Analysis of NP tetramer specific and virus specific CD8 and CD4 T cells in blood, lung, lung draining lymph nodes, and broncho-alveolar lavage (BAL) showed clear differences in cytokine production between these tissues. BAL contained the most highly activated CD8, CD4, and γδ T cells producing large amounts of cytokines, which likely contribute to elimination of virus. The weak response in blood did not reflect the powerful local lung immune responses. The immune response in the Babraham pig following H1N1pdm09 influenza infection was comparable to that of outbred animals. The ability to utilize these two swine models together will provide unparalleled power to analyze immune responses to influenza.We have used the pig, a large natural host animal for influenza with many physiological similarities to humans, to characterize αβ, γδ T cell and antibody (Ab) immune responses to the 2009 pandemic H1N1 virus infection. We evaluated the kinetic of virus infection and associated response in inbred Babraham pigs with identical MHC (Swine Leucocyte Antigen) and compared them to commercial outbred animals. High level of nasal virus shedding continued up to days 4 to 5 post infection followed by a steep decline and clearance of virus by day 9. Adaptive T cell and Ab responses were detectable from days 5 to 6 post infection reaching a peak at 9 to 14 days. γδ T cells produced cytokines  Influenza viruses are a global health threat to humans and pigs, causing considerable morbidity and mortality. Frequent zoonotic crossover between pigs and humans contributes to the evolution of influenza viruses and can be a source for novel pandemic strains –3. HumanAnimal models are essential to develop better vaccines and control strategies and to provide insight into human disease. Most models have limitations in recapitulating the full range of disease observed in humans. Mice, guinea pigs and non-human primates are not generally susceptible to natural routes of influenza infection and may require adapted strains, physiologic stressors and/or unnatural inoculation procedures –9. In coSeveral inbred miniature pig breeds have been developed, including NIH and Yucatan, with defined swine leukocyte antigens  , 17. HowDespite extensive knowledge of the role of T cells in protection against IAV in mice and humans, few studies in pigs have evaluated this in depth. The duration and magnitude of T cell and humoral responses has been assessed after swine H1N1, H1N2, and H3N2 challenges in pigs –26. The Here we characterized αβ, γδ T cell and Ab immune responses to H1N1pdm09 in local lung and systemic tissues in Babraham pigs and compared them to commercial outbred animals. These two pig models together will allow fine grain dissection of immune responses to IAV in a species which is a natural host for the virus and similar in many respects to humans.The animal experiments were approved by the ethical review processes at the Pirbright Institute and Bristol University and conducted according to the UK Government Animal (Scientific Procedures) Act 1986 under project license P47CE0FF2. Both Institutes conform to the ARRIVE guidelines.7 PFU of MDCK grown swine A(H1N1)pdm09 isolate, A/swine/England/1353/2009, derived from the 2009 pandemic virus, swine clade 1A.3. (H1N1pdm09) in a total of 4 ml (2 ml per nostril) using a mucosal atomization device MAD300 . Two experiments with outbred (OB) pigs (referred to as OB1 and OB2) and two with inbred Babraham (BM) pigs (referred to as BM1 and BM2) were performed (post-mortem examination performed with collection of tissue samples. Uninfected controls were sampled: two on the day prior to infection and two at day 8 post infection. Two naïve pigs (referred to as in-contact) were co-housed with the directly challenged pigs in experiments OB1, OB2, BM1, BM2 and culled at days 11 and 13 post infection together with the last two directly challenged pigs. A fifth experiment was performed with Babraham pigs (experiment BM3) in which three were culled on days 6, 7, 13, 14, 20, and 21 post infection  and 56 inbred Babraham pigs  were screened for absence of influenza A infection by matrix gene real time RT-PCR and for antibody-free status by HAI using four swine influenza virus antigens - H1N1pdm09, H1N2, H3N2, and avian-like H1N1. The average age of the outbred pigs 7 days before the challenge was 8.7 weeks and of the Babrahams 8.3 weeks. Pigs were challenged intra-nasally with 1 × 10Two nasal swabs (one per nostril) were taken from all surviving pigs following infection with H1N1pdm09 , 29. TheVirus titer in nasal swabs was determined by plaque assay on MDCK cells . Samples were 10-fold serially diluted in Dulbecco’s Modified Eagle’s Medium (DMEM) and 200 µl overlayered on confluent MDCK cells in 12 well tissue culture plates. After 1 h, the plates were washed and overlayered with 2 ml of culture medium containing 0.66% Agar. Plates were incubated at 37°C for 48 to 72 h and plaques visualized using 0.1% crystal violet. plaques were counted at the appropriate dilution and expressed as plaque forming units (PFU) per ml of nasal swab.6 cells after subtraction of the average number of spots in medium control wells.Frequencies of IFNγ spot forming cells (SFC) were determined using cryopreserved cells a previously described , 29. Cel6 cells/well. Cells were stimulated with live MDCK-grown H1N1pdm09 (MOI 1) or medium control and incubated at 37°C for 18 h. Golgi plug (BD Biosciences) was added for the last 4 h of stimulation. PMA Ionomycin (Biolegend) was added to appropriate control wells as a positive control at the same time as the Golgi plug. Following incubation cells were washed at 1,000g for 5 min and re-suspended followed by addition of primary antibodies, Near-Infrared Fixable LIVE/DEAD stain (Invitrogen) and secondary antibodies  as per the manufacturer’s instructions prior to the addition of internal cytokine antibodies. Cells were washed and re-suspended in PBS prior to analysis using a MACSquant analyser10 (Miltenyi).Cryopreserved single cell suspensions from blood, TBLN, BAL and lung were thawed, rested for 1 to 2 h and aliquoted into 96 well plates at 1 × 10290-298 SLA tetramer binding was performed as previously described (The NPescribed . BrieflyELISA was performed using live H1N1pdm09 virus or recombinant hemagglutinin from H1N1pdm09 (pH1) containing a C-terminal thrombin cleavage site, a trimerization sequence, a hexahistidine tag and a BirA recognition sequence as previously described . Cut-offThe porcine sera were also tested for binding to MDCK-SIAT1 cells stably expressing pH1 from H1N1pdm09 (A/England/195/2009), H1 from A/Puerto Rico/8/1934  and H5 HA (A/Vietnam/1203/2004). Confluent cell monolayers in 96-well microtiter plates were washed with PBS and 50 μl of the serum dilution was added for 1 h at room temperature. The plates were washed three times with PBS and 100 μl of horseradish peroxidase (HRP)-conjugated goat anti-pig Fc fragment secondary antibody  was added for 1 h at room temperature. The plates were washed three times with PBS and developed with 100 µl/well TMB high sensitivity substrate solution (Biolegend). After 5 to 10 min the reaction was stopped with 100 µl 1 M sulfuric acid and the plates were read at 450 and 570 nm with the Cytation3 Imaging Reader (Biotek). The cut off value was defined as the average of all blank wells plus three times the standard deviation of the blank wells.2SO4 and the plates were read at 450 and 630 nm using a Biotek Elx808 reader. Samples were measured as end titer representing the highest dilution with signal greater than cut-off. The cut off value was defined as the average of all blank wells plus three times the standard deviation of the blank wells.Neuraminidase inhibiting Ab titers were determined in serum and BAL fluid using an Enzyme-linked lectin assay (ELLA). Ninety six-well microtiter plates  were coated with 50 μl/well of 25 μg/ml fetuin and incubated at 4°C overnight. Heat inactivated sera samples were serially diluted in a separate 96-well plate. An equal volume of (H7(Net219) N1(Eng195) S-FLU (H7N1 S-FLU)  was added to each well and incubated at room temperature on a rocking platform for 20 min. The H7N1 S-FLU was titered beforehand in the absence of serum to determine optimal concentration for the assay. Fetuin plates were washed with PBS four times before 100 μl/well of the serum/virus mix was transferred and incubated overnight at 37°C. The serum/virus mix was removed, and the plate washed four times with PBS before adding 50 μl/well of Peanut Agglutinin-HRP at 1 μg/ml and incubating for 90 min at room temperature on a rocking platform. Plates were washed and 50 μl/well of TMB High Sensitivity substrate solution  was added. Plates were developed for 6 min, the reaction stopped with 50 μl of 1 M H2 incubator. After incubation the cell suspension was removed, the plates washed once with ice-cold sterile H2O and thereafter with PBS/0.05% Tween 20, before incubation with 100 µl per well of 0.5 µg/ml biotinylated anti porcine IgG  diluted in PBS/0.5% FBS for 2 h at room temperature. Plates were washed with PBS/0.05% Tween 20 and incubated with streptavidin – alkaline phosphatase conjugate . After a final wash, the plates were incubated with AP Conjugate Substrate  for a maximum of 30 min. The reaction was stopped by rinsing the plates in tap water and dried before spots were counted.B cell ELISpots were performed for the detection and enumeration of antibody-secreting cells in single cell suspensions prepared from different tissues and peripheral blood. ELISpot plates  were coated with 100 µl per well of appropriate antigen or antibody diluted in carbonate/bicarbonate buffer for 2 h at 37°C. To detect HA-specific spot-forming cells, plates were coated with 2.5 µg per well of recombinant pHA from H1N1pdm09 (A/England/195/2009) and for the enumeration of total IgG-secreting cells with 1 µg per well of anti-porcine IgG  or with culture medium supplemented with 10% FBS (media background control). The coated plates were washed with PBS and blocked with 200 µl/well 4% milk (Marvel) in PBS. Frozen cell suspensions from different tissues were filtered through sterile 70 µM cell strainers, plated at different cell densities in culture medium  on the ELISPOT plates and incubated for a minimum of 18 h at 37°C in a 5% CO10 PFU/ml) as the response variable, day post infection  and pig type (OB or BM) and an interaction between them as fixed effects and pig ID nested in experiment as random effects. The model was implemented using the lme4 package (https://www.R-project.org/).All statistical analyses were performed using Prism 8.1.2. The kinetics of viral shedding were analyzed using a linear mixed model. The model included viral titer , source  and pig type (OB or BM) and two- and three-way interactions between them