Journal: Molecular Cell

Article Title: Histone Methylation by SETD1A Protects Nascent DNA through the Nucleosome Chaperone Activity of FANCD2

doi: 10.1016/j.molcel.2018.05.018

Figure Lengend Snippet:

Article Snippet: BLM , Bethyl Labs , Cat# A300-110A; RRID: AB_2064794.

Techniques: Virus, Recombinant, In Situ, Mass Spectrometry, Control, Luciferase, Negative Control, Software

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: a, Enrichment of Gene Ontology (GO) terms (dot plot) in genes regulated by 3’UTR-APA upon depletion of Sam68 or XRN2. Dot size and color indicate the number of genes and statistical significance (Fisher’s exact test, two-sided), respectively, b, Cytometric analyses showing DNA content versus BrdU incorporation upon stable depletion of Sam68 (sh-Sam68) and XRN2 (sh-XRN2) in LNCaP cells. The bar graph shows the percentage of BrdU-positive (S phase) cells, c. Percentage (mean + s.d.) of BrdU-positive LNCaP cells described in b at the indicated time points after release from G1/S synchronization. d,e, Western blot (d) and qPCR (e) analyses of MCM10 and ORC2 expression level in sh-Sam68 and sh-XRN2 LNCaP cells (n = 3). f, PCR strategy used to evaluate 3’UTR-APA isoforms distribution on a 15–50% sucrose gradient, g, sqPCR analysis of the indicated p-pA and d-pA isoform abundance within the polysomal and non-polysomal fractions obtained from sucrose gradient. The graphs show the densitometric analysis of the band signal in each fraction, expressed as a percentage of that detected in all fractions, h, Relative luciferase activity (Renilla/Firefly ratio) of long and short MCM10 3’UTR in LNCaP cells. i, Representative western-blot analysis (n = 3) of the indicated proteins performed in LNCaP cells depleted for the indicated genes, j, Cytometric analyses showing DNA content versus BrdU incorporation in control (si-scr), si-MCMlO and si-ORC2 LNCaP cells. The bar graph shows the percentage of S-phase BrdU-positive cells, k, Kaplan-Meier curves comparing progression-free survival of494 patients with PC (Prostate Adenocarcinoma, TCGA, PanCancer Atlas; https://www.cbioportal.org) stratified according to MCM10 (right), ORC2 (middle) and MCM10/ORC2 (left) expression level. I, Schematic model showing the impact of the functional interaction between Sam68 and XRN2 on cell cycle regulation. The Sam68/XRN2 complex promotes 3’UTR shortening of cell cycle-related genes, increasing their mRNA translation efficiency and cell proliferation. Conversely, Sam68/XRN2 knockdown induces 3’UTR lengthening, reduces translation efficiency of transcripts and causes cell cycle arrest. In b, e, h and j, the bar graphs represent the mean + s.d. In b, c, e, g, h and j, statistical significance was calculated by unpaired Student’sf-test, two-sided (n = 3; *P < 0.05, **P < 0.01,***P < 0.001; NS, not significant; exactPvalues are reported in the source data). In d and I, β-actin was used as loading control.

Article Snippet: The following antibodies were used: Sam68 (Bethyl Laboratories, cat. no. A302-110A), XRN2 (Bethyl Laboratories, cat. no. A301-103A), MYC (Cell Signaling, cat. no. 9402), β-actin (Merck, Sigma-Aldrich, cat. no. A2066), CPSF160 (Bethyl Laboratories, cat. no. A301-580A), CPSF100 (Novus, cat. no. NB100-79823), CPSF73 (Bethyl Laboratories, cat. no. A301-091A), CPSF30 (Novus, cat. no. NB100-79826), WDR33 (Bethyl Laboratories, cat. no. A301-152A), CFIM68 (Bethyl Laboratories, cat. no. A301-358A), CSTF50 (Bethyl Laboratories, cat. no. A301-250A), CSTF64 (Bethyl Laboratories, cat. no. A301-092A), PCF11 (Bethyl Laboratories, cat. no. A303-706A), POLR2A (Cell Signaling, cat. no. 14958), H3 (Abcam, cat. no. ab1791), MCM10 (Bethyl Laboratories, cat. no. A300-131A), ORC2 (Bethyl Laboratories, cat. no. A302-734A), Lamin B1 (Santa Cruz, cat. no. sc-30264), GFP (Santa Cruz, cat. no. sc-9996), Flag (Merck, Sigma-Aldrich, cat. no. F3165), BrdU (BD Biosciences, cat. no. 347580) and Alexa-488 (Thermo Fisher Scientific, cat. no. A-11001).

