Journal: Nucleic Acids Research

Article Title: The SMC5/SMC6 complex is critical for resolving R-loop-induced transcription–replication conflicts

doi: 10.1093/nar/gkaf1537

Figure Lengend Snippet: CRISPR DDR library screening identifies the synthetic lethal effect between SETX and the SMC5/6 complex. ( A ) DDR screening results from MAGeCK analysis as described in the “Materials and methods” section. Genes are ranked by the RRA score of negative selection. ( B, C ) U2OS WT cells were infected with lentivirus encoding shRNA to knock down indicated genes. Three days after infection, cells were resuspended and seeded on new plates for colony formation assay (top) and monitoring growth speed (bottom). ( D ) U2OS WT cells were infected with shRNA-expressing lentivirus to deplete the indicated proteins. Three days after infection, cells were subjected to immunostaining with antibody against γH2AX. ( E ). U2OS WT cells with or without RNaseH1 expression were infected with SETX shRNA and SMC5 shRNA. Three days after infection, cells were subjected to immunostaining with antibody against γH2AX. ( F ). U2OS WT and SETX -KO cells were infected with SMC5 shRNA or vector. Three days after infection, cells were subjected to quantitative image-based cytometry (QIBC) analysis described in the “Materials and methods” section. The γH2AX level was displayed in different cell cycles (left). The percentage of cells with medium and high γH2AX signal was calculated and normalized to WT + vector control (right).

Article Snippet: Antibodies used for western blotting were listed below: γH2AX (07164, Upstate), FLAG (F1804, Sigma–Aldrich), SMC5 (sc-393282, Santa Cruz), SETX (NB100-57542, Novus Biologicals), KU70 (sc-17789, Santa Cruz), V5 ( R96025 , Invitrogen), and TOP3A (kindly provided by Dr Ian David Hickson).

Techniques: CRISPR, Library Screening, Selection, Infection, shRNA, Knockdown, Colony Assay, Expressing, Immunostaining, Plasmid Preparation, Cytometry, Control

Journal: Nucleic Acids Research

Article Title: The SMC5/SMC6 complex is critical for resolving R-loop-induced transcription–replication conflicts

doi: 10.1093/nar/gkaf1537

Figure Lengend Snippet: SMC5/6 is recruited to the sites of TRC in proximity to SETX and plays an important role in suppressing TRC. ( A, B, C, D, E ) U2OS WT cells were treated with vehicle or HU (2 mM) for 2 h and then subjected to PLA analysis showing colocalization between SETX and R-loops (A), SETX and PCNA (B), SETX and FANCD2 (C), SMC5-Flag and R-loop (D, left), SMC5-Flag and FANCD2 (D, right), and SMC5-Flag and SETX (E). ( F ) U2OS WT and SETX -KO cells with or without RNaseH1 expression were subjected to PLA analysis showing colocalization between replication (PCNA) and transcription (pPOLR2A). ( G ) U2OS WT and SETX -KO cells, with or without RNaseH1 expression, were subjected to PLA analysis showing colocalization between SMC5-Flag and R-loops (S9.6). ( H ) U2OS WT and SETX -KO cells with or without RNaseH1 expression were subjected to PLA analysis showing colocalization between SMC5-Flag and FANCD2. ( I ) U2OS WT and SETX -KO cells were infected with SMC5 shRNA or vector, with or without RNaseH1 expression. Three days after infection, cells were used for PLA analysis showing colocalization between replication (PCNA) and transcription (pPOLR2A).

Article Snippet: Antibodies used for western blotting were listed below: γH2AX (07164, Upstate), FLAG (F1804, Sigma–Aldrich), SMC5 (sc-393282, Santa Cruz), SETX (NB100-57542, Novus Biologicals), KU70 (sc-17789, Santa Cruz), V5 ( R96025 , Invitrogen), and TOP3A (kindly provided by Dr Ian David Hickson).

