thus providing a possible explanation for why CST decreases origin licensing." Finally, text was added to the Discussion to address these differences: "Further biochemical studies are needed to determine the mechanism by which CST or STN1 prevents origin licensing and to understand the contribution of each CST subunit, as CTC1 and TEN1 were needed to maximally inhibit the interaction between MCM and CDT1 ( Figure 4) but depletion of either CTC1 or TEN1 did not increase origin licensing ( Figure S4)." Other points: The introduction part about pre-RC assembly needs to be revised. "OCCM complex, also known as pre-RC" is not correct. Pre-RC refers to the MCM double hexamer assembled at the replication origin. "Following formation of the OCCM, two MCM hexamers are sequentially loaded onto the DNA." is also not correct. In the OCCM, the first MCM complex has already been loaded, encircling dsDNA. We thank the reviewer for bringing this to our attention and have changed the text, as follows: "Loading of the first MCM hexamer by ORC and CDC6 leads to formation the ORC-CDC6-CDT1-MCM (OCCM) complex. A second MCM hexamer is then recruited and loaded onto the DNA for origin licensing to form the pre-replication complex (pre-RC)." Referee #2: Minor comments 1. Fig.1B and others. Authors should specify the colors for merged image, magenta/MCM7, blue/DAPI and green/EdU.
This has been corrected in the revised manuscript. The colors are now indicated in the figure legends and we have added color coding to the titles above the images in Figure 1B, 1E and 6A.
Can the authors present a data set in which controls are consistent with each other.
We believe that the reviewer is referring to differences in Figure. 1F and Figure 1C and their point is well taken. However, these are two different types (or subclones) of HeLa cells. In Fig 1C, HeLa1.2.11 cells are used, which were originally used in STN1 knockdown studies due to their long telomeres (Stewart et al. 2012, EMBO J). In Figure 3C, HeLa TetOn cells are used (Wang, et al. Cell Cycle, 2014). Based on subtle differences in the subclones, we would not necessarily expect the levels of MCM staining to be identical. This likely explains the differences observed. Also, while exposure time was kept constant for a given cell type (e.g. HeLa1.2.11, HeLa TetOn, HCT116), exposure times between cell types (HeLa1.2.11 vs HeLa TetOn) was not necessarily the same, which could also reflect differences in relative AFU.
3. Fig.1F and Fig. EV1D. When CST-OE decreased severely chromatin-bound MCM7 and MCM6, did it affect DNA replication and cell cycle progression?
We have now included growth curves and cell cycle profiles of synchronized HeLa CST-OE cells as well as HCT116 shSTN1 cells ( Figure S5 and S6). We previously reported growth curves and cell synchronization for the HeLa shSTN1 cells, which showed no defects in cell growth or cell cycle progression (Stewart et al. 2012, EMBO J) and have reconfirmed these findings (data not shown). We have added the following section titled "Altered CST expression leads to cell type specific changes in Sphase progression" to the manuscript, which describes these results: "Since changes in origin licensing (i.e. MCM loading) could alter genome replication, we determined whether STN1 depletion or CST overexpression altered cell growth and cell cycle progression. Interestingly, both HCT116 STN1 depleted and HeLa CST-OE cells exhibit decreased cell proliferation ( Figure S5). However, we previously showed that HeLa shSTN1 cells do not exhibit growth defects or defects in cell cycle progression (Stewart et al., 2012). To assess cell cycle progression in the HCT116 shSTN1 and HeLa CST-OE cells, we synchronized the cells by double thymidine block and released them into S-phase. The shSTN1 cells progressed more quickly through S-phase ( Figure S6A). In contrast, a minor delay in S-phase progression was observed in CST-OE cells ( Figure S6B). While the changes in S-phase progression cannot be directly attributed to the changes in origin licensing, this does fit with increased or decreased MCM chromatin association altering origin licensing and activation following STN1 depletion or CST-OE, respectively. However, such effects on cell cycle progression and growth may reflect cell type specific differences (e.g. p53 status, cellular MCM or CST levels) or relate to the level of STN1 knockdown, as HeLa shSTN1 cells do not exhibit accelerated S-phase progression or growth defects (Stewart et al., 2012)." The following text was also added to the discussion regarding these results: "Our data suggest that STN1 depletion or CST-OE can also alter cell proliferation in HCT116 or HeLa cells, respectively ( Figure S4). Interestingly, two Coats plus patients with STN1 mutations had intrauterine growth retardation and cell lines derived from these patients showed decreased proliferation and DNA damage (Simon et al., 2016). Thus, future research to determine whether changes in CST expression or mutation affect origin licensing under different conditions may help clarify the molecular etiology of CST-related diseases." Furthermore, previous results demonstrated that when the HeLa CST-OE cell line was subjected to exogenous replication stress new replication origins were activated and cell survival was increased compared to wild type cells (Wang et al. Cell Cycle. 