Techniques: BrdU Incorporation Assay, Western Blot, Expressing, Luciferase, Activity Assay, Control, Functional Assay, Knockdown

Journal: Communications Biology

Article Title: Condensin II interacts with BLM helicase in S phase to maintain genome stability

doi: 10.1038/s42003-025-07916-0

Figure Lengend Snippet: a SMC2 was identified by mass spectrometry in a co-immunoprecipitate of endogenous BLM from nuclear extracts of WT cells 3 h after release from G1/S arrest . Select known BLM interacting proteins are shown for comparison. Isogenic BLM-KO cells were subjected to the same analysis and yielded zero hits for the listed proteins. Experiment was performed in triplicate. b Reciprocal co-immunoprecipitation of endogenous BLM and SMC2 from nuclear extracts of WT and BLM-KO cells 3 h after release from G1/S arrest. c , Proximity ligation assay (PLA) using antibodies against endogenous BLM and SMC2 in WT, BLM-COMP, and BLM-KO cells 3 h after release from G1/S arrest and quantification of BLM-SMC2 PLA foci/nucleus (WT, n = 111 nuclei; BLM-COMP, n = 103 nuclei; BLM-KO, n = 104 nuclei). Scale bars, 10 μM. Significance of difference between WT and BLM-COMP was determined by a Mann–Whitney test (ns, not significant). For additional PLA controls, see Supplementary Fig. S . d Five-subunit condensin I and II complexes (H, NCAPH; D2, NCAPD2; G, NCAPG; H2, NCAPH2; D3, NCAPD3; G2, NCAPG2). e Co-immunoprecipitation of endogenous BLM from whole-cell extracts of WT cells 3 h after release from G1/S arrest was probed with antibodies against condensin I and II subunits. Condensin subunits are color-coordinated with their respective condensin from ( d ). f Co-immunoprecipitation of endogenous NCAPH from whole-cell extracts of WT cells 3 h after release from G1/S arrest was probed with antibodies against condensin I and II subunits and BLM. g Co-immunoprecipitation of endogenous NCAPH2 from whole-cell extracts of WT cells 3 h after release from G1/S arrest was probed with antibodies against condensin I and II subunits and BLM.