as fixed effects. Model simplification proceeded by stepwise deletion of non-significant (P>0.05) terms as judged by F-tests. The model was implemented in R (version 3.6.1).ELISpot data were analyzed using a linear model. The model included log290-298 CD8, IFNγ CD8β, IL-2 CD8β, TNF CD8β, IFNγ CD4, IL-2 CD4, TNF CD4, IFNγ CD2 γδ ex vivo, TNF CD2 γδ ex vivo, IFNγ/TNF CD2 γδ ex vivo, IFNγ CD2 γδ, TNF CD2 γδ, IL-17A CD2 γδ) from each source  and pig type (OB and BM) at each day post infection were analyzed using Kruskal-Wallis tests. If significant (P<0.05), pairwise Wilcoxon rank-sum  tests were used to compare groups. These analyses were implemented in R (version 3.6.1). A similar approach was used to compare the percentage of different T cells in all sources from inoculated and uninfected control pigs at each time point and in BAL from in-contact and experimentally inoculated pigs (in this case observations from 6 to 11 dpi were combined).Because of possible non-normality and non-constant variance the percentage of different T cells (NPy=κ/(1+exp(-β(t-δ)), where y is the log10 antibody titer, t is days post infection, κ is the upper asymptote, β is the rate of increase and δ is the time of maximum increase. The parameters  were allowed to vary between BM and OB pigs. Model fitting using the nlme package (https://CRAN.R-project.org/package=nlme) in R (version 3.6.1).The dynamics of antibody responses were analyzed by fitting logistic growth curves to the data, Five experiments were performed to characterize local and systemic immune responses. In the first four experiments ten pigs were infected intranasally with H1N1pdm09 virus and monitored for clinical signs. One infected pig was culled on each of days 1 to 7, 9, 11, and 13 post infection. A full post-mortem examination was performed and BAL, lung, TBLN and PBMC samples collected. Four uninfected controls were sampled in parallel, two on the day prior to infection and two at day 8 post infection. Two experiments with outbred (OB) pigs (referred to as experiments OB1 and OB2) and two with inbred Babraham (BM) pigs (referred to as BM1 and BM2) were performed , whereas from 14 to 21 DPI the highest number of IFNγ-secreting cells was detected in the lung, with SFC continuing to expand in this tissue (mean 368 SFC/106 cells at 14 DPI and 972/106 cells SFC at 21 DPI). The response in the BAL was lower than lung (p=0.04), due to the low proportion of T cells present in the BAL (6 cells at 13 DPI. No differences in responses between the same tissues in OB and BM were detected (p > 0.11).As T cells are crucial for control of virus replication, we examined in detail the CD8 and CD4 responses during H1N1pdm09 infection –36. Firs290-298 (DFEREGYSL) tetramer, which we have previously shown to be dominant in BM animals infected with H1N1pdm09  and there were no detectable responses at 20 to 21 DPI  using peptide NP1N1pdm09 . TetrameWe analyzed production of IFNγ, TNF and IL-2 by CD8β and CD4 T cells by intracellular staining (ICS)  and thus we focused on CD2+ γδ T cells. Ex vivo, BAL CD2+ γδ T cells, without H1N1pdm09 stimulation, secreted IFNγ and TNF early post infection with the highest frequency of 0.6% IFNγ and 1.6% TNF at 3 DPI. The cells co-produced IFNγ/TNF at low levels (ex vivo (data not shown).We measured IFNγ, TNF and IL-17A production in CD2in vitro and the highest proportion of IFNγ producing cells was detected in BAL and lung at 7 DPI and maintained until 13 DPI (0.4% at 11 DPI for BAL) while in lung the highest frequency was 0.4% at 7 DPI (+ cells was much lower compared to IFNγ and TNF with the greatest response in the BAL at 11 DPI (0.2%).We also measured cytokine production after H1N1pdm09 stimulation ex vivo early post infection, but that H1N1pdm09 in vitro stimulation increases cytokine production in CD2+ γδ T cells from 7 to 13 DPI. No difference between OB and BM pigs were detected of response of ex vivo or stimulated γδ T cells.Overall these data demonstrate that γδ T cells produce cytokines The antibody response after H1N1pdm09 infection was determined in serum, BAL and nasal swabs. Virus specific IgG and IgA were measured by end point titer ELISA against H1N1pdm09 virus or recombinant HA from H1N1pdm09/A/England/195/2009 (pH1)  and HA from PR8 in which, unlike in ELISA, the natural conformation of HA is maintained. There was strong binding to the MDCK expressing pH1 and weaker binding to H5 and HA from PR8 suggesting that H1N1pdm09 induces cross reactive responses to other group 1 H1 and H5 viruses  assessing inhibition of virus entry, inhibition of hemagglutination (HAI) and inhibition of neuraminidase activity by enzyme-linked lectin assay (ELLA)  , no virus was detectable. These kinetics suggest that innate mechanisms, including perhaps early γδ T cell cytokine secretion, contain viral replication at a plateau level in the first 4 to 5 days post infection, while adaptive T and Ab responses contribute to the complete clearance of virus after 5 DPI in primary infection and prevent future infections by a more rapid secondary immune response.In this study we investigated the kinetic and magnitude of T cell and Ab responses in respiratory tissues and blood in outbred Landrace x Hampshire cross and inbred Babraham pigs following H1N1pdm09 infection. The relationship between these parameters and the virus load is illustrated in Similar kinetics of adaptive T cell responses have been reported in mice, with antigen specific cells detected as early 4 to 5 days post infection, increasing in number between 5 and 12 DPI in lung tissues , 46. ExpFew studies have analyzed in depth the conventional T cell response in pigs. The most comprehensive study showed a low frequency of virus specific IFNγ producing CD4 and CD8 in the lung as early as 4 DPI after H1N2 intratracheal challenge, reaching a peak at 9 DPI, with the highest response in lung compared to TBLN or PBMC . Here, f7 TCID50/ml) intratracheally  in contrast to humans where they usually represent 1% to 5% of lymphocytes , 58. The in mice . γδ T ce in mice , 59, 60.eceptors . FurtherH1N1pdm09 infection was characterized by high IgG and IgA titers in serum and BAL, and a detectable antibody titer in nasal swabs. The IgG titer was higher than IgA in serum, while similar levels of IgA and IgG were detected in BAL and nasal swabs, suggesting local production of this isotype or more efficient translocation. Neutralization, HAI and neuraminidase inhibition titers peaked at 11 - 21 DPI. Our findings are in agreement with previous studies showing that in experimentally H1N1 infected pigs HA-specific antibodies peaked at 2 to 3 weeks . SimilarSus scrofa in the Immuno‐Polymorphism Database (IPD) MHC database to date, making analysis of the fine specificity of immune responses extremely difficult : Mario Aramouni, Mick Bailey, Amy Boyd, Sharon Brookes, Ian Brown, Becky Clark, Bryan Charleston, Catherine Charreyre, Margo Chase-Topping, Federica Di-Palma, Matthew Edmans, Graham Etherington, Helen Everett, Ore Francis, Simon Frost, Sarah Gilbert, Ross Harley, Barbara Holzer, Adam McNee, Angela Man, Veronica Martini, Sophie Morgan, Emily Porter, Jin Qi Fu, Amy Thomas, Elma Tchilian, Laurence Tiley, Pauline van Diemen, James Wood, Fei Xiang.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Pirbright Institute, and Bristol University. Written informed consent was obtained from the owners for the participation of their animals in this study.BC, MB, and ET conceived, designed and coordinated the study. BC, MB, ET, ME, AM, EP, EV, BP, VM OF, RH, AT, RB, and SM designed and performed experiments, processed samples, and analyzed the data. SG performed statistical analysis. AF and AS generated SLA tetramers. ET, ME, EV, and AM wrote and revised the manuscript and figures. All authors contributed to the article and approved the submitted version.This work was funded by the UKRI Biotechnology and Biological Sciences Research Council (BBSRC) grants: sLoLa BB/L001330/1, BBS/E/I/00007031, BBS/E/I/00007038, and BBS/E/I/00007039.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


O-acyltransferase 1 (SOAT1) is an endoplasmic reticulum (ER) resident, multi-transmembrane enzyme that belongs to the membrane-bound O-acyltransferase (MBOAT) family. It catalyzes the esterification of cholesterol to generate cholesteryl esters for cholesterol storage. SOAT1 is a target to treat several human diseases. However, its structure and mechanism remain elusive since its discovery. Here, we report the structure of human SOAT1 (hSOAT1) determined by cryo-EM. hSOAT1 is a tetramer consisted of a dimer of dimer. The structure of hSOAT1 dimer at 3.5 Å resolution reveals that a small molecule inhibitor CI-976 binds inside the catalytic chamber and blocks the accessibility of the active site residues H460, N421 and W420. Our results pave the way for future mechanistic study and rational drug design targeting hSOAT1 and other mammalian MBOAT family members.Sterol  O-acyltransferase 1  is an endoplasmic reticulum resident enzyme which catalyzes the esterification of cholesterol to generate cholesteryl esters. Here, authors report cryo-EM structures of human SOAT1 which reveal the binding site of the competitive inhibitor CI-976.Sterol  The concentration of cellular free cholesterol is highly regulated3. Excessive intracellular cholesterol may form cholesteryl esters, which are catalyzed by the enzyme, sterol O-acyltransferase (SOAT), also called acyl-coenzyme A: cholesterol acyltransferase (ACAT). SOAT catalyzes the reaction between long chain fatty acyl-CoA and intracellular cholesterol to form the more hydrophobic cholesteryl ester, which is then stored in lipid droplets within the cell or transported in secreted lipoprotein particles to other tissues that need cholesterol. In addition to cholesterol, SOAT can use multiple sterols as substrates and activators4. Because of its functional importance, SOAT1 is a potential drug target for Alzheimer’s disease5, atherosclerosis6 and several types of cancers10.Cholesterol is an essential lipid molecule in the cell membranes of all vertebrate. It is important for maintaining the fluidity and integrity of the membrane and is the precursor for the biosynthesis of other crucial endogenous molecules, such as steroid hormones and bile acids. In addition, cholesterol can modulate the activity of many membrane proteins such as GPCR11. There are two SOAT enzymes in mammals: SOAT1 and SOAT2, which have a protein sequence identity of 48% in human . SOAT1 is ubiquitously expressed in many types of cells12; while SOAT2 is mainly expressed in the small intestine and liver13. Due to the pathophysiological importance of SOAT, many SOAT inhibitors of various structural types have been made. The small molecule CI-976 is a prototypical member of SOAT inhibitors that belong to the fatty