Evidence of centromeric histone 3 chaperone involved in DNA damage repair pathway in budding yeast
- Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, India
Figures
Figure 1 with 3 supplements
Depletion of Scm3 causes sensitivity to MMS.
(A) Wild-type (SCM3), SCM3-AID (three independent transformants), and rad52∆ cells, grown till mid-log, were serially diluted and spotted on the indicated plates. The plates were incubated at 30 °C for 24–48 hr before imaging. (B) An equal number of mid-log grown SCM3-AID cells were spread on plates containing the indicated supplements. Cell viability was estimated from the number of colonies that appeared after incubation at 30 °C for 2 days (see Materials and methods). The colony number on the plate without auxin and MMS was taken as 100% viability. Error bars represent the standard error of the mean. The p-values were estimated by a one-way ANOVA test followed by Tukey’s multiple comparison test from three independent experiments. (C) Wild-type (SCM3), SCM3-AID, SCM3-AID rad52∆, and rad52∆ cells, grown till mid-log, were serially diluted and spotted in the presence of the indicated auxin concentrations. The plates were incubated at 30 °C for 24–48 hr before imaging. (D) The cells of the strains used in (C) were grown till mid-log and treated with 0.75 mM auxin for the indicated duration before they were washed, serially diluted, and spotted on YPD plates that were incubated at 30 °C for 24–48 hr before imaging.
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Figure 1—source data 1
Source data for Figure 1B.
- https://cdn.elifesciences.org/articles/104431/elife-104431-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
NLS domain and the C-terminal amino acids of Scm3 are dispensable for the DDR function.
(A) The domain organization of HJURP and Scm3. HCTD, HJURP C-terminal domain; NES, nuclear export signal; BR/NLS, bromodomain, or nuclear localization signal; D/E, D/E rich region. (B) The domain organization of the Scm3 mutants used for the DNA damage sensitivity assay. Yeast cells harboring indicated Scm3 versions as the sole source of Scm3 were grown till mid-log and were serially diluted and spotted on the indicated plates. The plates were incubated at 30 °C for 24–48 hr before imaging.
Figure 1—figure supplement 2
Cells lacking Scm3 are sensitive to various DNA damaging agents.
(A)SCM3, SCM3-AID, and rad52∆ cells grown till mid-log were serially diluted and spotted on the indicated plates. The plates were incubated at 30 °C for 24–48 hr before imaging. (B) SCM3 and SCM3-AID cells were grown till mid-log and were treated with auxin for 2 hr. The cells were harvested and analyzed for DAPI staining and bud morphology to visualize the cell cycle stages. At least 100 cells were analyzed from two independent experiments. (C) Western blot analysis for the detection of Scm3 in a no-tag strain (NT) or in strains harboring SCM3-6HA or SCM3-6HA-AID in the absence or presence of auxin. (D) SCM3, SCM3-AID (three independent transformants), and rad52∆ cells, grown till mid-log, were pretreated with 0.75 mM auxin for 2 hr before they were washed, serially diluted, and spotted on the indicated plates. (E) The graph shows the cell viability percentage of the indicated strains treated with 0.01% MMS. An equal number of cells pretreated with 0.75 mM auxin were spread on YPD plates supplemented without or with 0.01% MMS. Cell viability was estimated from the number of colonies that appeared after incubation at 30 °C for 2 days (see Materials and methods). The colony number on the plate without MMS was taken as 100% viability. Error bars represent the standard error of the mean. The p-values were determined by a one-way ANOVA test followed by Tukey’s multiple comparison test from three independent experiments. (F) The cells from the indicated strains were grown till mid-log, serially diluted, and spotted on the indicated plates. The plates were incubated at 30 °C for 24–48 hr before imaging.
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Figure 1—figure supplement 2—source data 1
Original files for western blot analysis displayed in Figure 1—figure supplement 2C.
- https://cdn.elifesciences.org/articles/104431/elife-104431-fig1-figsupp2-data1-v1.zip
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Figure 1—figure supplement 2—source data 2
Original files for western blot analysis displayed in Figure 1—figure supplement 2C indicating relevant bands.
- https://cdn.elifesciences.org/articles/104431/elife-104431-fig1-figsupp2-data2-v1.zip
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Figure 1—figure supplement 2—source data 3
Source data for Figure 1—figure supplement 2E.