Article Snippet: Commercially available antibodies against the following proteins are as follows with the application and concentration used: β-actin (Santa Cruz, cat. no. sc-69879, WB, 1:5000), Biotin (Jackson Labs, cat. no. 200-002-211, Immunofluorescence (IF), 1:1000; Bethyl, cat. no. A150-109A, IF, 1:1000), BLM (Santa Cruz, cat. no. sc-365753, WB and IF, 1:200 and 1:250; Bethyl, cat. no. A300-110A, IP, 5 μg/mL; Abcam, cat no. ab2179, IF, 1:2000), BrdU (BD Biosciences, cat. no. 347580, IF, 1:50; Abcam, cat. no. ab6326, IF, 1:100), CREST (Antibodies Inc., cat. no. 15-234-0001, IF, 1:50), Cyclin A (Santa Cruz cat. no. sc-239, WB, 1:500), Cyclin B1 (Santa Cruz cat. no. sc-245, WB, 1:1000), Cyclin E (Santa Cruz cat. no. sc-377100, WB, 1:500), GAL4-TA (Santa Cruz, cat. no. sc-1663, WB, 1:200), γ-H2AX (Abcam, cat. no. ab81299, WB, 1:5000), Histone H2A (Invitrogen, cat. no. MA5-24662, WB, 1:1000), Histone H4 (Santa Cruz, cat. no. sc-25260, WB, 1:200), MCM6 (Santa Cruz, cat. no. sc-393618, WB, 1:200), NCAPD3 (Santa Cruz, cat. no. sc-81597, WB, 1:200), NCAPG (Santa Cruz, cat. no. sc-515297, WB, 1:200), NCAPH (Santa Cruz, cat. no. sc-101013, WB, 1:500; Bethyl cat. no. A300-603A, IP, 5 μg/mL), NCAPH2 (Santa Cruz, cat. no. sc-393333, WB and IF, 1:500 and 1:250; Bethyl cat. no. A302-275A, IP, 5 μg/mL), PCNA (Cell Signaling, cat. no. 2586, WB, 1:5000), p-DRP1 (Cell Signaling, cat. no. 4494S, WB, 1:500), p-Histone H3 (Cell Signaling, cat. no. 9701S, WB, 1:500), RAD51 (Santa Cruz, cat. no. sc-398587, WB, 1:200), RPA2 (Santa Cruz, cat. no. sc-56770, WB, 1:200), SMC2 (Bethyl, cat. no. A300-058A, IF and Immunoprecipitation (IP), 1:1000 and 5 μg/mL; Invitrogen, cat. no. GT4312, WB, 1:1000; Cell Signaling, cat. no. 5329, IP, 5 μg/mL; Abcam, cat. no. ab10412, IF, 1:250) SMC4 (Bethyl, cat. no. A300-063A, WB, 1:1000), TOP2A (Santa Cruz, cat. no. sc-365916, WB, 1:200), TOP3A (Abcam, cat. no. ab108493, WB, 1:1000).

Techniques: Mass Spectrometry, Comparison, Immunoprecipitation, Proximity Ligation Assay, MANN-WHITNEY

Journal: Communications Biology

Article Title: Condensin II interacts with BLM helicase in S phase to maintain genome stability

doi: 10.1038/s42003-025-07916-0

Figure Lengend Snippet: a BLM truncations tested in mammalian-two-hybrid (M2H) assay against full-length SMC2. Interaction is indicated by secreted alkaline phosphatase (SEAP) activity as ++ (strong), + (mild), and - (absent). See Supplementary Fig. S for measurements of SEAP activity from three experiments. Red box indicates BLM region required for interaction with SMC2. b Alignment of BLM residues 150-184 with BLM from other vertebrates. Residues W154, M157, and F160 were mutated to lysine, referred to as BLM-WMF mut . c SEAP activity detected in M2H assay between two independently generated pVP16-BLM-WMF mut clones (#1, #2) and pM-SMC2. pM3-VP16 is a positive control from the manufacturer expressing a fusion of the GAL4 DNA-binding domain to the VP16 activation domain. Significance of differences between means ± SD was determined by a t -test and is reported as **** p ≤ 0.0001. d Plasmid expressing BLM-WMF mut was stably transfected into BLM-KO cells. Western blot of nuclear extracts from two independently generated BLM-WMF mut clones (#1, #2) and of WT, BLM-KO, and BLM-COMP cells was probed with BLM antibody. PCNA was used as a loading control. Expression levels were analyzed 3 h after release from G1/S arrest. e Co-immunoprecipitations of BLM and BLM-WMF mut #1 were probed with antibodies against SMC2, condensin II-specific subunit NCAPH2, and known BLM-interacting proteins. Whole-cell extracts were prepared from cells 3 h after release from G1/S arrest. f PLA using antibodies against endogenous BLM and NCAPH2 in BLM-COMP, BLM-WMF mut #1, and BLM-KO cells 3 h after release from G1/S arrest. Scale bars, 10 μm. BLM-NCAPH2 PLA foci/nucleus were quantified (BLM-COMP, n = 102 nuclei; BLM-WMF mut #1, n = 130 nuclei; BLM-KO, n = 100 nuclei). Significance of differences was determined by a Mann–Whitney test and is reported as **** p ≤ 0.0001. For additional PLA controls, see Supplementary Fig. S .