acyl amide analog family. Studies showed that feeding CI-976 reduced atherosclerotic plaques as well as reduced plasma cholesterol levels in animals fed with high cholesterol diet14. CI-976 exhibits competitive inhibition against fatty acyl-CoA16.Previous studies have shown that SOAT1 is an ER-localized multi-transmembrane protein that is evolutionary conserved from yeast to humansO-acyltransferase (MBOAT) enzyme family, which transfers the acyl chain onto various substrates, including lipids, peptides and small proteins. There are 11 MBOAT family members in humans17, which participate in many physiological processes, such as the last step of triglyceride biosynthesis catalyzed by acyl-CoA: diacylglycerol acyltransferase 1 (DGAT1), the maturation of hedgehog morphogen catalyzed by hedgehog acyltransferase (HHAT), and the acylation of peptide hormone ghrelin catalyzed by ghrelin O-acyltransferase (GOAT). Recently, the X-ray crystal structure of DltB from Streptococcus thermophilus was determined and is the only available structure of an MBOAT family member18. The low-sequence conservation between DltB and SOAT (14.6% identity) hinders the accurate modeling of the human SOAT1 structure. Therefore, despite the important physiological functions of human SOAT enzymes, their architecture and mechanism remain elusive due to a lack of high-resolution structures. In this study, several human SOAT1 (hSOAT1) structures were determined. These structures reveal the architecture of hSOAT1 and the binding site of the competitive inhibitor CI-976, and provide a structural basis to understand the inhibition mechanism of these enzymes by small molecules.SOAT1 is the founding member of the membrane-bound 19)  and that hSOAT1 became predominantly dimeric with the deletion of the N-terminal tetramerization domain21. We compared the recombinant SOAT1 enzyme activities in HEK293F suspension cell after infection by the recombinant BacMam viruses, versus those expressed in the stable transfectant of AC29 cell line22, or the adherent HEK293 cell23. We used the crude cell extracts solubilized by CHAPS as the enzyme source22, added the 3H-oleoyl-CoA and cholesterol present in taurocholate/phospholipid/cholesterol mixture as the substrates. The crude cell extracts from uninfected/untransfected HEK293 cells, or from AC29 cells (a CHO cell mutant devoid of endogenous SOAT1 protein24 were used as controls). The results  and N-terminal truncated hSOAT1 (66–550), solubilized in detergent micelles migrated before and after mouse TPC1 channel, which is a well-characterized dimeric channel with a molecular weight of 189 kDa  of the dimeric hSOAT1 were invisible in the cryo-EM map, presumably due to their high flexibility  without severely lowering the cellular hSOAT1 protein expression in transfected cells  is inside the central cavity and surrounded by F258 and R262 on M4, F384 and W388 on M6, P304 on αC-M5 linker and V424 on αE  is close to the dimer interface. It is surrounded by L129, L132 and L133 on the hydrophobic side of αA, F145 on M1, C333 on M5, F382 on M6 and W408 on M6-αD loop  . Instead, the BiSAS map most likely represents the apo resting state of hSOAT1. Interestingly, all three additional non-protein densities (density A−C) present in the CI-976 map were also observed in the BiSAS map  , with the active site His 460 located at the luminal half of M7. This is consistent with previous biochemical work2 and 70% humidity in Free Style 293 medium (Thermo Fisher Scientific) supplemented with 1% fetal bovine serum (FBS). HEK293S cell line was kindly generated and provided by Professor Gobind Khorana at MIT42. The CHO mutant cell line AC29, deficient in ACAT1/SOAT1 activity, was isolated in Chang Lab43.Sf9 insect cells (Thermo Fisher Scientific #12659017) were cultured in Sf-900 III serum-free medium  or in SIM SF  at 27 °C. HEK293F (Thermo Fisher Scientific #R79007) cells were cultured at 37 °C with 6% CO44 (Please see Supplementary Table 7-strep-GFP tags45.We cloned human SOAT1 (Hs_SOAT1), human SOAT2 (Hs_SOAT2), Xenopus laevis SOAT1 (Xl_SOAT1), chicken SOAT1 (Gg_SOAT1) and zebrafish SOAT1 (Dr_SOAT1) into NGFP tagged BacMaM vector for screening by FSEC method22, by using solubilized cell extracts as the enzyme source and preformed taurocholate/cholesterol/phosphatidylcholine mixture and tritium-labeled oleoyl-CoA as the substrates. This assay provides large amounts of exogenous phospholipids and cholesterol at optimized concentrations, such that the membrane phospholipids and cholesterol endogenously associated with SOAT1 become significantly diluted and can no longer affect the enzyme activity in vitro. Cells were harvested by spinning down at 800 × g for 5 min at room temperature. After two washes with PBS, cell pellets were solubilized in 2.5% CHAPS, 1 M KCl in 50 mM KH2PO4 at pH 7.4 with protease inhibitors. 5 µl of cell homogenates at protein concentration of 2–5 mg/ml were diluted into 50 µl of preformed 9.3 mM taurocholate/1.6 mM cholesterol/11.2 mM phosphatidylcholine mixture and incubated on ice for 10 min. The reactions were initiated by adding 10 nmole of 3H-oleoyl-CoA. The reactions were continued at 37 °C for 10 min (ref.23).The assay was conducted as described in−1 leupeptin, 2 µg ml−1 pepstatin A, 2 µg ml−1 aprotinin and 1 mM PMSF) at 4 °C for 30 min. Cell debris was removed by centrifugation at 13,000 × g for 10 min and centrifuged at 60,000 × g for 30 min. Each supernatant was split into two samples. One sample was kept at 4 °C as unheated sample and the other one was heated at 45 °C for 10 min using a thermo cycler. After heating, each sample was centrifuged at 60,000 × g for additional 30 min to remove precipitates and the supernatants were analyzed on a Superose 6 increase 5/150  column running in 20 mM Tris pH 8.0 at 4 °C, 150 mM NaCl and 0.5 mM DDM. The fluorescence peak height at the hSOAT1 dimer elution position was divided by the peak height at the monomer elution position to generate the ratio which was used to quantify the thermostability. The enhancement effect of CI-976 on hSOAT1 dimer thermostability was quantified by dividing the ratio in the presence of CI-976 by the ratio in the presence of DMSO alone.The hSOAT1 dimer mutants were generated by Quick Change methods . Sodium butyrate (10 mM) was added to the culture 12 h post-infection to promote protein expression and the temperature was lowered to 30 °C. Cells were harvested 60 h post-infection and washed with TBS buffer. Cell pellets were frozen at −80 °C for later use.−1 leupeptin, 2 µg ml−1 pepstatin A, 2 µg ml−1 aprotinin and 1 mM PMSF). Unless stated otherwise, all buffers used for purification were supplemented with inhibitor either 1 μM inhibitors CI-976 or BiSAS. The cells were broken by sonication and centrifuged at 5000 × g for 10 min with JA25.5 rotor (Beckman) to remove cell debris. The supernatant was centrifuged at 60,000 × g for 1 h in Ti45 rotor (Beckman) to harvest cell membrane in pellets. The membrane pellets were homogenized in TBS, solubilized by 1% digitonin for 2 h at 4 °C, and centrifuged at 60,000 × g for 1 h. The supernatant was loaded onto a 1 ml prepacked strep-tactin superflow high-capacity column (IBA) and washed using TBS buffer with 0.1% digitonin. The binding protein was eluted using TBS buffer with 0.1% digitonin and 10 mM desthiobiotin. The eluted proteins were digested with PreScission protease to remove tags and further purified by superose-6 increase column  in TBS buffer with 0.1% digitonin or 40 μM GDN. Buffers were not supplemented with inhibitors during the purification of proteins used for the activity assay. Purified protein were either directly used for experiments or flash frozen in liquid nitrogen for storage at −80 °C. Stored protein was thawed on ice and centrifuged to remove precipitates before further experiments. The original gels for protein purification were provided as a Source Data file.The cell pellets were resuspended in TBS buffer supplemented with protease inhibitors . Mutant CHO AC29 cells that lack endogenous SOAT activity were cultured in 6-well plates at 37 °C and transiently transfected with DNAs from various single amino acid substitutions of hSOAT1 as indicated. Unless stated otherwise, each construct contained the 6x His tag at the N-terminus. We used hSOAT1 that contained the C92A substitution as the wild type hSOAT1. Control experiments showed that the SOAT activity of the C92A mutant remained the same as the wild type27 but the C92A substitution greatly diminished the SOAT1 protein aggregation that occurred in vitro during the SDS-PAGE process. At the third day after transfections, the cells were split into three equal parts by trypsinization. One part was used to monitor the hSOAT protein expression by western blot analysis, using the SOAT1 specific antibodies DM10 as the probe, as described46, with the intensity of the WT hSOAT protein set as 1.0. The other two parts of the transfected cells were used to monitor the SOAT enzyme activity, by incubating the intact cells to 20 μl of 3H-oleate/fatty acid free BSA (7.5 × 106 dpm μl−1) for 20 min. The amount of 3H-cholesteryl oleate produced was determined by the procedure previously described36. Briefly, after 3H-oleate/BSA pulse, cells were rinsed with PBS, harvested in 1 mL per well of 0.1 M NaOH, incubated at RT for 30 min. The cell homogenates were transferred to13 × 100 mm size glass tubes; then neutralized by adding 67 μl of 3 M HCl, and buffered with 50 μl of 1 M K2HPO4. 