EMBO/SourceData huggingface dataset:
Figure 5Superose 6 gel filtration elution profile of His-MBP-IHO11-281 mixed with Strep-REC114-His-MEI41-127. Overlay with individual elution profiles of His-MBP-IHO11-281 and the Strep-REC114-His-MEI41-127 complexSDS-PAGE analysis of fraction 1-13 of the Superose 6 gel filtration elution profile shown in A. L indicates input sample­­ loaded onto the column.Superose 6 gel filtration elution profile of His-MBP-IHO124-281 mixed with Strep-REC114-His-MEI41-127, showing lack of binding. Overlay with individual elution profiles of His-MBP-IHO124-281 and the Strep-REC114-His-MEI41-127 complexSDS-PAGE analysis of fraction 1-13 of the Superose 6 gel filtration elution profile shown in C. L indicates input sample­­ loaded onto the column.ITC measurement of the interaction affinity between of His-MBP-IHO11-281 and Strep-REC114-His-MEI41-127. ITC measurement of the interaction affinity between of His-MBP-IHO124-281 and Strep-REC114-His-MEI41-127 ITC measurement of the interaction affinity between of His-MBP-IHO11-23 and Strep-REC114-His-MEI41-127.
Figure 6ITC measurement of the interaction affinity between of His-MBP-IHO11-23 and the L104D mutant of Strep-REC1141-159.SDS-PAGE analysis of fractions 1-13 of Superose 6 gel filtration elution profiles of His-MBP-IHO11-281 mixed with His-MBP-ANKRD311808-1857, Strep-REC114 and His-MEI41-127 (upper panel); mixture of His-MBP-ANKRD311808-1857 with Strep-REC114-His-MEI41-127 (middle panel) and His-MBP-IHO11-281 mixed with Strep-REC114 and His-MEI41-127 (lower panel). L indicates input sample loaded onto the column. The red frame highlights the complex of IHO1 bound to REC114 and MEI4 (lower panel) and the lack of this complex in presence of ANKRD31 (upper panel). Orange square shows equivalent elution of ANKRD31, REC114 and MEI4 in presence or absence of IHO1.Superose 6 gel filtration elution profiles of His-MBP-IHO11-281 mixed with His-MBP-ANKRD311808-1857, Strep-REC114 and His-MEI41-127 (orange); His-MBP-ANKRD311808-1857, Strep-REC114 and His-MEI41-127 (blue) and His-MBP-IHO11-281 mixed with Strep-REC114 -His-MEI41-127 (green). Superose 6 gel filtration elution profiles of His-MBP-IHO11-281 mixed with His-MBP-ANKRD311808-1857, Strep-REC114 and His-MEI41-127 (orange); His-MBP-ANKRD311808-1857, Strep-REC114 and His-MEI41-127 (blue) and His-MBP-IHO11-281 (green).
Figure 1(B, Characterization of DONSON's chromatin association. Sperm nuclei (5,000/μL) were incubated for the indicated times in 20 μL of interphase egg extract. Chromatin fractions were isolated and analyzed by immunoblotting. His-p27 (100 μg/mL, p27 (CDKi)), PHA-767491 (50 μM, Cdc7i) were supplemented as indicated. Similar experiments with (B) were performed in triplicate and DONSON signal intensity was quantified by Image J.C, Characterization of DONSON's chromatin association. Sperm nuclei (5,000/μL) were incubated for the indicated times in 20 μL of interphase egg extract. Chromatin fractions were isolated and analyzed by immunoblotting. aphidicolin (10 μg/mL, aph), His-geminin (50 μg/mL, gem) were supplemented as indicated. Similar experiments with (C) were performed in triplicate and DONSON signal intensity was quantified by Image J.E) Characterization of DONSON's chromatin association. Sperm nuclei (5,000/μL) were incubated for the indicated times in 20 μL of interphase egg extract. Chromatin fractions were isolated and analyzed by immunoblotting. NMS-873 (100 μM, p97i), and MLN-4924 (20 μM, Culi) were supplemented as indicated.