- https://cdn.elifesciences.org/articles/104431/elife-104431-fig1-figsupp2-data3-v1.xlsx
Figure 1—figure supplement 3
MMS sensitivity of the cells lacking Scm3 is independent of its kinetochore function or effect on cell cycle progression.
(A) Wild-type, NDC10-AID, MIF2-AID, and rad52∆ cells grown till mid-log were serially diluted and spotted on the indicated plates. The plates were incubated at 30 °C for 24–48 hr before imaging. (B) Wild-type, ctf19∆, and rad52∆ cells grown till mid-log were serially diluted and spotted on the indicated plates. The plates were incubated at 30 °C for 24–48 hr before imaging.
Figure 2 with 2 supplements
Depletion of Scm3 causes more Rad52 foci in the presence or absence of MMS.
(A) Schematic of the experimental strategy for visualization of Rad52-GFP foci in the RAD52-GFP SCM3-AID cells either in the presence of DMSO or auxin (Scm3 depleted) and each either treated with MMS or left untreated. (B) Representative images showing different patterns of Rad52-GFP foci. Rad52-GFP produces a nuclear signal (row 1) in the absence of DNA damage, but with damage coalesces to form bright foci (rows 2–5) representing the DNA repair sites. (C) The percentage of indicated cells containing Rad52-GFP foci with or without MMS treatment for 90 min. (D) The average number of Rad52-GFP foci per MMS-treated cell from the experiment described in (C). At least 300 cells were analyzed from three independent experiments for each set in (C) and (D). Error bars represent the standard error of the mean. The p-values were estimated by an unpaired two-tailed Student’s t-test. Scale bar = 2 µm.
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Figure 2—source data 1
Source data for Figure 2.
- https://cdn.elifesciences.org/articles/104431/elife-104431-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Depletion of Scm3 leads to the formation of multiple DSB (Rad52-GFP) foci.
(A) Schematic of the experimental strategy that was followed for visualization of Rad52-GFP foci in the wild-type cells treated with DMSO or auxin (Scm3 depleted) for 2 hr. (B) The percentage of Rad52-GFP foci positive cells harvested from (A). (C) The percentage of wild type (SCM3-AID) and Scm3-depleted (SCM3-AID+Auxin) cells harboring indicated numbers of Rad52-GFP foci upon treatment with 0.02% MMS. At least 300 cells were analyzed from three independent experiments for each set in (C) and (D). Error bars represent the standard error of the mean. The p-values were determined by an unpaired two-tailed Student’s t-test.
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Figure 2—figure supplement 1—source data 1
Source data for Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/104431/elife-104431-fig2-figsupp1-data1-v1.xlsx
Figure 2—figure supplement 2
Depletion of Scm3 causes increased Rad52 foci along the cell cycle and cell death.
(A) The schematic of the experimental strategy that was followed for visualization of Rad52-GFP foci in the bar1∆ RAD52-GFP SCM3-AID cells at different stages of the cell cycle in the presence of DMSO (wild-type) or auxin (Scm3 depleted) and each either treated with 0.02% MMS or left untreated. All the cells were arrested eventually at metaphase using nocodazole. (B) Representative images showing different patterns of Rad52 foci at indicated time points following their release from alpha factor. (C, D) The percentage of Rad52-GFP foci in the indicated cells harvested as shown in (A). (E) Cells harvested, as shown in Figure 2A, were analyzed for cell viability using PI staining. Representative images show PI-positive cells (dead cell, arrow) and PI-negative cells (live cell, arrowhead). The percentage of dead cells with or without MMS treatment for 90 min is graphically shown. At least 250 cells were analyzed for each set in (C, D), and (E) from three independent experiments. Error bars represent the standard error of the mean. The p-values were determined by an unpaired two-tailed Student’s t-test. Scale bar = 2 µm.
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Figure 2—figure supplement 2—source data 1
Source data for Figure 2—figure supplement 2.
- https://cdn.elifesciences.org/articles/104431/elife-104431-fig2-figsupp2-data1-v1.xlsx
Figure 3 with 1 supplement
Localization of Scm3 in response to DNA damage.