80 μg per tube of non-radiolabeled cholesteryl oleate was added as carrier for identification purposes. Cellular lipids in each tube were extracted with 3 mL of CHCl3:MeOH at 2:1 followed by adding 1 mL of H2O. The bottom chloroform phase that contained the lipid samples were dried under N2, then redissolved in 80 μl of ethyl acetate and spotted onto TLC plates (Anatech), with petroleum ether:ether:acetic acid (90:10:1) as the solvent system. After TLC, the plates were air-dried, the cholesteryl oleate bands (at Rf 0.9) were identified by briefly staining the TLC plates with iodine vapor. The cholesteryl oleate bands were scraped into scintillation vials and counted in a scintillation counter after addition of 3 mL per vial of Ecoscint O. The enzyme activity of each mutant hSOAT1 was estimated relative to that of WT hSOAT1, with the protein content of each mutant hSOAT1 normalized with that of the WT hSOAT1.This assay is designed to measure the rate of 47. The tetrameric or dimeric hSOAT1 enzyme was prepared in GDN detergent. First, 10 μl 2 M KCl, 5 μl 5% BSA, 1 μl SOAT1 protein (A280 = 0.5), 5 μl micelles, 40 μl TBS buffer with 40 μM GDN were mixed with TBS buffer with 0.5% CHAPS to reach the volume of 100 μl and incubated at 37 °C for 2 min. In order to measure the IC50 of inhibitors, different concentrations of given inhibitors were added, as indicated. Then, 1.0 μl 0.2 mg ml−1 NBD-cholesterol  solubilized in 35% β-cyclodextrin  was added and the mixture was incubated at 37 °C for 2 min. To start the enzymatic reaction, 1 μl 2.5 mM oleoyl-CoA  was added and the reaction mixture was incubated at 37 °C for 15 min. The reaction was terminated by adding 2:1 chloroform/methanol, the extract was separated on an HPLC column at 0.2 ml/min  running in 100% ethanol and detected via fluorescence detector on an HPLC (SHIMADZU). NBD-cholesterol eluted at 1.3 min and its ester eluted at 1.9 min. The peak areas of the NBD-cholesteryl ester products and remaining NBD-cholesterol were integrated separately to obtain the relative ratio of NBD-cholesterol that was converted into NBD-cholesteryl esters.2.8 mM cholesterol/11.2 mM PC/18.6 mM taurocholate were mixed to generate the cholesterol donor6 tag and HRV 3C site. The MSP2X and MSP2N2 proteins were purified by Talon resin48. The eluted SOAT1 protein in TBS buffer with 0.1% digitonin and 10 mM desthiobiotin from strep-tactin column was concentrated by a 100-kDa cut-off ultrafiltration device (Millipore) and exchanged into buffer without desthiobiotin. The SOAT1 protein was mixed with soybean polar lipids extract  and purified MSP  at a molar ratio of SOAT1: MSP: SPLE = 1:7:100. For the hSOAT1 dimer in nanodisc that contained cholesterol, SPLE and cholesterol (at 4:1 ratio) were supplemented in GDN detergent micelles. The SOAT1 protein was mixed with micelles and purified MSP2X at a molar ratio of SOAT1: MSP2X: SPLE = 1:4:100. After incubating at 4 °C for 30 min, Bio-beads SM2 (Bio-Rad) were added and rotated at 4 °C for 1 h to initiate the reconstitution. Another batch of fresh bio-beads was added and rotated at 4 °C overnight. The next day, the Bio-beads were removed and the mixture was loaded into a streptactin column to remove the empty nanodisc. The eluted hSOAT1 in the nanodisc was concentrated and cleaved by Prescission protease to remove GFP tags. The nanodisc was centrifuged at 60,000 × g for 30 min, and then loaded onto a superose-6 increase column running in TBS buffer containing 0.5 mM TCEP. The collected fractions were detected by SDS−PAGE and peak fractions were concentrated to A280 = 1.2. Because the hSOAT1 tended to aggregate after nanodisc reconstitution, fractions of each peak were combined and concentrated for cryo-EM grids preparations and screening and only fractions containing high ratio of SOAT1 dimer were used for data collection.MSP2X was constructed by linking two MSP1E3D1 by PCR overlap extension, with Gly-Thr as the linker. The MSP2X gene was constructed into a pET vector, with N-terminal His−pixel−1s−1 using K2 Summit direct electron camera (Thermo Fisher Scientific) at a nominal magnification of 130,000 ×, equivalently to a calibrated super-resolution pixel size of 0.5225 Å, and with defocus ranging from −1.3 μm to −2.3 μm. All data acquisition was performed automatically using SerialEM49.The nanodisc samples were loaded onto glow-discharged GiG R1/1 holey carbon gold grids (Lantuo) and plunged into liquid ethane by Vitrobot Mark IV (Thermo Fisher Scientific). Cryo-grids were screened by Talos Arctica electron microscope (Thermo Fisher Scientific) operated at the voltage of 200 kV using a Ceta 16 M camera (Thermo Fisher Scientific). Optimal grids were transferred to Titan Krios electron microscope (Thermo Fisher Scientific) operated at the voltage of 300 kV, with an energy filter set to a slit width of 20 eV. Super-resolution movies (50 frames per movie) were collected with a dose rate of 5.4 e50. Micrographs with ice or ethane contamination were manually removed. Contrast transfer function (CTF) parameters were estimated using Gctf v1.06 (ref.51). Particles were picked by Gautomatch (developed by Kai Zhang) and subjected to reference-free 2D classification. Unless otherwise stated, all classification and reconstruction were performed with Relion 2.0 (ref.52). Initial model was generated by cryoSPARC53 using the selected particles from 2D classification. The selected particles were further subjected to 3D classification using C1 symmetry. The particles selected from good 3D classes were re-centered and their local CTF parameters were determined using Gctf v1.06. These particles were further refined by cisTEM54 using C2 symmetry imposed. The resolution estimation was based on the Part.FSC curve in cisTEM at FSC = 0.143 cut-off. Local resolution estimation for hSOAT1 dimer and CI-976 complex was calculated using blocres55. For the apo state, images were processed in the same way, except the finial refinement were done using Relion 3.0 (ref.56) with a soft mask that excluded the MSP and lipids. The resolution estimations of the apo state were based on the gold standard FSC of 0.143 cut-off after correction of the masking effect57. Local resolution estimation for hSOAT1 dimer in the apo state was calculated using Resmap58.For the CI-976 complex, super-resolution movie stacks were motion-corrected, dose-weighted and 2-fold binned by MotionCor2 1.1.0 using 9 × 9 patches59. The model was manually built de novo in Coot60. The assignments of transmembrane domain helices were based on their connectivity aided by the less-sharpened map. The register assignment and modeling building were based on the features of large aromatic side chains and partly aided by the further sharpened map. The manually built model was refined by Phenix59. The sterol-like ligand in non-protein density A was built as cholesterol for visualization. The model in the presence of CI-976 were fitted into the map in the presence of BiSAS by Chimera61 and further refined by Phenix.The sharpened map in the presence of CI-976 from cisTEM was converted into mtz file by Phenix62, aligned by Clustal Omega and uploaded into the Consuf server63 together with the CI-976 pdb files. The figures were prepared by UCSF Chimera61.The SOAT1 sequences (TF105767) were download from Treefam1H NMR  δ 5.39 , 4.67 , 4.17 , 2.54 , 2.40–2.32 , 2.12 , 2.08–1.84 , 1.75–1.58 , 1.56–1.39 , 1.37–1.11 , 1.03 , 0.88 , 0.63 . 13C NMR  δ 209.69, 169.49, 139.39, 122.83, 77.48, 77.16, 76.84, 75.51, 63.81, 56.96, 49.99, 46.72, 44.12, 38.92, 37.69, 37.04, 36.74, 35.04, 32.08, 31.94, 31.91, 31.71, 29.84, 29.83, 29.81, 29.73, 29.61, 29.52, 29.45, 28.97, 27.61, 27.40, 24.62, 22.97, 22.84, 21.18, 19.46, 14.28, 13.37. HRMS(ESI): m/z calcd for C39H66BrO3+ [M + H]+: 661.418985, found 661.420600To the solution of α-bromo stearic acid , pregnenolone  and dicyclohexylcarbodiimide  in dichloromethane  was added 4-dimethylaminopyridine . The solution was stirred at room temperature for 24 h. The crude product was purified by column chromatography (Hexanes: Ethyl acetate = 10:1) to obtain the α-bromo ester  as a white solid. N,N-dimethyllformamide  was added triethylamine . The solution was stirred under nitrogen atmosphere at 35 °C overnight. The crude product was purified by reverse phase HPLC (Water: Acetonitrile = 50: 50 to 5: 95) to obtain BiSAS in bis(triethylammonium) salt form (23.8 mg) as a colorless solid. 1H NMR  δ 8.44 , 8.06 , 6.04 , 5.27 , 4.89–3.72 , 3.65–3.20 , 3.10 , 2.89–1.73 , 1.42–1.00 , 0.88 , 0.77 , 0.72–0.60 , 0.50 . HRMS(ESI): m/z calcd for C60H99N7O19P3S− [M - H]−: 1346.593480, found 1346.591330.To the solution of α-bromo ester obtained above  and CoA-SH  in 64. Fluorescence values were plotted versus the log of the concentration of inhibitor, and GraphPad Prism 6 was used to generate a curve fit with dose-response inhibition equation: Y = 100/1 + 10[Log(IC50−X) *HillSlope]. IC50 values were calculated from the curve fit using GraphPad Prism software. The number of biological replicates (N) and the relevant statistical parameters for each experiment (such as mean or standard error) are described in the figure legends. No statistical methods were used to pre-determine sample sizes.Global resolution estimations of cryo-EM density maps are based on the 0.143 Fourier Shell Correlation criterionFurther information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Australian jarrah  honey produces particularly high levels of H2O2 and has been found to be potently antifungal. This study characterized the activity of jarrah honey on fungal dermatophyte species. Jarrah honey inhibited dermatophytes with minimum inhibitory concentrations (MICs) of 1.5–3.5% (w/v), which increased to ≥25% (w/v) when catalase was added. Microscopic analysis found jarrah honey inhibited the germination of Trichophyton rubrum conidia and scanning electron microscopy of mature T. rubrum hyphae after honey treatment revealed bulging and collapsed regions. When treated hyphae were stained using REDOX fluorophores these did not detect any internal oxidative stress, suggesting jarrah honey acts largely on the hyphal surface. Although H2O2 appears critical for the antifungal activity of jarrah honey and its action on fungal cells, these effects persisted when H2O2 was eliminated and could not be replicated using synthetic honey spiked with H2O2, indicating jarrah honey contains agents that augment antifungal activity.Superficial dermatophyte infections, commonly known as tineas, are the most prevalent fungal ailment and are increasing in incidence, leading to an interest in alternative treatments. Many floral honeys possess antimicrobial activity due to high sugar, low pH, and the production of hydrogen peroxide (H Dermatophytes are filamentous fungi that cause superficial infections of the hair, skin, and nails. These infections, collectively described as tineas, are some of the most prevalent fungal ailments, and it has been estimated that almost everyone will acquire an infection at some point of their lifetime . Over thCurrently, around 500 million USD is spent annually worldwide on antifungal therapies for tineas . Treatme2O2) or to “non-peroxide” nectar-derived chemicals. H2O2, a powerful oxidant, is produced by glucose oxidase, a bee-derived enzyme that is activated upon dilution of honey with water ) to be ~28 μM (Jarrah (Barnes 10+) honey had a high level of antifungal activity  but reladuction  from ½–4× MIC with artificial honey added to a final concentration of 10% (w/v) (2O2 Honey solutions from ½–4× MIC for pure H2O2 (where the MIC = 340 µM), a second series of 0, 25, 50, 100, 200, and 400 µM H2O2 to test lower H2O2 levels, and a solution containing 56 µM H2O2  honey using artificial honey. A final set of Jarrah (Barnes 10+) + Catalase + Artificial honeys were created where catalase (7000 U/mL) was added to Jarrah (Barnes 10+) honey and adjusted to 10% (w/v) honey using artificial honey. The figures in the results where these different honey solutions were used are listed in In order to explore the effect of jarrah honey and the production of H0% (w/v) . Similar2O2 and honey affected conidia and their germination. T. rubrum