Figure 2(A) Immunodepletion from the interphase egg extract was performed using control IgG beads (mock) or anti-DONSON antibody beads (ΔDNS). Recombinant Xenopus DONSON (untagged) was added to the mock- and DONSON-depleted extracts at 0 μM (-), 0.06 μM (×1), 0.18 (×3), and 0.36 μM (×9) as indicated. The extracts (1 μL) were analyzed by immunoblotting, except lanes 9-11, where 0.4 (.4), 0.2 (.2), and 0.1 (.1) μL were used. Since the anti-DONSON antibody was recognized by multiple bands, the presumable DONSON band was indicated by dotted lines in a vertically expanded blot. Asterisk (*), non-specific band.(B) DONSON requirement for the replisome assembly. Sperm nuclei (5,000/μL) were incubated in 20 μL of the mock- and DONDON-depleted extracts as prepared in (A) for 60 min in the presence of 10 μg/mL aphidicolin. The extracts (0.5 μL) and isolated chromatin fractions were analyzed by immunoblotting.(C) DONSON requirement for replisome assembly. Sperm nuclei were incubated for the indicated times and conditions in the egg extract and the chromatin fractions were analyzed by immunoblotting. Each of two types of anti-DONSON antibodies (α-DNS #1, α-DNS #2) was added at 14 μg/mL to suppress DONSON function.
Figure 3(A) DONSON requirement for DNA replication. Sperm nuclei were incubated for 60 min in egg extract with no IgG (non), control IgG (α-control), or one of the two kinds of anti-DONSON antibodies (α-DNS #1, α-DNS #2) in a final concentration of 14 μg/mL. Cy3-dCTP was added to all of the reactions at 1 μΜ to visualize the DNA replication. After incubation, the nuclei were fixed, stained with Hoechst 33258, and observed under a fluorescent microscope. Representative images are shown above. Scale bar; 50 µm. The relative Cy3 intensity of >100 nuclei was quantified using Image J and is shown in the box plot below. The boxes define the upper and lower quartiles; the whiskers define max to min values; the X-mark indicates each mean value; the central band indicates each median; the circles indicates outliers. P-values were calculated using the Mann-Whitney test. ns, not significant.(B) DONSON requirement for DNA replication. Sperm nuclei (5,000/μL) were incubated for 60 min in 10 μL of the untreated (control), mock- (mock), and DONDON-depleted (ΔDNS) extracts with 2 μM Cy5-dUTP. Aphidicolin (aph) was added to the mock-depleted extract (2.5 μg/mL) as a negative control. Various versions of the recombinant Xenopus DONSON (1-6) were added to the DONSON-depleted extracts at about 0.06 μM. 1; untagged DONSON, 2 and 3; GST-DONSON, 4 and 5; GST-DONSON (residues 155-579) (ΔN154), 6; His-DONSON. In the reactions corresponding to lanes 3 and 5, 3C protease was added to separate the GST from DONSON. After incubation, the genomic DNA was isolated and subjected to 0.8% TAE agarose gel electrophoresis, followed by SYBR Gold staining. The Cy5 and SYBR Gold fluorescent signals show replicated DNA and total DNA, respectively. The same experiment was performed in triplicate; the relative Cy5 intensities to the control reaction were quantified using Image J, as shown in the bar graph below. Error bar, mean ± standard deviation (S.D.). P-values were calculated using the unpaired t-test (two-tailed). ns, not significant.
Figure 4(A) Salt-sensitivity of chromatin-bound DONSON. Sperm nuclei (5,000/μL) were incubated in 20 μL of egg extract under each of the conditions as indicated, and the chromatin fractions were isolated with normal buffer plus 0 (-), 0.1, or 0.2 M NaCl, and analyzed by immunoblotting. In addition, 0.5 μL of each extract (ext) was analyzed for comparison.(C) The chromatin fractions were isolated and analyzed by immunoblotting.(E) (left) The isolated genomic DNA was analyzed in the same manner as in Figure 3B. (right) The same experiment was repeated six times. The relative Cy5 intensities to the control reaction (normal α-C) were quantified using Image J, as shown in the bar graph. Error bar, mean ± standard deviation (S.D.). P-values were calculated using the unpaired t-test (two-tailed). ns, not significant.