(A) Representative images showing the localizations of Ndc10-6HA and Scm3-13Myc using chromatin spreads. The mid-log grown NDC10-6HA SCM3-13MYC cells were treated with 0.02% MMS or mock-treated for 90 min prior to imaging. The arrows show the co-localization of Ndc10-6HA and Scm3-13Myc at the kinetochore cluster, and the arrowhead shows the rDNA loop. Scale bar = 2 µm. (B) The intensities of Scm3-13Myc and Ndc10-6HA after subtracting background intensity were quantified and plotted. (C) Mean values from three independent experiments. At least 30 spreads were analyzed for each set, as shown in (B). The p-values were determined by unpaired two-tailed student’s t-test. (D) Whole genome view of the enriched peaks at the centromeric and non-centromeric regions in cells treated with 0.02% MMS or mock-treated. (E) Representative snapshot of chromosome XII, showing the peaks in the input and the samples (Scm3 IP). In the sample tracks (MMS-treated or mock-treated), the corresponding input signal is overlapped on the sample signal to clearly show the enrichment at the peaks.
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Figure 3—source data 1
Source data for Figure 3.
- https://cdn.elifesciences.org/articles/104431/elife-104431-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Analysis of non-centromeric binding sites (peaks) of Scm3.
(A)SCM3-6HA cells were grown till mid-log and were treated with MMS or left untreated for 90 min following nocodazole treatment. The cells were harvested and analyzed for DAPI staining and bud morphology to visualize the cell cycle stages. Scale bar = 2 µm. (B) The peak statistics and (C) features within the peaks in MMS-treated and untreated samples from Figure 3. The GO term analysis of the non-centromeric peaks from (D) MMS-treated or (E) untreated samples (X axis: Gene count, Y axis: GO terms). (F) The motifs observed in the non-centromeric peaks from MMS-treated and untreated samples.
Figure 4 with 2 supplements
Scm3 colocalizes with DDR proteins in response to DNA damage.
(A) Representative images showing the localizations of Rad52-6HA and Scm3-13Myc using chromatin spreads. The mid-log grown RAD52-6HA SCM3-13MYC cells were treated with 0.02% MMS or mock-treated for 90 min prior to imaging. (B) Quantification of the colocalization between Rad52-6HA and Scm3-13Myc using Pearson’s correlation coefficient (PCC). (C) Mean values from three independent experiments. (D) Representative images showing the localizations of γ-H2A and Scm3-13Myc using chromatin spreads. The mid-log grown SCM3-MYC cells were treated with 0.02% MMS or mock-treated for 90 min prior to imaging. (E) Quantification of the colocalization between γ-H2A and Scm3-13Myc using PCC. (F) Mean values from three independent experiments. At least 30 spreads from three independent experiments were analyzed for each set in (B) and (E). The p-values were determined by unpaired two-tailed student’s t-test. Scale bar = 2 µm.
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Figure 4—source data 1
Source data for Figure 4.
- https://cdn.elifesciences.org/articles/104431/elife-104431-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Localization of proteins in presence or absence of DNA damage.
(A) Representative images showing the localization of γ-H2A using chromatin spreads. The mid-log grown wild type cells were treated with 0.02% MMS or mock-treated for 90 min prior to imaging. (B) The intensities of γ-H2A after subtracting background intensity were quantified and plotted. (C) Mean values from three independent experiments. (D) Representative images showing the localizations of γ-H2A and Rad52-6HA using chromatin spreads. The mid-log grown RAD52-6HA cells were treated with 0.02% MMS or mock-treated for 90 min prior to imaging. (E) Quantification of the co-localization between γ-H2A and Rad52-6HA was estimated using Pearson’s correlation coefficient (PCC). (F) Mean values from three independent experiments. (G) Representative images showing the localization of γ-H2A and Scm3-13Myc using chromatin spreads after rotating the γ-H2A image by 180° from Figure 4D. (H) Quantification of the co-localization between γ-H2A and Scm3-13Myc was estimated using Pearson’s correlation coefficient (PCC). (I) Mean values from three independent experiments. (J) Representative images showing the localization of γ-H2A and Scm3-13Myc using chromatin spreads. Wild type or sml1∆mec1∆tel1∆ cells were grown till mid-log and treated with 0.02% MMS for the assay. (K) The intensity of Scm3-13Myc subtracting with the background in the indicated strains was quantified and plotted. (L) Mean values from three independent experiments. At least 30 spreads were analyzed for each set in (B), (E), (H), and (K) from three independent experiments. The p values were determined by unpaired two-tailed student’s t-test. Scale bar = 2 µm.