Article Snippet: Commercially available antibodies against the following proteins are as follows with the application and concentration used: β-actin (Santa Cruz, cat. no. sc-69879, WB, 1:5000), Biotin (Jackson Labs, cat. no. 200-002-211, Immunofluorescence (IF), 1:1000; Bethyl, cat. no. A150-109A, IF, 1:1000), BLM (Santa Cruz, cat. no. sc-365753, WB and IF, 1:200 and 1:250; Bethyl, cat. no. A300-110A, IP, 5 μg/mL; Abcam, cat no. ab2179, IF, 1:2000), BrdU (BD Biosciences, cat. no. 347580, IF, 1:50; Abcam, cat. no. ab6326, IF, 1:100), CREST (Antibodies Inc., cat. no. 15-234-0001, IF, 1:50), Cyclin A (Santa Cruz cat. no. sc-239, WB, 1:500), Cyclin B1 (Santa Cruz cat. no. sc-245, WB, 1:1000), Cyclin E (Santa Cruz cat. no. sc-377100, WB, 1:500), GAL4-TA (Santa Cruz, cat. no. sc-1663, WB, 1:200), γ-H2AX (Abcam, cat. no. ab81299, WB, 1:5000), Histone H2A (Invitrogen, cat. no. MA5-24662, WB, 1:1000), Histone H4 (Santa Cruz, cat. no. sc-25260, WB, 1:200), MCM6 (Santa Cruz, cat. no. sc-393618, WB, 1:200), NCAPD3 (Santa Cruz, cat. no. sc-81597, WB, 1:200), NCAPG (Santa Cruz, cat. no. sc-515297, WB, 1:200), NCAPH (Santa Cruz, cat. no. sc-101013, WB, 1:500; Bethyl cat. no. A300-603A, IP, 5 μg/mL), NCAPH2 (Santa Cruz, cat. no. sc-393333, WB and IF, 1:500 and 1:250; Bethyl cat. no. A302-275A, IP, 5 μg/mL), PCNA (Cell Signaling, cat. no. 2586, WB, 1:5000), p-DRP1 (Cell Signaling, cat. no. 4494S, WB, 1:500), p-Histone H3 (Cell Signaling, cat. no. 9701S, WB, 1:500), RAD51 (Santa Cruz, cat. no. sc-398587, WB, 1:200), RPA2 (Santa Cruz, cat. no. sc-56770, WB, 1:200), SMC2 (Bethyl, cat. no. A300-058A, IF and Immunoprecipitation (IP), 1:1000 and 5 μg/mL; Invitrogen, cat. no. GT4312, WB, 1:1000; Cell Signaling, cat. no. 5329, IP, 5 μg/mL; Abcam, cat. no. ab10412, IF, 1:250) SMC4 (Bethyl, cat. no. A300-063A, WB, 1:1000), TOP2A (Santa Cruz, cat. no. sc-365916, WB, 1:200), TOP3A (Abcam, cat. no. ab108493, WB, 1:1000).

Techniques: Activity Assay, Generated, Clone Assay, Positive Control, Expressing, Binding Assay, Activation Assay, Plasmid Preparation, Stable Transfection, Transfection, Western Blot, Control, MANN-WHITNEY

Journal: Communications Biology

Article Title: Condensin II interacts with BLM helicase in S phase to maintain genome stability

doi: 10.1038/s42003-025-07916-0

Figure Lengend Snippet: Endogenous BLM was immunoprecipitated after release from G1/S arrest in ( a ) the absence of λ protein phosphatase (λPPase) and in ( b ) the presence of λPPase and probed with antibodies against SMC2, NCAPH2, RPA, and TOP2A. Cells were synchronized by double-thymidine, released, and BLM immunoprecipitated every hour for 12 h. IgG was used as a negative control for immunoprecipitation. Endogenous BLM was immunoprecipitated after release from G1/S arrest in ( c ) the absence of λ protein phosphatase (λPPase) and in ( d ) the presence of λPPase and probed with antibodies against SMC2, SMC4, and NCAPH2. Cells were synchronized by double-thymidine, released, and BLM immunoprecipitated every hour for 12 h. IgG was used as a negative control for immunoprecipitation. Experiment was performed at least twice with similar results. e Endogenous NCAPH2 was immunoprecipitated from WT, BLM-KO, and BLM-WMF mut #1 cells and probed with antibodies against BLM, SMC2, and SMC4. Cell cultures were arrested at G1/S by double-thymidine treatment, released, and NCAPH2 immunoprecipitated every hour for 7 h. IgG was used as a negative control for immunoprecipitation. Experiment was performed twice with similar results. f Endogenous NCAPH2 and SMC2 was immunoprecipitated from the same WT lysate used for hours 3–7 in ( e ) and probed with antibodies against NCAPH2, BLM, SMC2, and SMC4. IgG was used as a negative control for immunoprecipitation. NCAPH2 immunoprecipitation was performed three times with similar results and the SMC2 reciprocal immunoprecipitation once.