Figure 5(A) Ability of DONSON deletions to support replisome assembly. The DONSON depletion experiment, as that in Figure 2B, was performed using 3×FLAG-tagged versions of the full length (amino acid residues 1-579) or N-terminal/C-terminal deletions (amino acid residues 1-157, 133-579, 1-476, 1-369) of Xenopus (x) DONSON, whose final concentrations were about 0.06 μM. The signals derived from DONSON are indicated with dotted lines. His-p27 and His-geminin were used as negative controls. Asterisk (*), non-specific bands. (B) Interaction of DONSON with the replication factors. 3×FLAG-tagged full length (xF), N-terminal region residue 1-157 (xN), middle region residue 133-369 (xM), and C-terminal region residue 358-579 (xC) versions of Xenopus DONSON were added to an interphase egg extract at about 8.8 μM. After incubation for 15 min, the whole extract was subjected to a pull-down assay using FLAG-M2 beads, and the precipitated proteins were analyzed by immunoblotting (FLAG-IP). The extracts (2.5%) were analyzed for comparison (Input).(D) Identification of GINS- and Polε-interacting motifs in the N-terminal region of DONSON. The FLAG-IP experiment, as in (B), was performed with 2 μM of 3×FLAG-tagged versions of wild-type (xN-WT), P6A/Y8A (xN-2A), N67A/P68A/F69A (xN-3A), and P6A/Y8A/N67A/P68A/F69A (xN-5A) of Xenopus DONSON each with an N-terminal with 157 amino acids. The extracts (6.7%) were analyzed for comparison (Input). (E) Interaction of human DONSON with Xenopus GINS and Polε in egg extract. The FLAG-IP experiment, as in (B), was performed with 1 μM of 3×FLAG-tagged versions of wild-type (hF-WT), P6A/Y8A (hF-2A), N78A/P79A/F80A (hF-3A), P6A/Y8A/N78A/P79A/F80A (hF-5A), K489T (hF-KT) of full length human DONSON each as well as 3×FLAG-tagged wild-type Xenopus DONSON (xF-WT). The extracts (5%) were analyzed for comparison (Input).
Figure 6(A) Requirement of GINS- and Polε-interacting motifs within DONSON for replisome assembly. This DONSON depletion experiment, as that in Figure 2B, was performed using the same recombinant proteins as in Figure 5E. Human proteins were added at 0.3 μM each and Xenopus protein at 0.06 μM. His-p27 (p27) and His-geminin (gem) were used as negative controls.(B) Requirement of GINS- and Polε-interacting motifs within DONSON for replication activity. The same experiment procedure was used as in Figure 3A for the mock- and DONSON-depleted extracts with the same recombinant proteins as in Figure 5E. Xenopus proteins were added at 0.06 μM each while human proteins were added at 0.3 μM. The relative Cy3 intensity of >100 nuclei was quantified using Image J and is shown in the box plot. The boxes define the upper and lower quartiles; the whiskers define max to min values; the X-mark indicates each mean value; the central band indicates each median; the circles indicates outliers. P-values were calculated using the Mann-Whitney test. ns, not significant.
Figure 7(A, Requirement of GINS, Cdc45 for the DONSON chromatin association. Sperm nuclei (5,000/μL) were incubated in 20 μL of mock-, GINS-, Cdc45- depleted egg extract ΔGINS, ΔCdc45 for the indicated times and conditions, and the chromatin fractions were isolated and analyzed by immunoblotting. The extracts (0.5 μL) were also analyzed. Replication proteins were categorized into three groups (Group-1, -2, -3) based on their dependency on GINS, Cdc45B) Requirement of TopBP1 for the DONSON chromatin association. Sperm nuclei (5,000/μL) were incubated in 20 μL of mock-, or TopBP1- depleted egg extract , ΔTopBP1) for the indicated times and conditions, and the chromatin fractions were isolated and analyzed by immunoblotting. The extracts (0.5 μL) were also analyzed. Replication proteins were categorized into three groups (Group-1, -2, -3) based on their dependency on TopBP1.(C) (above) Requirement of DONSON for CDK and DDK function in the replisome assembly. Sperm nuclei (5,000/μL) were incubated for 50 min in 120 μL of egg extract with control IgG (α-cont) or anti-DONSON antibody #1 (α-DNS). Each part of this first reaction (1st) was divided equally into six parts, and the nuclear fractions were isolated and transferred to 20 μL of flesh extract with control IgG (α-cont) or anti-DONSON antibody #1 (α-DNS) in the presence or absence of CDK inhibitors (Ci: 100 μM roscovitine, 100 μg/mL His-p27) and Cdc7 inhibitor (7i: 50 μM PHA-767491). Aphidicolin was added to the first and second extracts at 10 μg/mL to suppress replication termination. After incubation for 40 min in the second reaction (2nd), the chromatin fractions were isolated and analyzed by immunoblotting. In addition, the extracts (0.5 μL) were analyzed (ext). (below) It is summarized whether or not replisome assembly was allowed under each condition, where DONSON, CDK, and Cdc7 were either functional (+) or non-functional (-) in the 1st and 2nd incubations.