2014). We suggest that the role of CST in replication restart may partially compensate for decreased origin licensing following CST-OE and thus lead to only minor changes in cell cycle progression and proliferation. Other groups have also shown that >75% depletion of MCM does not affect replication in the absence of replication stress, Thus, the decrease in origin licensing observed with CST-OE may not reach the threshold required to drastically alter S-phase progression (Ge, et al. Genes Dev. 2007). Future work will focus on untangling how different CST functions affect origin licensing, S-phase progression and DNA synthesis.
4. Fig.2C. Authors need to add rationale and justification for the dot line at MCM6 intensity value 2 and for "high intensity MCM6". What happens if intensity above the background similarly to Fig.2B is compared for MCM6?
This line was originally chosen to highlight increased MCM intensity of the STN1 knockdown samples and later the percentage of cells in this population was quantified. We agree with the reviewer that this is an arbitrary designation and have now removed the line, omitted the graph and replaced it with a new graph, which compares the mean signal intensity of MCM positive cells between cell lines ( Figure 2C and S3C). This new analysis shows that there is still a significant increase in the mean intensity of MCM positive cells with STN1 depletion. The text has been altered to describe this new analysis in the section "STN1 depletion leads to increased MCM in G1 and S-phase": "However, the intensity of MCM positive cells in the G1 population of shSTN1 cells was significantly increased compared to the controls, suggesting increased origin licensing after STN1 depletion ( Figure  2C-D)." 5. Fig.4B. Expression of CST severely impaired the CDT1-MCM interaction. I wonder if DNA replication is delayed under the conditions.
We also considered that DNA replication and cell growth was significantly delayed in cells overexpressing CST. However, we only observe a minor cell growth defect and delay S-phase progression in these cells compared to wild type (see comment for #3). Fig.6C. To justify the comparison, the fold change of IB: H3 should be presented. Alternatively the values should be normalized using IB: H3.

5.
We have now normalized the levels of AND-1 and pol  to H3 in Figure 6C, which shows similar results to normalizing to Ponceau S.

Several references including Bhattacharjee et al, Higa et al, are incomplete.
We thank the reviewer for their careful reading of the manuscript. The references have been corrected.
Referee #3: 2. The overexpression and depletion experiments analyze cells that have had long-term changes -shRNA or tet-induced stable expression (for how long?). Can they authors rule out the possibility that the MCM loading changes are indirect effects of cell cycle distribution, a secondary effect of S phase perturbations on subsequent G1 phases, etc?
The stable HeLa shSTN1 cell line is from a single clone so were originally cultured until sufficient cells were obtained for freeze down. The passage number beyond that is maintained to as few as possible with new stocks frozen shortly after unthawing. Profiles of the stable HeLa STN1 knockdown cells have been previously published (Stewart et al. EMBO J. 2012) and showed no significant changes in cell cycle profile. However, the HCT116 shSTN1 cells show growth defects and changes in cell cycle ( Figure S4). This cell line was derived from a pool of cells following drug selection. HCT116 cells are also have functional p53, which would affect cell growth under conditions of DNA damage or replication stress. The CST-OE cells were stably selected and TEN1 is under a doxycycline-inducible promoter (Wang et al. Cell Cycle. 2014). CTC1 and STN1 are constitutively expressed but increase significantly when doxycycline is added. Doxycycline is added 24 h prior to each experiment so these higher levels are not present until the day prior to collection. We also have now analyzed cell growth and S-phase progression, which showed defects for HCT116 shSTN1 and HeLa CST-OE cell lines (see Referee #2, comments #2 and Figure S5 and S6).