Article Snippet: Commercially available antibodies against the following proteins are as follows with the application and concentration used: β-actin (Santa Cruz, cat. no. sc-69879, WB, 1:5000), Biotin (Jackson Labs, cat. no. 200-002-211, Immunofluorescence (IF), 1:1000; Bethyl, cat. no. A150-109A, IF, 1:1000), BLM (Santa Cruz, cat. no. sc-365753, WB and IF, 1:200 and 1:250; Bethyl, cat. no. A300-110A, IP, 5 μg/mL; Abcam, cat no. ab2179, IF, 1:2000), BrdU (BD Biosciences, cat. no. 347580, IF, 1:50; Abcam, cat. no. ab6326, IF, 1:100), CREST (Antibodies Inc., cat. no. 15-234-0001, IF, 1:50), Cyclin A (Santa Cruz cat. no. sc-239, WB, 1:500), Cyclin B1 (Santa Cruz cat. no. sc-245, WB, 1:1000), Cyclin E (Santa Cruz cat. no. sc-377100, WB, 1:500), GAL4-TA (Santa Cruz, cat. no. sc-1663, WB, 1:200), γ-H2AX (Abcam, cat. no. ab81299, WB, 1:5000), Histone H2A (Invitrogen, cat. no. MA5-24662, WB, 1:1000), Histone H4 (Santa Cruz, cat. no. sc-25260, WB, 1:200), MCM6 (Santa Cruz, cat. no. sc-393618, WB, 1:200), NCAPD3 (Santa Cruz, cat. no. sc-81597, WB, 1:200), NCAPG (Santa Cruz, cat. no. sc-515297, WB, 1:200), NCAPH (Santa Cruz, cat. no. sc-101013, WB, 1:500; Bethyl cat. no. A300-603A, IP, 5 μg/mL), NCAPH2 (Santa Cruz, cat. no. sc-393333, WB and IF, 1:500 and 1:250; Bethyl cat. no. A302-275A, IP, 5 μg/mL), PCNA (Cell Signaling, cat. no. 2586, WB, 1:5000), p-DRP1 (Cell Signaling, cat. no. 4494S, WB, 1:500), p-Histone H3 (Cell Signaling, cat. no. 9701S, WB, 1:500), RAD51 (Santa Cruz, cat. no. sc-398587, WB, 1:200), RPA2 (Santa Cruz, cat. no. sc-56770, WB, 1:200), SMC2 (Bethyl, cat. no. A300-058A, IF and Immunoprecipitation (IP), 1:1000 and 5 μg/mL; Invitrogen, cat. no. GT4312, WB, 1:1000; Cell Signaling, cat. no. 5329, IP, 5 μg/mL; Abcam, cat. no. ab10412, IF, 1:250) SMC4 (Bethyl, cat. no. A300-063A, WB, 1:1000), TOP2A (Santa Cruz, cat. no. sc-365916, WB, 1:200), TOP3A (Abcam, cat. no. ab108493, WB, 1:1000).