Techniques: Expressing, Infection, shRNA, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: The SMC5/SMC6 complex is critical for resolving R-loop-induced transcription–replication conflicts

doi: 10.1093/nar/gkaf1537

Figure Lengend Snippet: SMC5/6 is required for recruitment of BLM/TOP3A/RMI (BTRR) complex to TRC. ( A, B, C, D ) U2OS WT and SETX -KO cells with or without RNASEH1 expression were subjected to PLA analysis showing colocalization between TOP3A and FANCD2 (A), TOP3A and SMC5-Flag (B left), TOP3A and R-loops (B right), BLM and SMC5-Flag (C left), RMI1 and SMC5-Flag (C right), BLM and R-loops (D left), and RMI1 and R-loops (D right). ( E ) U2OS SETX -KO cells were infected with SMC5 shRNA or vector. Three days after infection, cells were used for PLA analysis showing colocalization between BLM and R-loops (left), TOP3A and R-loops (middle), and RMI1 and R-loops (right). ( F ) U2OS WT cells were infected with shRNA to knock down indicated genes. Three days after infection, cells were used for PLA analysis showing colocalization between SMC5-Flag and R-loops (S9.6). ( G ) U2OS WT cells were infected with shRNA to knock down indicated genes. Three days after infection, cells were subjected to PLA analysis showing colocalization between replication (PCNA) and transcription (pPOLR2A).

Article Snippet: Antibodies used for western blotting were listed below: γH2AX (07164, Upstate), FLAG (F1804, Sigma–Aldrich), SMC5 (sc-393282, Santa Cruz), SETX (NB100-57542, Novus Biologicals), KU70 (sc-17789, Santa Cruz), V5 ( R96025 , Invitrogen), and TOP3A (kindly provided by Dr Ian David Hickson).

Techniques: Expressing, Infection, shRNA, Plasmid Preparation, Knockdown

Journal: Nucleic Acids Research

Article Title: The SMC5/SMC6 complex is critical for resolving R-loop-induced transcription–replication conflicts

doi: 10.1093/nar/gkaf1537

Figure Lengend Snippet: BTRR is important for reducing supercoils at TRCs and exhibits a synthetic lethal interaction with SETX. ( A ) U2OS WT and SETX -KO cells were infected with SMC5 shRNA or vector. Three days after infection, cells were subjected to PLA analysis showing colocalization between TOP2A and R-loops (S9.6). ( B ) U2OS SETX -KO cells were infected with shRNA to knock down indicated genes. Three days after infection, cells were subjected to PLA analysis showing colocalization between TOP2A and R-loops (S9.6). ( C ) U2OS SETX -KO cells were infected with lentivirus to express TOP3A-WT or Y362F mutant. Then, cells were infected with TOP3A shRNA or vector. Three days after shRNA infection, cells were subjected to PLA analysis showing colocalization between TOP2A and R-loops (S9.6). ( D ) U2OS WT cells were infected with shRNA-expressing lentivirus to knock down indicated genes. Three days after infection, cells were subjected to immunostaining with antibody against γH2AX. ( E ) U2OS WT cells were infected with shRNA-expressing lentivirus to knock down indicated genes. Three days after infection, cells were resuspended and seeded on plates for colony formation assay.

Article Snippet: Antibodies used for western blotting were listed below: γH2AX (07164, Upstate), FLAG (F1804, Sigma–Aldrich), SMC5 (sc-393282, Santa Cruz), SETX (NB100-57542, Novus Biologicals), KU70 (sc-17789, Santa Cruz), V5 ( R96025 , Invitrogen), and TOP3A (kindly provided by Dr Ian David Hickson).

Techniques: Infection, shRNA, Plasmid Preparation, Knockdown, Mutagenesis, Expressing, Immunostaining, Colony Assay

Journal: Nucleic Acids Research

Article Title: The SMC5/SMC6 complex is critical for resolving R-loop-induced transcription–replication conflicts

doi: 10.1093/nar/gkaf1537

Figure Lengend Snippet: SLF2 is required for SMC5/6 loading to TRCs upon SETX loss. ( A ) U2OS SETX-KO cells were infected with SLF2 shRNA or vector. Three days after infection, cells were subjected to PLA analysis showing colocalization between SMC5-Flag and FANCD2. ( B ) U2OS WT cells were infected with SLF2 shRNA or vector. Three days after infection, cells were subjected to PLA analysis showing colocalization between replication (PCNA) and transcription (pPOLR2A). ( C ) U2OS WT and SLF2 -KO cells were infected with SETX shRNA or vector. Three days after infection, cells were harvested for western blotting using the antibodies indicated. ( D ) U2OS WT cells were infected with shRNA to knock down indicated genes. Three days after infection, cells were resuspended and seeded on new plates for colony formation assay (left) and monitoring growth speed (right).