conidia treated for 48 h with Jarrah (Barnes 10+) honey  (p < 0.001). Representative images showing the effect of Jarrah (Barnes 10+) and Synthetic H2O2 Honey on conidial germination are shown in As the MIC for Synthetic H 430 µM;  was subs2O2 (2O2 and other intracellular reactive oxygen species (ROS) that exceed cellular antioxidant defences can result in temporary or permanent oxidative changes to cellular structures including lipids, proteins and DNA  honey); for all treatments osmolarity was standardized to a final concentration of 10% (w/v) honey (w/v) artificial honey + 7000 U catalase. Following a 24-h incubation at 30 °C, treatment solutions were removed by aspiration and coverslips were then washed in 0.1 M phosphate buffer (PB). Slides were then fixed by adding 250 µL of primary fixative containing 2.5% glutaraldehyde  and 2% paraformaldehyde  in 0.1 M PB to each well, with overnight incubation at 4 °C.Scanning electron microscopy (SEM) was used to reveal hyphal changes in greater detail. Round Thermanox coverslips (13 mm diameter)  were first sterilized by washing in 90% acetone then Milli-Q Hv) honey . A contrv/v) OsO4  in 0.1 M PB, for 1 h. This was removed and followed with three × 5-min washes with 250 µL of Milli-Q H2O. An ethanol concentration gradient was used to dehydrate fixed samples consisting of two treatments of 30, 50 and 70% (v/v) ethanol, then three treatments of 95 and 100% (v/v) ethanol. Further chemical desiccation of samples was conducted by adding 250 µL hexamethyldisilazane (HMDS)  for 2 min. HMDS was removed and samples were allowed to dry in the fume hood. Coverslips containing desiccated hyphae were mounted onto metallic stubs and an Emitech K550X sputter coater  covered them in gold at 2 mA for 2 min. Samples were visualized under 15 kV using a JEOL JCM-6000 benchtop scanning electron microscope . The primary fixative solution was removed by aspiration and coverslips were then rinsed three times for 5-min with 500 µL of 0.1 M PB. Rinsed coverslips were incubated in ~250 µL (enough to completely submerge coverslip) of secondary fixative containing 2% . The D’Agostino-Pearson omnibus normality test was conducted on hyphal germination and fluorescence data sets to determine whether the data exhibited a Gaussian distribution. As all data had a non-Gaussian distribution, they were analyzed using the non-parametric Kruskal–Wallis test. Significant results were followed up with a Dunn’s multiple comparison test that determined the treatment groups that were significantly different to each other. In all figures, asterisks denote the following: *


Dia2. This mechanism pushes CMG beyond a ‘5-ubiquitin threshold’ that is inherent to Cdc48, which specifically unfolds ubiquitylated Mcm7 and thereby disassembles CMG. These findings explain the exquisite regulation of CMG disassembly and provide a general model for the disassembly of ubiquitylated protein complexes by Cdc48.The eukaryotic replisome assembles around the CMG helicase, which stably associates with DNA replication forks throughout elongation. When replication terminates, CMG is ubiquitylated on its Mcm7 subunit and disassembled by the Cdc48/p97 ATPase. Until now, the regulation that restricts CMG ubiquitylation to termination was unknown, as was the mechanism of disassembly. By reconstituting these processes with purified budding yeast proteins, we show that ubiquitylation is tightly repressed throughout elongation by the Y-shaped DNA structure of replication forks. Termination removes the repressive DNA structure, whereupon long K48-linked ubiquitin chains are conjugated to CMG-Mcm7, dependent on multiple replisome components that bind to the ubiquitin ligase SCF Eukaryotic chromosomes are duplicated just once per cell cycle, by a large molecular machine called the replisome . ReplisoCdc45-Mcm2-7-GINS) helicase complexes , to an extent that reflected their contribution to CMG-Mcm7 ubiquitylation  of such protein complexes, or whether unmodified protomers are also unfolded. This is an important question, since Cdc48/p97 has the ability to unfold both ubiquitylated and unmodified proteins . The recWe found that CMG disassembly disrupts the association of Mcm2-7 with Cdc45 and the 4-protein GINS complex, but GINS remains intact and can still interact with Cdc45 to some degree  and 7–8.Degradation by Cdc48-FtsH is dependent upon translocation of peptides through the central channel of Cdc48, thereby providing a proxy for the unfolding of polypeptide substrates by Cdc48-Ufd1-Npl4 . The CdcAs noted above, the ubiquitylated Mcm7 subunit of CMG remains associated with Cdc48-Ufd1-Npl4 after helicase disassembly . As obseBuilding on previous in vivo approaches and those that used cell extracts, the fully reconstituted assays in this study have enabled us to define the minimal components and molecular basis for the regulation of CMG helicase ubiquitylation, together with the mechanism by which the ubiquitylated CMG is disassembled. As discussed above and summarised in Dia2 and Cdc34. At a pair of converged replication forks, the repressive DNA structure is removed when the two CMG helicases unwind the final base pairs of parental dsDNA and then bypass each other. From this point onwards, each CMG helicase moves along a single parental DNA strand that is no longer connected to the other. Whilst it is likely that CMG encounters the 5’ end of the opposing fork’s lagging strand and moves onto dsDNA in the few minutes before CMG is disassembled  of Dia2, which comprise the canonical substrate-binding domain,  was transformed with linearised plasmids using standard procedures to generate protein expression strains, as detailed in the Appendix 1-key resources table. For the protein expression strains, the codon usage of the synthetic gene constructs was optimised for high-level expression in Saccharomyces cerevisiae, as described previously  were transformed into Rosetta (DE3) pLysS cells (Novagen) (F-ompT hsdSB(rB- mB-) gal dcm (DE3) pLysSRARE (CamR)).For expression of proteins in ORC, Cdc6, Cdt1-Mcm2-7, DDK, S-CDK, Sld3-7, Cdc45, Dpb11, Pol ε, Sld2, GINS, Mcm10, Ctf4, RFC, PCNA, RPA, Mrc1, Pol α - primase, Pif1, Tof1-Csm3 and Top1 were purified based on previously established protocols . A briefOne protease inhibitor tablet  was used per 25 ml of lysis buffer where indicated.1 ml of Sigma protease inhibitor cocktail  was used per 100 ml of lysis buffer where indicated.600 of 0.15. The culture was left to grow at 30°C until an OD600 of 1 was reached. 0.8 mM IPTG was added to induce expression, and cells were incubated for 2.5 hr at 30°C. Cells were harvested by centrifugation at 5000 rpm for 10 min in an JLA-9.1000 rotor (Beckman).Rosetta cells were transformed with the Cdc34 expression vector (pTDK7). Transformant colonies were inoculated into a 250 ml LB/kanamycin (50 µg/ml)/chloramphenicol (35 µg/ml) culture, which was grown overnight at 37°C with shaking at 200 rpm. The following morning, the culture was diluted into 1 l of LB/kanamycin (50 µg/ml)/chloramphenicol (35 µg/ml) to a final ODFor lysis, cell pellets were resuspended in 20 ml of buffer containing 25 mM Hepes-KOH pH 7.6, 10% glycerol, 0.02% NP-40, 0.5 M NaCl, 20 mM imidazole, Roche protease inhibitor tablets, 1 mM DTT (Cdc34 buffer/20 mM imidazole). Lysozyme was added to a final concentration of 500 µg/ml and the mixture then left for 20 min on ice. Subsequently, the sample was sonicated for 90 s  at 40% on a Branson Digital Sonifier. Insoluble material was removed by centrifugation at 15,000 rpm for 30 min in an SS-34 rotor .2+ affinity purification by incubation with 2 ml packed bead volume of Ni-NTA resin (Qiagen) for 90 min at 4°C. Beads were recovered in a disposable gravity flow column and washed extensively with Cdc34 buffer/20 mM imidazole. Cdc34 was eluted with 16 ml of Cdc34 buffer/0.4 M imidazole without protease inhibitors.The supernatant was subjected to NiCdc34-containing fractions were pooled, concentrated to 4 ml, then loaded onto a 120 ml Superdex 75 column in 25 mM Hepes-KOH pH 7.6, 10% glycerol, 0.2 M NaCl, 1 mM DTT. Cdc34-containing fractions were pooled, concentrated, aliquoted and snap froze. About 1.5 mg Cdc34 was purified from a 1 l culture.14-SUMO-Cdc48 expressing plasmid was transformed into Rosetta cells. Transformant colonies were inoculated into a 50 ml LB/kanamycin (50 µg/ml)/chloramphenicol (35 µg/ml) culture, which was grown overnight at 37°C with shaking at 200 rpm. The following morning, each culture was diluted into 1 l of LB/kanamycin (50 µg/ml)/chloramphenicol (35 µg/ml) to a final OD600 of 0.15. The culture was left to grow at 30°C until an OD600 of 0.7 was reached. 0.5 mM IPTG was added to induce expression, and cells were incubated for 2 hr at 30°C.His2, 0.1 mM ATP, 0.5 mM TCEP, Roche protease inhibitor tablets (Cdc48 buffer/40 mM imidazole). Cells were lysed by addition of lysozyme (1 mg/ml) and 250 U Pierce Universal Nuclease  followed by incubation at room temperature for 30 min. Insoluble material was removed by centrifugation at 15,000 rpm for 30 min in an SS-34 rotor .Cells were harvested by centrifugation at 5000 rpm for 10 min in an JLA-9.1000 rotor (Beckman). Bacterial pellets were then resuspended in 20 ml buffer containing 50 mM Tris-Cl pH 8, 0.5 M NaCl, 40 mM imidazole, 5 mM Mg(OAc)2+ affinity purification by incubation with 1 ml packed bead volume of Ni-NTA resin (Qiagen) for 2 hr at 4°C. Beads were recovered in a disposable gravity flow column and washed extensively with Cdc48 buffer/40 mM imidazole. Cdc48 was eluted with 6 ml of Cdc48 buffer/0.5 M imidazole without protease inhibitors.The supernatant was subjected to Ni14-SUMO tag from Cdc48) and the sample incubated at 4°C for 30 min. The sample was then concentrated to 500 µl and loaded onto a 24 ml Superose 6 column in 20 mM Hepes-KOH (pH 7.4), 0.3 M sorbitol, 0.15 M NaCl, 5 mM magnesium acetate, 0.1 mM ATP, 0.5 mM TCEP. Cdc48-containing fractions were pooled, concentrated, aliquoted and snap frozen. About 0.5 mg Cdc48 was routinely purified from a 1 l culture.Ulp1 protease (10 µg/ml) was added . The cell pellets were then resuspended in 0.3–0.4 volumes of CMG buffer/0.3 M KCl/Roche protease inhibitor tablets and the resulting suspensions were frozen dropwise in liquid nitrogen. The frozen cells were freezer milled (SPEX CertiPrep 6850 Freezer/Mill) with 4 cycles of 2’ at a rate of 15. The resulting powders were stored at −80°C.Yeast cells (yTDK20) were grown at 30°C in YP + 2% raffinose to 2–3 × 10CMG buffer/0.3 M KCl/Roche protease inhibitor tablets was added to thawed powder and the sample was centrifuged . The soluble extract was recovered and mixed with 8–10 ml anti-FLAG M2 affinity resin (Sigma) and the mixture incubated at 4°C for 3 hr with rotation.Resin was collected and washed extensively with CMG buffer/0.3 M KCl/Roche protease inhibitor tablets then CMG buffer/0.3 M KCl without protease inhibitors. CMG was eluted in 1 column volume CMG buffer/0.3 M KCl/0.5 mg/ml 3FLAG peptide, then 1 column volume CMG buffer/0.3 M KCl/0.25 mg/ml 3FLAG peptide.The eluate fraction was loaded onto a 0.2 ml MiniQ column in CMG buffer/0.3 M KCl. CMG was eluted with a 4 ml gradient from 0.3 to 0.6 M KCl in CMG buffer. CMG containing fractions were then pooled and loaded onto a 24 ml Superose 6 in CMG buffer/0.3 M KCl. Peak fractions containing CMG were pooled and re-loaded onto a 0.2 ml MiniQ column in CMG buffer/0.3 M KCl. CMG was eluted with a 2.5 ml gradient from 0.3 to 0.6 M KCl in CMG buffer.Peak fractions containing CMG were pooled and dialysed against CMG buffer/0.2 M KOAc at 4°C for 4 hr. The dialysed sample was recovered, aliquoted and snap frozen. A 12 litre culture routinely yielded ~0.3 mg purified CMG.14-SUMO-FtsH expressing plasmid was transformed into Rosetta cells. A 100 ml LB/kanamycin (50 µg/ml)/chloramphenicol (35 µg/ml) culture was innoculated with transformant colonies and grown overnight at 37°C with shaking at 200 rpm. The following morning, the culture was diluted into 1 l of LB/kanamycin (50 µg/ml)/chloramphenicol (35 µg/ml) to a final OD600 of 0.15. The culture was left to grow at 37°C until an OD600 of 1 was reached. 