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Figure 4—figure supplement 1—source data 1
Source data for Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/104431/elife-104431-fig4-figsupp1-data1-v1.xlsx
Figure 4—figure supplement 2
Hsf1 does not co-localize with γ-H2A in response to MMS or heat shock.
(A) Representative images showing the localization of Hsf1-13Myc using chromatin spread. (B) Box plots showing the number of Hsf1-13Myc foci per nuclear spread in cells treated at 37 °C for 15 min or untreated. (C) Mean values from three independent experiments. (D) Representative images showing the localization of γ-H2A and Hsf1-13Myc using chromatin spreads. The mid-log grown HSF1-13MYC cells were treated with 0.02% MMS or mock-treated for 90 min or given heat shock at 37 °C for 15 min prior to imaging. (E) Quantification of the co-localization between γ-H2A and Hsf1-13Myc was estimated using Pearson’s correlation coefficient (PCC). (F) Mean values from three independent experiments. At least 30 spreads were analyzed for each set in (B) and (E), from three independent experiments. The p values were determined by unpaired two-tailed student’s t-test. Scale bar = 2 µm.
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Figure 4—figure supplement 2—source data 1
Source data for Figure 4—figure supplement 2.
- https://cdn.elifesciences.org/articles/104431/elife-104431-fig4-figsupp2-data1-v1.xlsx
Figure 5 with 2 supplements
Scm3 is recruited to a site-specific double-stranded break (DSB).
(A) Schematic representation of the HO endonuclease cut site (HOcs) integrated at the URA3 locus in the NA14 strain. Upon galactose induction, the HO endonuclease cleaves the DNA at the specific site, generating a DSB. The positions of the primers (OSB289 and kanB1), used to detect the DSB, are shown along with primers to detect binding of Scm3 near and distal (−1 to –3 kb) to the DSB (B) ChIP analyses were performed to quantify the association of Scm3-6HA in the NA14 strain. The PCR-amplified DNA bands following ChIP were run on an agarose gel and stained with ethidium bromide. (C) ChIP analyses for measuring the association of Scm3-6HA with the indicated loci were performed at the indicated time points of HO induction. The ChIP efficiency as percentage enrichment per input obtained by qPCR analysis of the immunoprecipitated DNA is plotted. (D) The same analysis as in (C) using no antibody (-Ab) control. Error bars represent the standard error of the mean. The p-values in (C) and (D) were determined by two-way ANOVA test followed by Dunnett’s multiple comparison test from two independent experiments.
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Figure 5—source data 1
Original files for gels displayed in Figure 5B.
- https://cdn.elifesciences.org/articles/104431/elife-104431-fig5-data1-v1.zip
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Figure 5—source data 2
Original files for gels displayed in Figure 5B indicating relevant bands.
- https://cdn.elifesciences.org/articles/104431/elife-104431-fig5-data2-v1.zip
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Figure 5—source data 3
Source data for Figure 5.
- https://cdn.elifesciences.org/articles/104431/elife-104431-fig5-data3-v1.xlsx
Figure 5—figure supplement 1
Dynamics of HO-induced DSB formation and protein binding at centromere and DSB sites.
(A) NA14 yeast cells were grown till mid-log in 3% glycerol, following which galactose was added for HO induction. Cells were harvested at indicated time points, and genomic DNA was extracted. PCR was performed to get the band (DSB) to estimate the extent of DNA damage using the primers OSB289 and kanB1, as shown in Figure 4A. PCR was also performed using primers against TUB2 ORF as a loading control band (Tubulin). (B) The intensity of the DSB band normalized with the tubulin band in (A) was quantified using ImageJ software. The value at 0 hr was taken as 100% DSB band remaining (uncut), and accordingly, the % uncut DNA remaining for other time points was calculated. (C) ChIP analyses for measuring the association of Scm3-6HA and Rad51 with the CEN3 locus were performed at the indicated time points of HO induction. (D) ChIP analyses for measuring the association of Scm3-6HA at the CEN3 and TUB2 loci (E) were performed at the indicated time points of HO induction. (F) ChIP analyses for measuring the association of Rad51 with the indicated loci were performed at the indicated time points of HO induction. Error bars represent the standard error of the mean. The p values in (F) were determined by a two-way ANOVA test followed by Dunnett’s multiple comparison test from two independent experiments.
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Figure 5—figure supplement 1—source data 1
Original files for gels displayed in Figure 5—figure supplement 1A.