Figure 1B. Quantification of time-course nucleosome H2A-Ub assays using the indicated BRCA1/BARD1 truncations. Data are presented as the normalized fraction of H2A consumed during a time-course nucleosome ubiquitylation assay. Data points represent mean values and error bars are ± 1-s.d. of n=3 independent technical replicate experiments. The residue bounds of BRCA1RING are 1-112, and BARD1RING are 26-140. Additional details about all reagents used in these studies can be found in the methods section.C. Nucleosome binding curves from fluorescence-quenching-based measurements using the indicated BRCA1/BARD1 constructs. Data show mean values and error bars are ± 1-s.d. of n=4 independent technical replicate experiments. The reported difference in affinities to BRCA1RING/BARD1RING is likely underestimated, as the minimal RING/RING heterodimer binding data was collected at a lower ionic strength than the other constructs to obtain well-behaved binding data (50 mM vs. 100 mM NaCl).
Figure 2B. 1H15N-HSQC NMR amide chemical shift perturbations (CSPs) observed for 15N-BARD1 141-216 signals when bound to a 36-bp dsDNA fragment (1:1 molar equivalent complex).C. Representative SDS-PAGE gel monitoring in-gel fluorescence (labelled DNA) of UV-induced Bpa crosslinking of the E3 ligase fusion BRCA1-ƒ-BARD1221 (BCƒBD) to nucleosomes (left). In this construct, the C-terminus of BRCA11-104 is genetically fused to the N-terminus of BARD126-221 via a 12-residue GlySer-linker. Quantification of Bpa crosslinking experiments with nucleosomes (right). The intensity of each crosslinked band was normalized to the intensity of the L120Bpa crosslinked band for each replicate experiment. Graph bars show the mean; error bars are ± 1-s.d. and the open circles are the values of individual replicates for of n=3 independent technical replicate experiments.
Figure 3Quantification of H2A-Ub time-course assays using the indicated BRCA1/BARD1 constructs. Data show the mean; error bars are ± 1-s.d. of n=3 independent technical replicate experiments.Two single time-points from panel C from the BRCA1RING/BARD1RING curve were excluded from analysis due to their extremely low activities coupled with quenching/loading errors yielding normalized fraction of H2A consumed values of <0. Values for these excluded points are included as source data.
Figure 4C. Inhibition of UV-induced Bpa crosslinking between the indicated BRCA1-f-BARD1221 Bpa-incorporated constructs and NCP185 substrates in the presence of increasing amounts of dsDNA or bubble-DNA competitor. Data show the mean; error bars are ± 1-s.d. of n=3 (W146Bpa) or n=4 (A195Bpa) independent technical replicate experiments.D. 1H15N-HSQC NMR CSPs observed to 15N-BARD1 141-216 signals when bound to a 36-mer dsDNA (blue bars) or bubble-DNA (gray bars with black outlines) fragment (1:1 molar equivalent complex). Signals for residues 143-157 are broadened beyond detection in the bubble-DNA-bound spectrum; for visualization purposes, gray bars are set equal to the CSP value for the W146Ne CSP that was observed; the real CSP values are likely larger that this valueF. Single time-point H2A-Ub inhibition assays using heterodimers containing BRCA1RING/BARD1FL (left, 12 min endpoint) or the indicated BARD1 internal deletion mutant (middle and right, 20 min endpoint) and increasing amounts of dsDNA or bubble-DNA competitor. The same E3 concentration (50 nM) was used for each BRCA1/BARD1 truncation. A longer time point was used for the double-deletion mutant as the intrinsic H2A-Ub activity of this mutant was lower, likely due to the deletion of DNA binding regions.
Figure 5A. Western blot analysis to detect HA-BARD1 in nuclei isolated from HeLa-shBARD1 cells stably expressing wild-type or Δ194-216 mutant of HA-BARD1, where endogenous BARD1 was depleted by doxycycline-induced shBARD1 expression. The nuclei were salt-fractionated into NS100, NS300, NS420, and NP420 to assess the amount of HA-BARD1 associated with chromatin. Data are representative of n=2 biological replicate experiments.B. Quantification of cells with >5 HA-BARD1 foci (left panel) and number of HA-BARD1 foci per nucleus (right panel) with and without olaparib treatment. *p<0.05, **p<0.01, ***p<0.001, by Student's t-test. Data show the individual points from n=3 biological replicate experiments.C. Clonogenic survival of HeLa-shBARD1 cells stably expressing wild type or Δ194-216 mutant of HA-BARD1 upon treatment with indicated amount of olaparib and cisplatin. Data points show the mean; error bars represent SEM of n=3 biological replicate experiments.