0.5 mM IPTG was added to induce expression, and cells were incubated 16 hr at 18°C.HisCells were harvested by centrifugation at 5000 rpm for 10 min in an JLA-9.1000 rotor (Beckman). Bacterial pellets were then resuspended in 20 ml buffer containing 50 mM Tris-Cl pH 8, 0.2 M NaCl, 30 mM imidazole (FtsH buffer/30 mM imidazole). Cells were lysed by addition of lysozyme (0.5 mg/ml) and 250 U Pierce Universal Nuclease  followed by incubation on ice for 10 min, followed by sonication for 90 s  at 40% on a Branson Digital Sonifier. Insoluble material was removed by centrifugation at 15,000 rpm for 30 min in an SS-34 rotor .2+ affinity purification by incubation with 1 ml packed bead volume of Ni-NTA resin (Qiagen) for 90 min at 4°C. Beads were recovered in a disposable gravity flow column and washed extensively with FtsH buffer/30 mM imidazole. FtsH was eluted with 10 ml of 50 mM Tris-Cl pH 8, 0.1 M NaCl, 0.4 M imidazole, 1 mM TCEP.The supernatant was subjected to Ni14-SUMO tag from FtsH) and the sample incubated on ice overnight. The sample was next loaded onto a 1 ml HiTrap Q HP column pre-equilibrated in 50 mM Tris-Cl pH 8, 0.1 M NaCl. The flow-through was collected and dialysed against 50 mM Tris-Cl pH 7.5, 0.1 M NaCl at 4°C for 3 hr. The dialysed sample was recovered and the HIS-tagged Ulp1 and cleaved HIS-SUMO tag was removed by incubation with 2 ml packed bead volume of Ni-NTA resin (Qiagen) for 1 hr at 4°C. The Ni-NTA flow-through, containing pure FtsH, was collected, aliquoted and snap frozen. The yield of FtsH from 1 l of cells was 9 mg.Ulp1 protease (10 µg/ml) was added .Rosetta cells were transformed with the Otu1 expression vector (pTDK35). Transformed colonies were inoculated into a 200 ml LB/kanamycin (50 µg/ml)/chloramphenicol (35 µg/ml) culture, which was grown overnight at 37°C with shaking at 200 rpm. The following morning, the culture was diluted into 2 l of LB/kanamycin (50 µg/ml)/chloramphenicol (35 µg/ml) to a final ODFor lysis, cell pellets (corresponding to 1 litre of culture) were resuspended in 20 ml of buffer containing 50 mM Tris-Cl pH 7.5, 0.5 M NaCl, 40 mM imidazole, Roche protease inhibitor tablets and 0.5 mM TCEP (Otu1 buffer/40 mM imidazole). Lysozyme was added to a final concentration of 500 µg/ml and the mixture then left for 15 min on ice. Subsequently, the sample was sonicated for 45 s  at 40% on a Branson Digital Sonifier. Insoluble material was removed by centrifugation at 15,000 rpm for 30 min in an SS-34 rotor .2+ affinity purification by incubation with 1 ml packed bead volume of Ni-NTA resin (Qiagen) for 90 min at 4°C. Beads were recovered in a disposable gravity flow column and washed extensively with Otu1 buffer/40 mM imidazole. Otu1 was eluted with 8 ml of Otu1 buffer/0.5 M imidazole without protease inhibitors.The supernatant was subjected to Ni14-SUMO tag from Otu1) and the sample was dialysed for 16 hr at 4°C vs. 50 mM Tris-Cl pH 7.5, 0.1 M NaCl and then loaded onto a 1 ml HiTrap Q HP column pre-equilibrated in 50 mM Tris-Cl pH 7.5, 0.1 M NaCl. The flow-through was collected, concentrated to 500 µl, then loaded onto a 24 ml Superdex 75 column in 25 mM Hepes-KOH pH 7.6, 0.2 M NaCl, 0.5 mM TCEP. Otu1-containing fractions were pooled, aliquoted and snap frozen. The yield of purified Otu1 from 1 l of cells was ~1.2 mg.Ulp1 protease (10 µg/ml) was added . Protein expression was induced for 3 hr at 30°C with 2% galactose. 12 litres of cells were typically used for each purification. Cells were harvested and lysed as described above for purification of CMG, except buffer containing 25 mM Hepes KOH pH 7.6, 10% glycerol, 0.02% NP-40-S, 0.4 M KOAC, 1 mM DTT, Sigma protease inhibitor cocktail and Roche protease inhibitor tablets (SCFDia2 buffer/protease inhibitors) was used in place of CMG buffer.Yeast cells (yTDK5) were grown to 2 × 10Dia2 buffer/protease inhibitors was added to thawed cell powder and the sample was centrifuged . The soluble extract was recovered and mixed with 4 ml IgG Sepharose 6 Fast Flow  and the mixture incubated at 4°C for 1 hr with rotation.SCFDia2 buffer/protease inhibitors then SCFDia2 buffer without protease inhibitors. Washed beads were resuspended in 8 ml SCFDia2 buffer without protease inhibitors and 400 µg TEV protease was added, followed by incubation at 4°C for 3 hr with rotation.Resin was collected and washed extensively with SCFDia2 buffer without protease inhibitors. Peak fraction containing SCFDia2 were pooled, concentrated, aliquoted and snap frozen. A 12-l culture routinely yielded ~0.2 mg purified SCFDia2.The TEV eluate fraction was collected, concentrated to 5 ml, then loaded onto a 120 ml Superdex 200 column in SCFHIS-tagged Uba1, expressed in SF21 insect cells and partially purified by Ni-NTA affinity chromatography, was kindly provided by Dr. Axel Knebel. Uba1 was subsequently purified further over a 24 ml Superdex 200 column in 25 mM Hepes-KOH pH 7.6, 0.15 M NaCl, 10% glycerol, 1 mM DTT. Peak fractions containing Uba1 were pooled, aliquoted and snap froze. The yield of purified protein from 1 l of cells was about 22 mg.14-SUMO-Ufd1 were expressed separately as follows. Npl4 or His14-SUMO-Ufd1 expressing plasmids were transformed into Rosetta cells. Transformant colonies were innoculated into a 50 ml LB/chloramphenicol (35 µg/ml) culture (containing kanamycin (50 µg/ml) for His14-SUMO-Ufd1 and ampicillin (50 µg/ml) for Npl4), which was grown overnight at 37°C with shaking at 200 rpm. The following morning, each culture was diluted into 1 l of LB/chloramphenicol (35 µg/ml) (plus kanamycin or ampicillin) to a final OD600 of 0.15. The culture was left to grow at 18°C until an OD600 of 0.7 was reached. 0.5 mM IPTG was added to induce expression, and cells were incubated for 16 hr at 18°C.Untagged Npl4 and HisCells were harvested by centrifugation at 5000 rpm for 10 min in an JLA-9.1000 rotor (Beckman). Bacterial pellets were then mixed at a 6:1 ratio (Npl4:Ufd1) and resuspended in 40 ml buffer containing 50 mM Tris-Cl pH 8, 0.5 M NaCl, 40 mM imidazole, 0.5 mM TCEP, Roche protease inhibitor tablets (UN buffer/40 mM imidazole). Cells were lysed by passing through a C5 Emulsiflex Cell Disruptor (BioPharma Process Systems) three times at 15 kPsi, and insoluble material was removed by centrifugation at 15,000 rpm for 30 min in an SS-34 rotor .2+ affinity purification by incubation with 1 ml packed bead volume of Ni-NTA resin (Qiagen) for 2 hr at 4°C. Beads were recovered in a disposable gravity flow column and washed extensively with UN buffer/40 mM imidazole. Ufd1-Npl4 was eluted with 8 ml of UN buffer/0.5 M imidazole without protease inhibitors.The supernatant was subjected to Ni14-SUMO tag from Ufd1) and the sample was dialysed for 3 hr at 4°C vs. 50 mM Tris-Cl pH 8, 0.1 M NaCl, 0.5 mM TCEP and then loaded onto a 6 ml Resource Q column pre-equilibrated in 50 mM Tris-Cl pH 8, 0.1 M NaCl. Protein was eluted with a 10-column volume gradient from 0.1 to 0.5 M NaCl. Ufd1-Npl4 containing fractions were pooled, concentrated to 500 µl, then loaded onto a 24 ml Superose 6 column in 20 mM Hepes-KOH (pH 7.4), 0.2 M NaCl, 5 mM magnesium acetate, 0.5 mM TCEP. Ufd1-Npl4-containing fractions were pooled, aliquoted and snap frozen. 1 l of cells yielded 0.6 mg of purified Ufd1-Npl4.Ulp1 protease (10 µg/ml) was added (to cleave the HIS2 and 5 mM ATP at 30°C for 10 min.Mcm2-7 loading and DDK phosphorylation was performed by incubating 6 nM 3.2 kb plasmid DNA template (pBS/ARS1WTA), 5–10 nM ORC, 20 nM Cdc6, 40 nM Cdt1/Mcm2-7 and 20 nM DDK in 25 mM Hepes-KOH (pH 7.6), 100 mM potassium acetate, 0.02% NP-40-S, 0.1 mg / ml BSA, 1 mM DTT, 10 mM Mg(OAc)2, 3.75 mM ATP, 30 µM dATP-dCTP-dGTP-dTTP, 33 nM γ-[32P]-dCTP, 400 µM CTP-GTP-UTP, 20 µM creatine phosphate, 50 µg/ml creatine phospho-kinase, 6 µM ubiquitin, 20 nM S-CDK, 30 nM Dpb11, 8 nM GINS, 40 nM Cdc45, 30 nM Pol ε, 5 nM Mcm10, 5 nM RFC, 20 nM PCNA, 20 nM Top1, 20 nM Pol α-primase, 6.25 nM Sld3-7, 40 nM Ctf4, 100 nM RPA, 10 nM Csm3-Tof1, 40 nM Mrc1, 50 nM Sld2 and 5 nM Pif1 (unless otherwise indicated). The extra contribution from protein storage buffers to the final reaction was approximately 22 mM chloride and 50–60 mM acetate, and the corresponding potassium counter-ions. Pol δ was omitted from replication reactions to avoid strand displacement synthesis, which has previously been shown to be enhanced by Pif1 , 100 mM potassium acetate, 0.02% NP-40-S, 0.1 mg/ml BSA, 1 mM DTT, 10 mM Mg(OAc) by Pif1 . Top2 is by Pif1  and was Dia2 were added after the replication step, and the incubation continued at 30°C for a further 20 min. For the experiment in Dia2 were added during the replication step, which was conducted at 30°C for 10 min. Pif1 (5 nM) was then added and the incubation continued at 30°C for a further 20 min. For the experiment in Dia2 were then added and the incubation continued as in The replication step was routinely conducted at 30°C for 20 min. For the experiment in 2 (Wash buffer/700 mM KOAc). The bound proteins were then eluted by the addition of SDS-PAGE sample loading buffer and boiling for 5 min at 95°C.Ubiquitylation reactions were stopped by the addition of KOAc to 700 mM. Next, plasmid DNA was digested by addition of 125 U Pierce Universal Nuclease  and incubation on ice for 30 min. Each sample was then incubated for 30 min at 4°C with 2.5 µl magnetic beads  that had been coupled to antibodies raised against Sld5. After the incubation, protein complexes bound on antibody-coupled magnetic beads were washed twice with 190 µl of buffer containing 