- https://cdn.elifesciences.org/articles/104431/elife-104431-fig5-figsupp1-data1-v1.zip
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Figure 5—figure supplement 1—source data 2
Original files for gels displayed in Figure 5—figure supplement 1A indicating relevant bands.
- https://cdn.elifesciences.org/articles/104431/elife-104431-fig5-figsupp1-data2-v1.zip
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Figure 5—figure supplement 1—source data 3
Source data for Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/104431/elife-104431-fig5-figsupp1-data3-v1.xlsx
Figure 5—figure supplement 2
DSB repair is delayed in the absence of Scm3.
(A) NA14 yeast cells harboring SCM3-AID degron allele were grown till mid-log in 3% glycerol in the presence of DMSO (wild type), or auxin (Scm3 depleted), following which galactose was added for HO induction. Cells were harvested at the indicated time points, and genomic DNA was extracted. PCR was performed to get the band (DSB) to estimate the extent of DNA damage using the primers OSB289 and kanB1, as shown in Figure 4A. PCR was also performed using primers against TUB2 ORF as a loading control band (Tubulin). (B) The intensity of the DSB band normalized with the tubulin band in (A) was quantified using ImageJ software. The value at 0 hr was taken as 100% DSB band remaining (uncut), and accordingly, the % uncut DNA remaining for other time points was calculated.
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Figure 5—figure supplement 2—source data 1
Original files for gels displayed in Figure 5—figure supplement 2A.
- https://cdn.elifesciences.org/articles/104431/elife-104431-fig5-figsupp2-data1-v1.zip
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Figure 5—figure supplement 2—source data 2
Original files for gels displayed in Figure 5—figure supplement 2A indicating relevant bands.
- https://cdn.elifesciences.org/articles/104431/elife-104431-fig5-figsupp2-data2-v1.zip
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Figure 5—figure supplement 2—source data 3
Source data for Figure 5—figure supplement 2.
- https://cdn.elifesciences.org/articles/104431/elife-104431-fig5-figsupp2-data3-v1.xlsx
Figure 6 with 1 supplement
Evidence of a DNA damage checkpoint defect in the absence of Scm3.
(A) Schematic of the experimental strategy to detect Rad53 in Scm3- or Cdc20-depleted cells treated with MMS for different time points. (B) Western blots from indicated cells according to the protocol in (A). (C) mad2∆, SCM3-AID, and SCM3-AID mad2∆ cells were first treated with 0.75 mM auxin for 2 hr to deplete Scm3 and then either treated with 0.02% MMS for 90 min or left untreated. The cells were harvested and scored for the presence of multibudded cells (shown on the left). At least 150 cells from three independent experiments were analyzed for each set. Error bars represent the standard error of the mean. The p-values were determined by a one-way ANOVA test followed by Tukey’s multiple comparison test. Scale bar = 2 µm. (D) Schematic of the experimental strategy to detect Pds1 in mad2∆ SCM3-AID cells in the presence of DMSO or auxin (Scm3-depleted) and each treated with 0.02% MMS for different time points. (E) Western blot showing Pds1 and TIR1 (as loading control). Cells were harvested following the strategy given in (D). (F) The intensities of the Pds1 measured by ImageJ were plotted after normalizing with the corresponding loading control (TIR1). (G) The percentage of anaphase cells judged by DAPI staining and bud morphology for each time point of cells harvested in (D) is presented graphically.
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Figure 6—source data 1
Original files for western blot analysis displayed in Figure 6B and E.
- https://cdn.elifesciences.org/articles/104431/elife-104431-fig6-data1-v1.zip
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Figure 6—source data 2
Original files for western blot analysis displayed in Figure 6B and E indicating relevant bands.
- https://cdn.elifesciences.org/articles/104431/elife-104431-fig6-data2-v1.zip
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Figure 6—source data 3
Source data for Figure 6.
- https://cdn.elifesciences.org/articles/104431/elife-104431-fig6-data3-v1.xlsx
Figure 6—figure supplement 1
H2A phosphorylation is not perturbed in the absence of Scm3.