To more directly address these concerns, we performed transient knockdown of STN1 with siRNA (see Reviewer #1, comment #2) and found a similar increase in chromatin-bound MCM levels compared to stable shRNA knockdown, indicating that increased MCM also arises with transient knockdown of STN1.
3. The genetic rescue experiments in Figure 2 are not particularly convincing. In particular, the red and grey signals in Figure 2E look the same whereas red should resemble the blue controls if STN is complementing well. (If this assay were working better, it could be the foundation to test CST mutants.) In addition, the gating of "high" MCM cells is an artificial designation that magnifies the phenotype quantified in 2D; there should be some justification for where this cutoff is placed, and it would be even better to not impose a binary classification on data that are largely continuous. Finally, this figure -while impressive in its technical sophistication -is just one biological replicate.
The cutoff line and graph for the high intensity MCM cells has been removed and new graphs depicting changes in mean intensity in all MCM positive cells is now included (see also Reviewer #2, #4).
While the shSTN1-RES cells do not fully rescue the phenotype, there is a substantial decrease in the MCM6 positive cells compared to our controls. In fact, analysis the signal intensity of MCM positive cells is similar between shNT and shSTN1-RES cells ( Figure 2C). In addition, incomplete rescue of the shSTN1-RES cell line has been seen with other phenotypes in our previous work and may arise from expression levels, influence from the N-terminal Flag-tag or subtle differences between the single clones isolated for the shNT and shSTN1 cell lines (Stewart et al. 2012, EMBO J). We have now replicated these results with siRNA knockdown of STN1 in the HeLa and HCT116 cells ( Figure S4), which shows similar results to stable knockdown of STN1. Together with the results from the HCT116 shSTN1 cells, these results provide strong evidence that STN1 depletion increases origin licensing.
The flow cytometry data are a representation of three biological replicates for both the HeLa and HCT116 shSTN1 cells. This is now clearly stated in the figure legend. These findings are also now replicated with transient siRNA knockdown of STN1, in three independent biological replicates ( Figure  S4).
4. The authors should demonstrate interaction of CST with MCM and interference with Cdt1-MCM association without the need for the very high expression typically associated with plasmid transfections. Is Cdt1-MCM binding enhanced by CST depletion? Analyzing endogenous proteins will also eliminate the need to normalize for differences in total MCM expression.
We made multiple attempts to detect the interaction with endogenous protein in different cell lines with and without STN1 depletion or CST overexpression. However, we were unable to co-IP sufficient levels of endogenous CDT1 or MCM to reliably detect interact, regardless of CST expression levels. We also tried expressing CDT1 in the HeLa or HCT116 shSTN1 cell lines but were unable to reliably express or detect CDT1 at sufficient levels for IP experiments. Due to these technical issues, we are not able to show the IP with endogenous CDT1 or in STN1 depleted cells. In our opinion, the best approach will be to perform in vitro binding experiments with recombinant, purified CDT1, MCM and CST. However, while we are pursuing such experiments, we feel that they are beyond the scope of the current study. For this reason, we have changed the text to suggest that disruption of MCM-CDT1 interaction as one possible explanation for the suppression of origin licensing. The following sentence of the section "CST disrupts the interaction between MCM and CDT1", has been altered to reflect this: "Disruption of the MCM-CDT1 interaction would directly affect origin licensing (i.e. MCM loading) by preventing MCM recruitment, thus providing a possible explanation for why CST decreases origin licensing." Minor: a) The claim that CST "directly" binds MCM is a minor overstatement. One would need to test the complexes in isolation since it is formally possible that an evolutionarily-conserved bridging protein mediates the binding.