25 mM Hepes-KOH (pH 7.6), 700 mM potassium acetate, 0.02 % NP-40-S, 0.1 mg / ml BSA, 1 mM DTT, 10 mM Mg(OAc)For native agarose gel analysis of replication products from these experiments, reactions were quenched by addition of 25 mM EDTA after the ubiquitylation step. SDS (0.1%) and proteinase K (1/100 volumes) were subsequently added and the incubation continued at 37°C for 30 min. An equal volume of phenol:chloroform:isoamyl alcohol 25:24:1 (Sigma-Aldrich P2069) saturated with TE  was next added and the DNA was extracted. Unincorporated nucleotides were removed and the aqueous phase buffer exchanged to TE with Illustra MicroSpin G-50 columns . For restriction digest, 17.75 µl of sample was incubated in 1x CutSmart buffer with 0.25 μl SpeI-HF at 37°C for 30 min. Digested samples were then separated in 0.8% native agarose gels at 20 V overnight in 1X TAE then dried directly onto chromatography paper . The dried gels were typically exposed to both Amersham Hyperfilm ECL  and BAS-MS Imaging Plates (Fujifilm), which were then developed on a Typhoon phosphorimager .Dia2, 30 nM Ctf4, 30 nM Pol ε, 45 nM Mrc1, 45 nM FACT, 45 nM Top1, 45 nM Pol α - primase, 45 nM Mcm10 and 45 nM Tof1-Csm3 were assembled on ice in 25 mM Hepes-KOH (pH 7.6), 75 mM potassium acetate, 0.02% NP-40-S, 0.1 mg / ml BSA, 1 mM DTT, 10 mM Mg(OAc)2, 6 µM ubiquitin and 5 mM ATP. From Dia2 was included at 2 nM, and Cdc34 was included at the indicated concentrations). Protein storage buffers typically contributed approximately 50 mM acetate, and the corresponding potassium counter-ions, to the final reaction. Ubiquitylation reactions were conducted at 30°C for 20 min unless otherwise indicated. Reactions were stopped by the addition of SDS-PAGE sample loading buffer and boiling at 95°C for 5 min.Reactions  containing 15 nM CMG, 30 nM Uba1, 15 nM Cdc34, 1 nM SCFForked DNA substrates were prepared by mixing equimolar amounts of partially complementary oligonucleotides (see Appendix 1-key resources table). The mixture  was incubated at 95°C for 10 min followed by gradual cooling to room temperature.2, 6 µM ubiquitin and 2 mM ATP for 1 hr on ice. Reactions were shifted to room temperature for 1 min before addition of 30 nM Ctf4, 30 nM Uba1, 15 nM Cdc34 and 1 nM SCFDia2 and incubation at 30°C for 8 min. Mrc1, Pol ε and other replisome components were omitted from these reactions to limit DNA unwinding by CMG. Reactions were stopped by the addition of SDS-PAGE sample loading buffer and boiling at 95°C for 5 min.Ubiquitylation reactions performed in the presence of forked DNA were prepared by incubating 15 nM CMG with 50 nM DNA in 25 mM Hepes-KOH (pH 7.6), 75 mM potassium acetate, 0.02% NP-40-S, 0.1 mg / ml BSA, 1 mM DTT, 10 mM Mg(OAc)To monitor DNA binding by CMG in these experiments as in , reactioFor CMG disassembly reactions in solution, CMG ubiquitylation reactions were assembled as above and conducted at 30°C for 20 min, followed by the addition of 50 nM Cdc48 and 50 nM Ufd1-Npl4, and incubation at 30°C for a further 20 min. Each sample was then incubated for 1 hr at 4°C with 2.5 µl magnetic beads  that had been coupled to antibodies raised against specific CMG subunits as indicated. After the incubation, protein complexes bound on antibody-coupled magnetic beads were washed twice with 190 µl of wash buffer/150 mM KOAc. The bound proteins were then eluted by the addition of SDS-PAGE sample loading buffer and boiling for 5 min at 95°C.For CMG disassembly reactions on beads , ubiquit2. Where indicated, 400 nM human Usp2b was added to reactions after the Cdc48-FtsH step, and the incubation continued at 30°C for a further 60 min.For CMG disassembly/proteolysis reactions using Cdc48-FtsH , reactioFor experiments to monitor the release of ubiquitylated Mcm7 from Cdc48-Ufd1-Npl4, dependent on Otu1 , reactioDia2, 30 nM Ctf4, 30 nM Pol ε and 45 nM Mrc1 were assembled in 25 mM Hepes-KOH (pH 7.6), 75 mM potassium acetate, 0.02% NP-40-S, 0.1 mg / ml BSA, 1 mM DTT, 10 mM Mg(OAc)2, 6 µM ubiquitin and 5 mM ATP and incubated on ice for 10 min. Input samples  were removed and the remainder of each sample was then incubated for 30 min at 4°C with 3.75 µl magnetic beads  that had been coupled to anti-Sld5 antibody. After the incubation, protein complexes bound on beads were washed twice with 190 µl of Wash buffer/150 mM KOAc. The bound proteins were then eluted by the addition of SDS-PAGE sample loading buffer and boiling for 5 min at 95°C.Reactions  containing 15 nM CMG, 30 nM Uba1, 15 nM Cdc34, 10 nM SCFProtein samples were resolved by SDS–polyacrylamide gel electrophoresis on NuPAGE Novex 4–12% Bis-Tris gels  with NuPAGE MOPS SDS buffer , or NuPAGE Novex 3–8% Tris-Acetate gels  with NuPAGE Tris-Acetate SDS buffer . Resolved proteins were either stained with colloidal Coomassie blue dye , or were transferred onto a nitrocellulose iBlot membrane (Invitrogen) with the iBlot Dry Transfer System (Invitrogen).Antibodies used for protein detection in this study are described in Appendix 1-key resources table. Conjugates to horseradish peroxidase of anti-sheep IgG from donkey , anti-rabbit IgG from donkey , or anti-goat IgG from rabbit  were used as secondary antibodies before the detection of chemoluminescent signals on Hyperfilm ECL  using ECL Western Blotting Detection Reagent .For quantification of immunoblots, ECL Western Blotting Detection Reagent  was applied as above, and the membrane then imaged using a ChemiDoc imaging system (Bio-Rad). The Tiff files generated were opened in ImageJ, boxes were drawn around each band, the background signal subtracted, and relative signal calculated as a percentage of the signal in the complete reaction  or reactThe experiments in As indicated in the Figure Legends, the mean and standard deviations for the experiments in  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Recent studies on eukaryotic DNA replication have shown that it is possible to reconstitute the entire replication process with purified proteins and the current paper extends this to reconstitute termination of DNA replication and removal of the replicative DNA helicase from the replicated DNA. In so doing, the authors have discovered a new mechanism for linking ubiquitylation of the helicase subunit, Mcm7, to the structure of the replicated DNA. This is an important contribution to understanding how the genome is copied only once per cell division cycle.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, including Bruce Stillman as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Jessica Tyler as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Dirk Remus (Reviewer #2); Philip Zegerman (Reviewer #3).Thank you for submitting your article "CMG helicase disassembly is controlled by replication fork DNA, replisome components and a ubiquitin threshold" 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.eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as Summary:This paper describes the reconstitution of replisome disassembly after the termination of DNA replication with purified budding yeast proteins. Replisome disassembly has been previously shown by the Labib lab and others to be mediated by the ubiquitylation-dependent extraction of Mcm7 by the Cdc48 ATPase. However, the mechanism that links replisome disassembly to the termination of DNA replication has been elusive. Here, the authors propose that the displaced lagging strand template inhibits replisome disassembly during replication elongation. Moreover, the authors propose that replisome disassembly requires conjugation of extended ubiquitin chains on Mcm7 as a failsafe mechanism against premature replisome disassembly due to potential stochastic Mcm7 ubiquitylation. In addition, the authors demonstrate novel roles for Ctf4 and Mrc1 in mediating Mcm7 ubiquitylation.This is a very interesting study that addresses an important biological problem and provides several critical mechanistic insights into an essential step of the DNA replication reaction. The experiments are generally well-controlled and of high quality. There are a number of minor clarifications and comments that the authors should address in a revised manuscript, none requiring new data.Minor points:1) The purification of Cdc34, Cdc48, FtsH, Otu1, SCFDAI2, Uba1, Ufd1-Npl4, and CMG are described in the Materials and methods. The authors should state the yield of purified protein from the amount of cells used.2) Discussion: The authors discuss why Mcm7 in the CMG is not ubiquitylated when it is an active helicase and the model is fine and consistent with the data. Perhaps they should also discuss why Mcm7 in all of the loaded Mcm2-7 double hexamers (DH) is not ubiquitylated. Two possibilities come to mind, one is the lack of elongation factors associated with the DH  or that the juxtaposition of the DH Mcm2-7 amino termini buries the Mcm7 N-terminal tails, making then only accessible after DH dissociation. The structures of the Mcm2-7 DH show some accessibility of the Mcm7 amino terminus, so it is either masked or elongation factors are required.3) The paper relies on a system that utilizes Pif1 to induce replication termination in reactions containing Top1 but lacking Top2. This may not represent the default termination pathway. For example, the authors had previously demonstrated that termination of plasmid replication in yeast cells is only slightly delayed after depletion of Pif1/Rrm3 (PMID 30850330), indicating that termination is not strictly dependent on Pif1/Rrm3 in vivo. Moreover, as demonstrated in PMID 32341532 and the authors' previous paper (PMID 30850330) termination is less efficient in the presence of Top1 compared to Top2. Therefore: Why are the reactions here performed in the absence of Top2? Moreover, termination efficiency in the reconstituted budding yeast system reported in PMID 32341532 is significantly higher than that reported by the authors' system here, indicating that specific reaction conditions significantly affect the termination process. Combined, it therefore seems relevant to point out at the beginning of the paper that the Top1/Pif1 system is being employed here as a useful tool to enrich for LRIs.4) Could the authors explain why the replication experiments are performed in the absence Pol-δ, which is required for normal DNA replication?5) Figure 4: The authors had previously shown that K29 ubiquitylation is not essential for replisome disassembly in vivo (PMID 28355556). Thus, while the specificity for K29 ubiquitylation observed in the in vitro system here is a useful tool to assess the ubiquitin chain length requirement for Cdc48-catalyzed replisome disassembly , it should be noted that alternative disassembly pathways remain possible. Minor points:1) The purification of Cdc34, Cdc48, FtsH, Otu1, SCFDAI2, Uba1, Ufd1-Npl4, and CMG are described in the Materials