Techniques: Immunoprecipitation, Negative Control

Journal: Communications Biology

Article Title: Condensin II interacts with BLM helicase in S phase to maintain genome stability

doi: 10.1038/s42003-025-07916-0

Figure Lengend Snippet: a Immunofluorescence microscopy images of endogenous BLM, NCAPH2, SMC2 and EdU-labeled nascent DNA in WT cells. Nucleus is outlined in merged image and box represents ROI for magnified image. Scale bars, 10 μM and 1 μM (magnified image). Quantification of BLM, NCAPH2, and SMC2 colocalization with EdU in WT cells. Colocalization percentage was determined by counting the number of BLM, NCAPH2, or SMC2 foci that overlapped with EdU in comparison to the total number of BLM, NCAPH2, or SMC2 foci in an ROI for each nucleus. 20 nuclei were analyzed for each protein and median percentage of colocalization is reported. b SIRF assay of SMC2 in BLM-COMP, BLM-WMF mut #1, and BLM-KO cells. Following nascent DNA labeling with EdU and EdU:biotin co-Click with biotin azide and Alexa Fluor 488 azide, PLA was performed using antibodies against biotin and endogenous SMC2. Nucleus is outlined in the merged image. Scale bars, 10 μM. Corrected total Cellular Fluorescence (ctCF) of the SMC2 SIRF signal/EdU ctCF is shown for individual nuclei (BLM-COMP, n = 150 nuclei; BLM-WMF mut #1, n = 156 nuclei; BLM-KO, n = 79 nuclei) and the median presented by a horizontal line. Significance of differences between cell lines was determined using a Mann-Whitney test (ns, not significant). c SIRF assay of NCAPH2 in BLM-COMP, BLM-WMF mut #1, and BLM-KO cells. Following nascent DNA labeling with EdU and EdU:biotin co-Click with biotin azide and Alexa Fluor 488 azide, PLA was performed using antibodies against biotin and endogenous NCAPH2. Nucleus is outlined in the merged image. Scale bars, 10 μM. Corrected total Cellular Fluorescence (ctCF) of the NCAPH2 SIRF signal/EdU ctCF is shown for individual nuclei (BLM-COMP, n = 99 nuclei; BLM-WMF mut #1, n = 85 nuclei; BLM-KO, n = 85 nuclei) and the median presented by a horizontal line. Significance of differences between cell lines was determined using a Mann-Whitney test (ns, not significant). d SIRF assay of BLM in BLM-COMP, BLM-WMF mut #1, and BLM-KO cells. Following nascent DNA labeling with EdU and EdU:biotin co-Click with biotin azide and Alexa Fluor 488 azide, PLA was performed using antibodies against biotin and endogenous BLM. Nucleus is outlined in the merged image. Scale bars, 10 μM. Corrected total Cellular Fluorescence (ctCF) of the BLM SIRF signal/EdU ctCF is shown for individual nuclei (BLM-COMP, n = 120 nuclei; BLM-WMF mut #1, n = 125 nuclei; BLM-KO, n = 140 nuclei) and the median presented by a horizontal line. Significance of differences between cell lines was determined using a Mann–Whitney test and is reported as **** p ≤ 0.0001. e BLM SIRF assay in the BLM-COMP and BLM-WMF mut #1 cell lines following transfection with NCAPH2 siRNA or mock siRNA. Following nascent DNA labeling with EdU and EdU:biotin co-Click with biotin azide and Alexa Fluor 488 azide, PLA was performed using antibodies against biotin and endogenous BLM. Nucleus is outlined in the merged image. Scale bars, 10 μM. Corrected total Cellular Fluorescence (ctCF) of the BLM SIRF signal/EdU ctCF was calculated for each nucleus (BLM-COMP siNCAPH2, n = 102 nuclei; BLM-COMP siMock, n = 89 nuclei; BLM-WMF mut #1 siMock n = 94 nuclei) with the horizontal line representing the median. Significance of differences was determined by a Mann-Whitney test and is reported as **** p ≤ 0.0001. For additional controls for Fig. 5b–e see supplementary Fig. S . f Immunofluorescence microscopy of calyculin-induced premature chromosome condensation (PCC) in BLM-COMP, BLM-KO, and BLM-WMF mut #1 cells. Following nascent DNA labeling with EdU and chromosome spreading, EdU:biotin Click-iT was performed with biotin azide and slides were hybridized with biotin antibody and stained with DAPI. Box represents ROI for magnified image. Scale bars, 10 μM. The chromosomes of three spreads from a single experiment for each cell line were analyzed to quantify the percentage of resolved and unresolved sister chromatids.