Article Snippet: Antibodies used for western blotting were listed below: γH2AX (07164, Upstate), FLAG (F1804, Sigma–Aldrich), SMC5 (sc-393282, Santa Cruz), SETX (NB100-57542, Novus Biologicals), KU70 (sc-17789, Santa Cruz), V5 ( R96025 , Invitrogen), and TOP3A (kindly provided by Dr Ian David Hickson).

Techniques: Infection, shRNA, Plasmid Preparation, Western Blot, Knockdown, Colony Assay

Journal: Nucleic Acids Research

Article Title: The SMC5/SMC6 complex is critical for resolving R-loop-induced transcription–replication conflicts

doi: 10.1093/nar/gkaf1537

Figure Lengend Snippet: FANCD2 activation depends on BTRR. ( A ) U2OS WT and SETX -KO cells with or without RNASEH1 expression were used for immunostaining with antibody against FANCD2. ( B ) SMC6-Flag-expressing U2OS WT and SETX -KO cells, with or without RNaseH1 expression, were subjected to PLA analysis showing colocalization between SMC6-Flag and FANCD2. ( C ) U2OS WT and SETX -KO cells with or without RNASEH1 expression were subjected to PLA analysis showing colocalization between BLM and FANCD2. ( D ) U2OS SETX -KO cells were infected with shRNA to knock down indicated genes. Three days after infection, cells were used for immunostaining with an antibody against FANCD2. ( E ) U2OS WT cells were infected with shRNA to knock down indicated genes. Three days after infection, cells were subjected to PLA analysis showing colocalization between FANCD2 and R-loops (S9.6). ( F ) U2OS WT cells were infected with FANCD2 shRNA or vector. Three days after infection, cells were subjected to PLA analysis showing colocalization between SMC5-Flag and BLM (left), SMC5-Flag and TOP3A (middle), and SMC5-Flag and RMI1 (right).

Article Snippet: Antibodies used for western blotting were listed below: γH2AX (07164, Upstate), FLAG (F1804, Sigma–Aldrich), SMC5 (sc-393282, Santa Cruz), SETX (NB100-57542, Novus Biologicals), KU70 (sc-17789, Santa Cruz), V5 ( R96025 , Invitrogen), and TOP3A (kindly provided by Dr Ian David Hickson).

Techniques: Activation Assay, Expressing, Immunostaining, Infection, shRNA, Knockdown, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: The SMC5/SMC6 complex is critical for resolving R-loop-induced transcription–replication conflicts

doi: 10.1093/nar/gkaf1537

Figure Lengend Snippet: BTRR is important for recruiting FANCM to activate FANCD2 at TRCs. ( A ) U2OS WT and SETX -KO cells with Flag-FANCM expression were subjected to PLA analysis showing colocalization between SMC6 and Flag-FANCM. ( B ) U2OS SETX -KO cells were infected with FANCM shRNA or vector. Three days after infection, cells were analyzed using PLA to assess colocalization between SMC5-Flag and FANCD2 (left) or subjected to immunostaining with an anti-FANCD2 antibody (right). ( C ) U2OS SETX -KO cells with Flag-FANCM expression were infected with shRNA to knock down indicated genes. Three days after infection, cells were subjected to PLA analysis showing colocalization between SMC6 and Flag-FANCM. ( D ) U2OS SETX -KO cells expressing flag-FANCM-WT or Flag-FANCM-MM2 with silent mutations resistant to its FANCM shRNA were infected with FANCM shRNA to deplete endogenous FANCM. Three days after infection, cells were subjected to PLA analysis showing colocalization between SMC6 and Flag-FANCM. ( E ) Illustration of SMC5/6-mediated resolution of TRC in SETX-deficient cells. Positive supercoiling accumulates at TRC sites due to SETX loss. The SMC5/6 complex recognizes the supercoiling signals and recruits BTRR to relieve the tension. Meanwhile, BTRR recruits FANCM through direct interaction, ultimately activating FANCD2 to resolve TRCs.