(A) The schematic of the experimental strategy that was followed for detecting Rad53 in SCM3-AID cells in the presence of DMSO (wild-type) or auxin (Scm3 depleted) and each either treated with MMS or left untreated. (B) Western blots showing p-Rad53/Rad53 and tubulin (as loading control) from indicated cells obtained from (A). (C) Loading control normalized intensities of the p-Rad53 were quantified and plotted. Error bars represent the standard error of the mean. The p-values were determined by an unpaired t-test. (D) CDC20-AID or SCM3-AID strains were grown till mid-log and treated with 0.75 mM auxin for depletion of Cdc20 or Scm3, respectively. The cells were then treated with MMS for 240 min, and samples were harvested at the indicated time points. The percentage of metaphase cells judged by DAPI staining and bud morphology for each time point is presented graphically. (E) The percentage of multibudded cells in the samples harvested in (D) is presented. (F) Western blots showing γ-H2A and tubulin (as loading control). Cells were harvested following the strategy given in Figure 6A.
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Figure 6—figure supplement 1—source data 1
Original files for western blot analysis displayed in Figure 6—figure supplement 1B, F.
- https://cdn.elifesciences.org/articles/104431/elife-104431-fig6-figsupp1-data1-v1.zip
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Figure 6—figure supplement 1—source data 2
Original files for western blot analysis displayed in Figure 6—figure supplement 1B, F indicating relevant bands.
- https://cdn.elifesciences.org/articles/104431/elife-104431-fig6-figsupp1-data2-v1.zip
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Figure 6—figure supplement 1—source data 3
Source data for Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/104431/elife-104431-fig6-figsupp1-data3-v1.xlsx
Figure 7 with 1 supplement
Post-translational modification of Scm3 in response to DNA damage.
(A) Left, western blot showing Scm3-6HA and its modified form (Scm3*) along with tubulin as loading control from the cells untreated or treated with indicated concentrations of MMS for 90 min. Middle, intensity of Scm3* with increasing MMS concentrations, normalized to tubulin. Right, the ratio of the modified to unmodified forms of Scm3. Error bars represent the standard error of the mean obtained from two independent experiments. The p-values were determined by a one-way ANOVA test followed by Dunnett’s multiple comparison test. (B) Schematic of the experimental strategy that was followed for detecting Scm3-6HA in G1 arrested cells and following their release from the arrest in the absence or presence of MMS. (C) Representative images showing the cell cycle stages by DAPI staining and bud morphology at indicated time points after release from G1 arrest. (D) Western blot showing Scm3-6HA along with tubulin as a loading control from the cells harvested, as shown in (B). The cell lysates were probed using anti-HA and anti-tubulin antibodies to detect Scm3-6HA and tubulin, respectively. Scale bar = 2 µm.
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Figure 7—source data 1
Original files for western blot analysis displayed in Figure 7A and D.
- https://cdn.elifesciences.org/articles/104431/elife-104431-fig7-data1-v1.zip
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Figure 7—source data 2
Original files for western blot analysis displayed in Figure 7A and D indicating relevant bands.
- https://cdn.elifesciences.org/articles/104431/elife-104431-fig7-data2-v1.zip
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Figure 7—source data 3
Source data for Figure 7.
- https://cdn.elifesciences.org/articles/104431/elife-104431-fig7-data3-v1.xlsx
Figure 7—figure supplement 1
Phosphorylation of Scm3 in response to DNA damage.
(A) Left, western blots showing Scm3-6HA and its modified form (Scm3*) along with tubulin as loading control from SCM3-6HA cells untreated or treated with 0.02% MMS for indicated time points. Middle, the graph showing the intensity of Scm3* with increasing duration of MMS treatment, normalized with respect to tubulin using ImageJ software. Right, the graph represents the ratio of the modified to unmodified forms of Scm3. (B, C) bar1∆ SCM3-HA cells were released from G1 arrest by alpha factor into medium in the absence (B) or presence (C) of MMS. The cell cycle stages of the cells at the indicated time points were determined using DAPI staining and bud morphology and are graphically shown. (D, E) SCM3-6HA cells were treated with the indicated concentration of MMS for 90 min, and total protein was extracted. The proteins were run on 10% SDS-PAGE without (D) or supplemented with 50 µm Phos-Tag reagent (E). The Scm3 band under untreated conditions (black dotted line) gradually shifts upwards with increasing concentration of MMS in the presence of Phos-tag reagent, shown by a basal black line. Two red dotted lines show an increase in the spread of Scm3* in the 0.1% MMS lane as compared to the same shown by two blue lines in the untreated lane. (F) SCM3-6HA cells were treated with MMS or left untreated for 90 min, and total protein was extracted. From the total protein, the immunoprecipitate with anti-HA antibodies was incubated in the presence or absence of alkaline phosphatase (AP) for 3 hr at 37 °C. The proteins were eluted from the beads, and immunoblotting was performed using the same antibodies. (G) Western blots showing Scm3-6HA and its modified form (Scm3*) in the cell lysates from the indicated strains untreated or treated with 0.02% MMS for 90 min. Error bars in (A) represent the standard error of the mean obtained from two independent experiments. The p-values were determined by a one-way ANOVA test followed by Dunnett’s multiple comparison test.