Article Snippet: Commercially available antibodies against the following proteins are as follows with the application and concentration used: β-actin (Santa Cruz, cat. no. sc-69879, WB, 1:5000), Biotin (Jackson Labs, cat. no. 200-002-211, Immunofluorescence (IF), 1:1000; Bethyl, cat. no. A150-109A, IF, 1:1000), BLM (Santa Cruz, cat. no. sc-365753, WB and IF, 1:200 and 1:250; Bethyl, cat. no. A300-110A, IP, 5 μg/mL; Abcam, cat no. ab2179, IF, 1:2000), BrdU (BD Biosciences, cat. no. 347580, IF, 1:50; Abcam, cat. no. ab6326, IF, 1:100), CREST (Antibodies Inc., cat. no. 15-234-0001, IF, 1:50), Cyclin A (Santa Cruz cat. no. sc-239, WB, 1:500), Cyclin B1 (Santa Cruz cat. no. sc-245, WB, 1:1000), Cyclin E (Santa Cruz cat. no. sc-377100, WB, 1:500), GAL4-TA (Santa Cruz, cat. no. sc-1663, WB, 1:200), γ-H2AX (Abcam, cat. no. ab81299, WB, 1:5000), Histone H2A (Invitrogen, cat. no. MA5-24662, WB, 1:1000), Histone H4 (Santa Cruz, cat. no. sc-25260, WB, 1:200), MCM6 (Santa Cruz, cat. no. sc-393618, WB, 1:200), NCAPD3 (Santa Cruz, cat. no. sc-81597, WB, 1:200), NCAPG (Santa Cruz, cat. no. sc-515297, WB, 1:200), NCAPH (Santa Cruz, cat. no. sc-101013, WB, 1:500; Bethyl cat. no. A300-603A, IP, 5 μg/mL), NCAPH2 (Santa Cruz, cat. no. sc-393333, WB and IF, 1:500 and 1:250; Bethyl cat. no. A302-275A, IP, 5 μg/mL), PCNA (Cell Signaling, cat. no. 2586, WB, 1:5000), p-DRP1 (Cell Signaling, cat. no. 4494S, WB, 1:500), p-Histone H3 (Cell Signaling, cat. no. 9701S, WB, 1:500), RAD51 (Santa Cruz, cat. no. sc-398587, WB, 1:200), RPA2 (Santa Cruz, cat. no. sc-56770, WB, 1:200), SMC2 (Bethyl, cat. no. A300-058A, IF and Immunoprecipitation (IP), 1:1000 and 5 μg/mL; Invitrogen, cat. no. GT4312, WB, 1:1000; Cell Signaling, cat. no. 5329, IP, 5 μg/mL; Abcam, cat. no. ab10412, IF, 1:250) SMC4 (Bethyl, cat. no. A300-063A, WB, 1:1000), TOP2A (Santa Cruz, cat. no. sc-365916, WB, 1:200), TOP3A (Abcam, cat. no. ab108493, WB, 1:1000).

Techniques: Immunofluorescence, Microscopy, Labeling, Comparison, DNA Labeling, Fluorescence, MANN-WHITNEY, Transfection, Staining

Journal: Communications Biology

Article Title: Condensin II interacts with BLM helicase in S phase to maintain genome stability

