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