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Figure 7—figure supplement 1—source data 1
Original files for western blot analysis displayed in Figure 7—figure supplement 1A, D, E, F, G.
- https://cdn.elifesciences.org/articles/104431/elife-104431-fig7-figsupp1-data1-v1.zip
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Figure 7—figure supplement 1—source data 2
Original files for western blot analysis displayed in Figure 7—figure supplement 1A, D, E, F, G indicating relevant bands.
- https://cdn.elifesciences.org/articles/104431/elife-104431-fig7-figsupp1-data2-v1.zip
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Figure 7—figure supplement 1—source data 3
Source data for Figure 7—figure supplement 1.
- https://cdn.elifesciences.org/articles/104431/elife-104431-fig7-figsupp1-data3-v1.xlsx
Figure 8
A working model summarizing the role of Scm3 in the DNA damage response (DDR).
Left, in wild-type cells, a subset of Scm3 present in the nucleus (green) is phosphorylated upon DNA damage. The phosphorylated Scm3 (p-Scm3) associates with DSB sites, colocalizes with γ-H2A, and spreads to chromatin flanking the DSB site. Possibly, p-Scm3 facilitates proper activation of Rad53 through its phosphorylation, leading to cell cycle arrest. Additionally, p-Scm3 co-localizes with Rad52 and assists in DSB repair. Right, in Scm3-depleted cells, there is an increase in endogenous DNA damage, corresponding to more Rad52 foci, likely due to the inability to repair DSBs, and cell viability drops. In parallel, reduced Rad53 activation causes improper DDR-mediated cell cycle arrest, generating multibudded cells and possibly aneuploid cells, leading to cell death. Phenotypes associated with the loss of Scm3 are shown in red. Graphical elements in the figure are partially adapted from the Servier Medical Art Repository (https://smart.servier.com), licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/).
Author response image 1
The schematic of the experimental strategy to be followed to elucidate the role of Scm3 in DNA damage checkpoint activation.
Author response image 2
The percentage of wild type (SCM3-AID) and Scm3 depleted (SCM3-AID+Auxin) cells harboring indicated numbers of Rad52-GFP foci upon treatment with 0.
02% MMS.
Tables
Author response table 1
The table summarising the occupancy of Scm3 in untreated or MMS treated conditions at the indicated regions.
| Region | Chromosome | Scm3 occupancy | |
|---|---|---|---|
| Untreated | MMS treated | ||
| Rrm3 binding sites | Chr II | YES | |
| Chr X | YES | ||
| Chr XII | YES | ||
| Chr XII | YES | YES | |
| Chr XIV | YES | YES | |
| LTRs | Chr XVI | YES | |
| Chr XVI | YES | ||
| tRNA | Chr XV | YES | |
| TERs | Chr IV | YES | |
| Chr V | YES | ||
| Chr VI | YES | YES | |
| Chr VII | YES | ||
| Chr X | YES | ||
| Chr X | YES | ||
| Chr XIV | YES | YES | |
| Chr XV | YES | YES | |
| Chr XVI | YES | ||
| Pol II binding sites | Chr II | YES | |
| Chr X | YES | ||
| Chr XII | YES | ||
| Chr XII | YES | ||
| Chr XV | YES |
Additional files
-
Supplementary file 1
Supplementary file related to main article file.
(A) List of strains used in this study. (B) List of primers used in ChIP-qPCR.
- https://cdn.elifesciences.org/articles/104431/elife-104431-supp1-v1.docx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/104431/elife-104431-mdarchecklist1-v1.pdf
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Evidence of centromeric histone 3 chaperone involved in DNA damage repair pathway in budding yeast
eLife 14:e104431.
https://doi.org/10.7554/eLife.104431