doi: 10.1038/s42003-025-07916-0

Figure Lengend Snippet: a Model of how BLM binding to condensin II increases efficiency of detection and repair of BLM substrates. Current models of loop extrusion posit that the hinge domain reels in new DNA through the SMC2-SMC4 lumen creating an intermediate loop with gate opening potentially allowing the intermediate loop to join the main loop , , , . BLM, through its interaction with condensin II at the WMF site, can scan this intermediate loop for its substrates such as R-loops, DNA breaks, or G-quadruplexes, leading to their resolution prior to becoming an impediment to the replication or transcription machinery, thus mediating normal DNA transactions and genome stability. The many DNA repair and replication factors that bind to BLM’s disordered tail likely contribute to resolution and repair. In the absence of BLM from condensin II due to the WMF mutation, the DNA lesions and replisome blocks are not resolved efficiently, thus increasing risk of collisions with the replisome or transcriptional machinery, leading to impaired replisome progression and fork stalling, impaired DNA-damage response, delayed DSB repair and, eventually, mitotic chromosome aberrations. Created in BioRender. Rodemoyer, B. (2025) https://BioRender.com/h40u746 . b Model of how BLM-condensin II interaction may contribute to suppression of crossovers. Extrusion of adjacent loops may aid BLM helicase-mediated branch migration of Holliday junctions between sister chromatids. BLM-condensin II interaction puts BLM in close proximity to the HR intermediate, increasing efficiency of dissolution as noncrossovers (NCO) in cooperation with interacting proteins. Loss of BLM-condensin II interaction due to BLM-WMF mut reduces dissolution of HR intermediates by BLM complexes and increases resolution via cleavage by structure-specific endonucleases, causing crossovers (CO). Created in BioRender. Rodemoyer, B. (2025) https://BioRender.com/k17g890 .

Article Snippet: Commercially available antibodies against the following proteins are as follows with the application and concentration used: β-actin (Santa Cruz, cat. no. sc-69879, WB, 1:5000), Biotin (Jackson Labs, cat. no. 200-002-211, Immunofluorescence (IF), 1:1000; Bethyl, cat. no. A150-109A, IF, 1:1000), BLM (Santa Cruz, cat. no. sc-365753, WB and IF, 1:200 and 1:250; Bethyl, cat. no. A300-110A, IP, 5 μg/mL; Abcam, cat no. ab2179, IF, 1:2000), BrdU (BD Biosciences, cat. no. 347580, IF, 1:50; Abcam, cat. no. ab6326, IF, 1:100), CREST (Antibodies Inc., cat. no. 15-234-0001, IF, 1:50), Cyclin A (Santa Cruz cat. no. sc-239, WB, 1:500), Cyclin B1 (Santa Cruz cat. no. sc-245, WB, 1:1000), Cyclin E (Santa Cruz cat. no. sc-377100, WB, 1:500), GAL4-TA (Santa Cruz, cat. no. sc-1663, WB, 1:200), γ-H2AX (Abcam, cat. no. ab81299, WB, 1:5000), Histone H2A (Invitrogen, cat. no. MA5-24662, WB, 1:1000), Histone H4 (Santa Cruz, cat. no. sc-25260, WB, 1:200), MCM6 (Santa Cruz, cat. no. sc-393618, WB, 1:200), NCAPD3 (Santa Cruz, cat. no. sc-81597, WB, 1:200), NCAPG (Santa Cruz, cat. no. sc-515297, WB, 1:200), NCAPH (Santa Cruz, cat. no. sc-101013, WB, 1:500; Bethyl cat. no. A300-603A, IP, 5 μg/mL), NCAPH2 (Santa Cruz, cat. no. sc-393333, WB and IF, 1:500 and 1:250; Bethyl cat. no. A302-275A, IP, 5 μg/mL), PCNA (Cell Signaling, cat. no. 2586, WB, 1:5000), p-DRP1 (Cell Signaling, cat. no. 4494S, WB, 1:500), p-Histone H3 (Cell Signaling, cat. no. 9701S, WB, 1:500), RAD51 (Santa Cruz, cat. no. sc-398587, WB, 1:200), RPA2 (Santa Cruz, cat. no. sc-56770, WB, 1:200), SMC2 (Bethyl, cat. no. A300-058A, IF and Immunoprecipitation (IP), 1:1000 and 5 μg/mL; Invitrogen, cat. no. GT4312, WB, 1:1000; Cell Signaling, cat. no. 5329, IP, 5 μg/mL; Abcam, cat. no. ab10412, IF, 1:250) SMC4 (Bethyl, cat. no. A300-063A, WB, 1:1000), TOP2A (Santa Cruz, cat. no. sc-365916, WB, 1:200), TOP3A (Abcam, cat. no. ab108493, WB, 1:1000).

Techniques: Binding Assay, Mutagenesis, Migration, Dissolution