Article Snippet: Antibodies used for western blotting were listed below: γH2AX (07164, Upstate), FLAG (F1804, Sigma–Aldrich), SMC5 (sc-393282, Santa Cruz), SETX (NB100-57542, Novus Biologicals), KU70 (sc-17789, Santa Cruz), V5 ( R96025 , Invitrogen), and TOP3A (kindly provided by Dr Ian David Hickson).

Techniques: Expressing, Infection, shRNA, Plasmid Preparation, Immunostaining, Knockdown

Journal: Cell Reports Methods

Article Title: Approaches for identification of 5′ UTR mutations impacting translation and protein production from neurodevelopmental disorder genes

doi: 10.1016/j.crmeth.2025.101247

Figure Lengend Snippet: 5′ UTR variants from autism probands alter translation in the native genomic context (A) Examples of polysome/80S enrichments altered by the alternate allele from the MPRA ( ∗∗∗ q value < 0.001, ∗∗∗∗ q value < 0.0001; for full results). (B) LCLs from these patients were subjected to polysome fractionation followed by targeted sequencing of the variant loci to quantify relative Ref and Alt allele abundance. (C) Differences in the relative abundance of each allele between total RNA and DNA (AEI) were tested. Variants in MKRN1 , SMC5 , and C12orf57 had an effect on transcript abundance. (D) ATI showed variants in SMC5 , MKRN1 , and USP48 had translational effects ( n = 2, significance of change in Alt allele abundance between fractions assessed by negative binomial generalized linear model fit; see STAR Methods and ; see also A). (E) Measurement of the relative allele abundance in 80S and 40S RNA fractions from proband-derived LCLs reveals an effect of proband variants in NSD2 and C12orf57. (F) Highly robust allelic effects in polysome/total RNA enrichment are found for proband variants in SMC5 and MKRN1. (G–I) Western blotting of MKRN1, NSD2, and SMC5 in proband-derived cell lines (light blue) alongside cell lines from matched sibling control (dark blue) and cell lines from unrelated control individuals (gray) ( n = 8 technical replicates per sample in G and I, n = 4 technical replicates per sample in H, Wilcoxon signed-rank test in G and I; Mann-Whitney U test in H; ∗ p < 0.05, ∗∗ p < 0.01).

Article Snippet: mouse anti-SMC5 primary antibody , Santa Cruz , sc-393282.

Techniques: Fractionation, Sequencing, Variant Assay, Derivative Assay, Western Blot, Control, MANN-WHITNEY

Journal: Cell Reports Methods

Article Title: Approaches for identification of 5′ UTR mutations impacting translation and protein production from neurodevelopmental disorder genes

doi: 10.1016/j.crmeth.2025.101247

Figure Lengend Snippet: 5′ UTR variants also alter protein production in reporter assays (A) Boxplots of dual-luciferase reporter assay in 51 variant reporters ( n = 4 per experiment, ∗FDR-adjusted p < 0.05, Mann-Whitney U test). Log 2 -normalized luciferase signal is adjusted to the reference allele for each pair. Purple rows are variants from probands. (B) A C>T transition in HPR introduces a stop codon downstream of an uAUG, terminating an uORF. (C) A deletion in SMC5 removes a series of guanine repeats (underlined) predicted to alter structure. (D) A G>A transition in NSD2 introduces an uAUG and a downstream oORF. (E–H) Pearson’s correlation between MPRA measures ( y axis) and dual luciferase measures for all variants (E), variants decreasing degree of 5′ UTR structure (F), variants increasing degree of 5′ UTR structure (G), and finally variants that alter uORFs (H).

Article Snippet: mouse anti-SMC5 primary antibody , Santa Cruz , sc-393282.