Figure 6B, C. Quantification of time-course H2A-Ub assays using the indicated BRCA1/BARD1 constructs and chromatin substrates. data show the mean; error bars are ± 1-s.d. of n=3 independent technical replicate experiments.D. Quantification of time-course H2A-Ub assays using homogenously modified H2A K15-Ub mono-nucleosome substrates and indicated BRCA1/BARD1 constructs. data show the mean; error bars are ± 1-s.d. of n=3 independent technical replicate experiments. One time-point from Panel D was determined to be an outlier due to a reaction quenching error and excluded from analysis. The excluded value is reported as source data.G, H. Quantification of time-course H2A-Ub assays using the indicated combinations of di-NCP substrate and BRCA1/BARD1 heterodimer. In each case, the y-axis reports on the fraction of H2Aobserve (as shown in Panel F). Lower E3 concentrations were used in these reactions than in those presented in Figure 1 using unmodified mono-nucleosome substrates (15 nM vs. 100 nM), accounting for the slower observed H2A-Ub kinetics for the unmodified di-nucleosome substrate. data show the mean; error bars are ± 1-s.d. of n=3 independent technical replicate experiments.
Figure 2A-B: Proximity ligation assay (PLA) of association of dynamin-2 with ERBB4 in primary mouse cardiomyocytes treated with either DMSO or the dynamin inhibitor dynasore for 5 hours. Panel A depicts a z projection of a stack of confocal images. PLA interactions are shown in red, nuclear stain DAPI in blue. Panel B depicts quantification of the data. One dot in the boxplot corresponds to the number of PLA signals in µm2 in one cell (Control: n = 6, DMSO buffer: n=11, dynasore: n=11 ; technical replicates from one of two biological replicate experiments). Non-parametric Kruskal-Wallis ANOVA and the Dunn's multicomparison test with multiple test correction was used for statistics. Scale bar 20µm. Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.G: Real-time RT-PCR analysis of expression of the indicated STAT5b target genes in primary mouse cardiomyocytes treated for 1-3 hours with the DMSO buffer, the ERBB kinase inhibitor AG1478, or dynasore, and stimulated or not for 30 minutes with NRG-1. One dot corresponds to the mean of technical repeats in one experiment and the whiskers the standard deviation (for each group in Igf-1 and Cdkn1a panel: n = 6, for each group expect the dynasore group in Myc panel: n=4, for the dynasore group in Myc panel: n=3 ; biological replicate experiments). One-way ANOVA and the Dunnett's multicomparison test with multiple test correction was used for statistics. *, P < 0.05; ** P < 0.01; ***, P < 0.001; against DMSO + NRG-1 treatment (Igf-1 panel: DMSO without NRG-1 P = 0.0041, AG1478 P = 0.0082, dynasore P = 6.61e-06; Myc panel: DMSO without NRG-1 P = 0.0386, AG1478 P = 0.0037, dynasore P = 0.0064; Cdkn1a panel: DMSO without NRG-1 P = 0.0123, AG1478 P = 0.0064, dynasore P = 0.0092). Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.H-I: Confocal images (H) and quantification (I) of immunofluorescence staining of myosin heavy chain in primary mouse cardiomyocytes. The cells were treated with either DMSO or dynasore and stimulated with NRG-1 for two days. The myosin immunoreactivity was used to quantify cell area. One dot in the boxplot corresponds to one cell (DMSO: n = 22, dynasore: n = 28; technical replicates combined from three biological replicate experiments). Non-parametric Mann-Whitney U-test was used for statistics. Scale bar 20µm. Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.
Figure 3Confocal images (A and quantification (B, of immunofluorescence staining of myosin heavy chain (green) and DAPI (blue) in the hearts of 4 dpf zebrafish embryos injected with either the buffer control or NRG-1 into the pericardial sac at 2 dpf. The myosin immunoreactivity was used to quantify both the average thickness of the ventricular wall as well as the cross-sectional area of the ventricles. The number of nuclei was estimated with the DAPI stain. The amount nuclei (red) in the ventricle and the ventricle area (yellow) was quantified to differentiate between hypertrophic and hyperplastic growth. One dot in the boxplots corresponds to one heart (Control (A-B): n = 10, NRG-1 (A-B): n = 7 biological replicates from two replicate experiments). Unpaired two-tailed T-test was used for statistics. Scale bars 20µm. Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.Western analysis of Stat5, Akt and Erk phosphorylation in zebrafish embryos. The embryos were treated as in A. (Control: n = 15, NRG-1: n = 16; biological replicates combined from three replicate experiments).densitometric quantification of Stat5, Akt and Erk phosphorylation in zebrafish embryos. The embryos were treated as in A. One dot in the boxplots corresponds to the relative densitometric value of one pooled sample of 3-4 zebrafish embryos (Control: n = 15, NRG-1: n = 16; biological replicates combined from three replicate experiments). Unpaired two-tailed T-test with Welch's correction (pStat5) or unpaired two-tailed T-test (pErk, pAkt) was used for statistics. Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.