We have added to following sentence to address this possibility and removed the work "directly" from this subsection, as follows: "Based on the yeast-two-hybrid data, we propose that this interaction is direct. However, it is possible that an evolutionary-conserved protein could bridge the interaction." b) The authors should report how many cells were analyzed in the dot plots and how many independent biological replicates were performed (co-IPs, FACS, etc.).
The number of biological replicates performed for each experiment has been added to the figure legends and the number of cells for the dot plots below the x-axis in the figures.
c) The interpretation that HU has no effect on binding doesn't necessarily rule out a role for replication stress in chromatin recruitment in Figures 5 and 6. Cells generate endogenous replication stress every S phase. Given that, the S phase chromatin association of CST in Figure 5 could have nothing to do with MCM loading and everything to do with its replication fork function.
We agree with the reviewer and were trying to make the point that HU does not affect the AND-1 chromatin association. We did not test how HU affected chromatin bound STN1 levels. We have sought to clarify this in the text with the following changes to the section "CST interacts with AND-1 and promotes AND-1 and pol  chromatin binding": "Since the AND-1 levels were not magnified with STN1 knockdown following HU treatment, this finding indicates that CST is unnecessary for AND-1 to associate with dormant origins that are fired in response to genome-wide replication fork stalling." d) The yeast spotting assays are spliced images; it would be better to not need that extra manipulation to present the data.
All the yeast-two-hybrid assays in original Figure 3C were performed at the same time and two biological replicates had been performed. However, other mutants were also tested so the MCM subunits were not on single plates with each CST subunit. We have re-run the experiment so that all MCM subunits are plated with each of the CST subunits ( Figure 3C). Interestingly, this time we did not observe the weak interaction previously observed between MCM5 and CTC1. Since this interaction appears weak and inconsistent (observed two out of three times) under the most stringent conditions (QDO media), we have removed reference to a potentially weak interaction between MCM5 and STN1 until future studies can confirm their interaction.
e) The two-hybrid reporters encode genes for adenine and histidine biosynthesis, not GAL4 itself (page 8, top).
This error has been corrected in the text. The sentence now reads: "The DDO media was used to select for plasmid transformations and QDO media for cells producing adenine and histidine, which indicates protein interaction.
f) The general protein stain Ponceau S is mis-spelled on several figures.
We thank the reviewer for identifying these mistakes and the figures have been updated. Thank you for submitting your revised manuscript entitled "Human CST suppresses origin licensing and promotes AND-1/Ctf4 chromatin association". I appreciate the introduced changes and would be happy to publish your paper in Life Science Alliance pending final revisions necessary to meet our formatting guidelines: -please note that we only have supplementary figures in LSA, there are still some callouts to EV figures, please fix -please add scale bars to Fig 1B, 1E, 6A -please link your ORCID iD to your profile in our submission system, you should have received an email with instructions on how to do so If you are planning a press release on your work, please inform us immediately to allow informing our production team and scheduling a release date.
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Reviews, decision letters, and point-by-point responses associated with peer-review at Life Science Alliance will be published online, alongside the manuscript. If you do want to opt out of this transparent process, please let us know immediately. ***IMPORTANT: If you will be unreachable at any time, please provide us with the email address of an alternate author. Failure to respond to routine queries may lead to unavoidable delays in publication.*** Scheduling details will be available from our production department. You will receive proofs shortly before the publication date. Only essential corrections can be made at the proof stage so if there are any minor final changes you wish to make to the manuscript, please let the journal office know now.

DISTRIBUTION OF MATERIALS:
Authors are required to distribute freely any materials used in experiments published in Life Science Alliance. Authors are encouraged to deposit materials used in their studies to the appropriate repositories for distribution to researchers.
You can contact the journal office with any questions, contact@life-science-alliance.org Again, congratulations on a very nice paper. I hope you found the review process to be constructive and are pleased with how the manuscript was handled editorially. We look forward to future exciting submissions from your lab.