Techniques: Luciferase, Reporter Assay, Variant Assay, MANN-WHITNEY

Journal: Nucleic Acids Research

Article Title: RAD18 directs DNA double-strand break repair by homologous recombination to post-replicative chromatin

doi: 10.1093/nar/gkae499

Figure Lengend Snippet: RAD18 promotes recruitment of SMC5 to DNA lesions. ( A ) Relative HR (right) and NHEJ (left) repair in traffic light reporter U2OS cells treated with indicated siRNAs. The repair efficiency was normalized to the total repair efficiency in control siRNA treated cells (mean + SD, n ≥ 3, two-tailed t -test). ( B ) Relative proliferation of parental U2OS and RAD18–KO cells treated with control or 53BP1 siRNA was evaluated using resazurin viability assay 7 days after IR-irradiation with indicated doses (mean ± SD, n ≥ 3, two-way ANOVA). ( C ) Colocalization of SMC5 with γH2AX foci in parental U2OS and RAD18–KO. Cells were incubated with EdU prior IR-irradiation, pre-extracted and fixed at indicated time points. Incorporated EdU was Click-iT-labelled and cells were stained for SMC5 and γH2AX. EdU positive cells are shown (arrows indicate SMC5 foci colocalizing with γH2AX foci, scale bar 10 μm). Right, quantification of cell fraction with >1 SMC5 foci colocalizing with γH2AX foci. EdU positive cells were analyzed (mean + SD, n = 3, two-tailed t -test). ( D ) Quantification of the mean nuclear SMC5 intensity in parental U2OS and RAD18–KO cells. Where indicated, cells were pre-extracted prior fixation (mean ± SD, n = 300, Mann–Whitney test). ( E ) Relative HR and NHEJ repair in traffic light reporter U2OS cells treated with indicated siRNAs. The repair efficiency was normalized to the total repair efficiency in control siRNA treated cells (mean + SD, n ≥ 3, two-tailed t -test). ( F ) Quantification of RAD51 foci in U2OS cells treated with indicated siRNAs for 48 h. Cells were incubated with EdU prior IR-irradiation, pre-extracted and fixed after 6 h. Incorporated EdU was Click-iT-labelled and cells were stained for RAD51. EdU positive cells were analyzed (mean + SD, n = 3, two-tailed t -test). ( G ) Relative proliferation of parental U2OS and RAD18–KO cells treated with control or SMC5 siRNA was evaluated using resazurin viability assay 7 days after IR-irradiation with indicated doses (mean ± SD, n ≥ 3, two-way ANOVA). ( H ) Model describing RAD18 function at DSBs. RAD18 is recruited to the post-replicative chromatin by bimodal recognition of H2AK15Ub and H4K20me0 with its UBZ domain and with ARD domain of its interacting partner SLF1, respectively. RAD18 then inhibits NHEJ by limiting 53BP1 activity and promotes HR by recruiting the SLF2/SMC5/6 complex. RAD18 accumulation at DSBs is inhibited by RAD6-mediated auto-ubiquitination.

Article Snippet: The following antibodies were used in this study: BRCA1 (clone D-9, sc-6954, 1:100 for IF), RAD6 (UBE2A, clone G-9, sc-365507, 1:1000 for WB), Ubiquitin (clone P4D1, sc-8017, 1:500 for WB), PCNA (clone PC10, sc-56, 1:1000 for WB) from Santa Cruz; FLAG (clone M2, F 1804, 1:300 for IF), γH2AX (clone JBW301, 05–636, 1:1000 for WB), 53BP1 (clone BP-13, MAB3802, 1:300 for IF), GFP (clones 7.1 and 13.1, #11814460001, 1:1000 for WB), BrdU (clone BU-33, B8434, 1:100 for IF), anti-conjugated ubiquitin (FK2, 04-263, 1:2000 for IF) from Merck; γH2AX (clone D7T2V, #80312, 1:300 for IF), γH2AX (clone 20E3, #9718, 1:300 for IF), RAD18 (clone D2B9, #9040, 1:400 for IF, 1:1000 for WB), PCNA ubiquityl-Lys164 (clone D5C7P, #13439, 1:100 for IF), H2A (clone D603A, #12349, 1:1000 for WB) from Cell Signaling Technology; 53BP1 (NB100-305, 1:400 for IF), SMC5 (NB100-469, 1:300 for IF, 1:1000 for WB) from Novus Biologicals; RAD51 (ab176458, 1:400 for IF) and H4K20Me0 (ab227804, 1:2000 for IF, 1:1000 for WB) from Abcam.

Techniques: Control, Two Tailed Test, Viability Assay, Irradiation, Incubation, Staining, MANN-WHITNEY, Activity Assay, Ubiquitin Proteomics