and methods. The authors should state the yield of purified protein from the amount of cells used.For each of the purified proteins mentioned by the reviewers, the yield has now been included at the end of the corresponding section of Materials and methods.2) Discussion: The authors discuss why Mcm7 in the CMG is not ubiquitylated when it is an active helicase and the model is fine and consistent with the data. Perhaps they should also discuss why Mcm7 in all of the loaded Mcm2-7 double hexamers (DH) is not ubiquitylated. Two possibilities come to mind, one is the lack of elongation factors associated with the DH  or that the juxtaposition of the DH Mcm2-7 amino termini buries the Mcm7 N-terminal tails, making then only accessible after DH dissociation. The structures of the Mcm2-7 DH show some accessibility of the Mcm7 amino terminus, so it is either masked or elongation factors are required.Dia2 . As the reviewers note, the failure to ubiquitylate Mcm2-7 double hexamers could either be due to a requirement for elongation factors, or else might reflect the occlusion within the double hexamer interface of important binding sites for SCFDia2.We previously showed that Mcm2-7 complexes are only ubiquitylated during S-phase in yeast cells, dependent upon Cdc45 . This indicated that only CMG complexes and not Mcm2-7 double hexamers are a substrate for SCFDia2 . Therefore, the failure to ubiquitylate Mcm2-7 double hexamers is unlikely to be due to the occlusion of important binding sites for SCFDia2 within the double hexamer interface. Instead, only CMG is a substrate for SCFDia2 and ubiquitylation is almost entirely dependent upon the Ctf4 and Mrc1 partners of CMG .However, our new data in the present manuscript show that free Mcm2-7 complexes (single hexamers) are not ubiquitylated by SCFDia2 by Mrc1 and Ctf4”).In the revised manuscript we now discuss why Mcm2-7 complexes cannot be ubiquitylated outside of S-phase (subsection “Efficient ubiquitylation of the CMG-replisome is dependent upon recruitment of SCF3) The paper relies on a system that utilizes Pif1 to induce replication termination in reactions containing Top1 but lacking Top2. This may not represent the default termination pathway. For example, the authors had previously demonstrated that termination of plasmid replication in yeast cells is only slightly delayed after depletion of Pif1/Rrm3 (PMID 30850330), indicating that termination is not strictly dependent on Pif1/Rrm3 in vivo. Moreover, as demonstrated in PMID 32341532 and the authors' previous paper (PMID 30850330) termination is less efficient in the presence of Top1 compared to Top2. Therefore: Why are the reactions here performed in the absence of Top2? Moreover, termination efficiency in the reconstituted budding yeast system reported in PMID 32341532 is significantly higher than that reported by the authors' system here, indicating that specific reaction conditions significantly affect the termination process. Combined, it therefore seems relevant to point out at the beginning of the paper that the Top1/Pif1 system is being employed here as a useful tool to enrich for LRIs.It is true, as the reviewers note, that we use Pif1-dependent DNA replication termination as a useful tool to compare ubiquitylation in the absence (-Pif1) or presence (+Pif1) of termination. We point this out at the beginning of the Results: “replication reactions in the presence or absence of Pif1 provide a model system, with which to study the regulation of CMG ubiquitylation during DNA replication termination”.However, our choice of topoisomerase was not related to the efficiency of termination. The reviewers correctly note that termination is slightly more efficient in our system in the presence of Top2, compared to reactions that only have Top1 . But even in the presence of Top2, more than 80% of replicated plasmids accumulate as ‘late replication intermediates’, due to a failure of DNA replication termination.Furthermore, Top2 is not essential for replication termination either in vivo  or in vitro . For this study, we omitted Top2 in order to avoid a slight inhibitory effect of Top2 on replication efficiency under our reaction conditions. We have now added a note to this effect in Materials and methods (subsection “In vitro replication-ubiquitylation assays”).4) Could the authors explain why the replication experiments are performed in the absence Pol-δ, which is required for normal DNA replication?Pol-δ was omitted to avoid strand displacement synthesis, which is driven by Pol-δ and was previously shown to be enhanced by Pif1 . Although this strand displacement activity of Pol-δ can be alleviated by higher salt concentrations, the latter conditions would also impair the efficiency of ubquitylation. We now state this and cite the relevant papers in Materials and methods (subsection “In vitro replication-ubiquitylation assays”).5) Figure 4: The authors had previously shown that K29 ubiquitylation is not essential for replisome disassembly in vivo (PMID 28355556). Thus, while the specificity for K29 ubiquitylation observed in the in vitro system here is a useful tool to assess the ubiquitin chain length requirement for Cdc48-catalyzed replisome disassembly , it should be noted that alternative disassembly pathways remain possible.Dia2-Cdc34 during in vitro ubiquitylation reactions in yeast cell extracts, under conditions that are relatively inefficient . Mass spectrometry analysis confirmed K29 as a bona fide ubiquitylation site in vivo, but we found that other sites on Mcm7 could also be ubiquitylated in yeast cells with high efficiency . Consistent with these findings, our new data in the present manuscript show that multiple sites on Mcm7 can be ubiquitylated in the reconstituted ubiquitylation system under optimal conditions . However, Mcm7-K29 is the only site that is modified if the efficiency of the reactions is reduced . We now state that more than one lysine on Mcm7 is modified under standard reaction conditions in the subsection “CMG ubiquitylation is inherently efficient in the presence of other replisome proteins”.Our previous work showed that Mcm7-K29 is the only residue that is ubiquitylated by SCFTogether with our previous in vivo findings , these data indicate that ubiquitylation of other sites on Mcm7, in addition to Mcm7-K29, can also support CMG disassembly. However, there is currently no reason to suggest the existence of alternative disassembly pathways that are independent of the ubiquitylation of CMG-Mcm7 (at least in budding yeast).


Fusarium fungi are the cause of an array of devastating diseases affecting yield losses and accumulating mycotoxins. Fungicides can be exploited against Fusarium and deoxynivalenol (DON) production. However, Fusarium resistance to common chemicals has become a therapeutic challenge worldwide, which indicates that new control agents carrying different mechanisms of action are desperately needed. Here, we found that a nonantibiotic drug, ethylenediaminetetraacetic acid disodium salt (EDTANa2), exhibited various antifungal activities against Fusarium species and DON biosynthesis. The infection of wheat seeding caused by F. graminearum was suppressed over 90% at 4 mM EDTANa2. A similar control effect was observed in field tests. Mycotoxin production assays showed DON production was significantly inhibited, 47% lower than the control, by 0.4 mM EDTANa2. In vitro experiments revealed a timely inhibition of H2O2 production as quickly as 4 h after amending cultures with EDTANa2 and the expression of several TRI genes significantly decreased. Chitin synthases of Fusarium were Mn2+-containing enzymes that were strongly inhibited by Mn2+ deficiency. EDTANa2 inhibited chitin synthesis and destroyed the cell wall and cytomembrane integrity of Fusarium, mainly via the chelation of Mn2+ by EDTANa2, and thus led to Mn deficiency in Fusarium cells. Taken together, these findings uncover the potential of EDTANa2 as a fungicide candidate to manage Fusarium head blight (FHB) and DON in agricultural production. Fusarium is a globally important genus of fungal pathogens, responsible for many devastating diseases of plants and various serious diseases of humans . Th. Th7/mL)4 mL−1) were cultured in SNA, SNA amended with 0.15 mM EDTA or SNA amended with 0.15 mM EDTANa2 for 7 days and were used for chitin determination as previously described [−1 of NaNO3, 1.31 g L−1 of K2HPO4, 0.5 g L−1 of KCl, 0.5 g L−1 of MgSO4·7H2O, 0.01 g L−1 of FeSO4·7H2O, 30 g L−1 of sucrose, pH 7.2) for 5 d were collected and finely ground with liquid nitrogen for chitin synthase extraction. Chitin synthase activity was extracted and measured according to Song et al. [14C) GlcNAc incorporation into the filter-retainable polymer in the presence of 0–1 mM EDTANa2 plus 5 mM Mg2+ or Mn2+. To measure the cell membrane permeability, macroconidia  were inoculated into SNA, SNA with 0.3 mM NaCl, SNA with 0.15 mM EDTA or SNA with 0.15 mM EDTANa2 at 25 °C for 7 days. The conductivity was measured with a conductometer  as described previously [Macroconidia  of F. graminearum PH-1 were plated in a 96-well flat bottom culture plate containing 0.15 mM EDTA, EDTANa2, EDTANa3 or EDTANa4 in 100 μL of SNA medium (pH 4.2). The plates were cultured at 25 °C for 24 h and observed using an inverted microscope. The growth and morphology of mycelia were photographed and compared among treatments using NIS-Elements AR software . The experiment was replicated three times.Ten microliters of spores  fungal inoculated in 96-well microtiter plates were treated with EDTANa2 at different concentrations and incubated for 24 or 36 h. The lowest concentration that demonstrated no visible growth was determined as the MIC. Measurements were repeated three times.The determination of the minimum inhibitory concentration (MIC) was performed using 96-well microtiter plates. The 2 on mycelial growth and chitin synthesis, an additional 0.3 mM MgCl2, 0.3 mM CaCl2, 0.3 mM MnCl2 or 0.15 mM FeCl3 was added into SNA medium separately to bind the preadded 0.15 mM EDTANa2, followed by inoculation at 25 °C for 24 h and subsequent micro-examination. Corresponding amounts of MgCl2, CaCl2, MnCl2 or FeCl3 were used as controls. Subsequently, a series of MgCl2, CaCl2 or MnCl2 concentrations  was added to the media to saturate the 0.15 mM EDTANa2 to different degrees, and the fungi were cultured for 52 h to measure the mycelial biomass at OD290. The mycelia that were treated with 0.3 mM cations were collected at 7 d, frozen and dried for chitin content and trace element measurements [To assess whether cations would ameliorate the inhibitory effects of EDTANaurements . Three it-test, all at a significance level of 0.05.Statistical analysis was performed using Duncan’s multiple comparison test (for multiple comparisons) and Student’s