Journal: Nucleic Acids Research

Article Title: The TIMELESS and PARP1 interaction suppresses replication-associated DNA gap accumulation

doi: 10.1093/nar/gkae445

Figure Lengend Snippet: siRNA-mediated TIM knockdown results in increased DNA damage and ssDNA gap accumulation. ( A ) Schematic of the DNA fiber assay with S1 endonuclease. ( B ) Western blotting (WB) confirming the knockdown of TIM in U2OS cells using two independent siRNA oligonucleotides (versus negative control, Ctrl). ( C ) Dot plot of the DNA fiber IdU track lengths from U2OS cells depleted of TIM by siRNA. Where indicated, 20 U/ml S1 nuclease was treated for 30 min. 10 μM olaparib was treated for 2 h as a positive control to induce DNA gaps. Red bars indicate the median value of at least 150 tracks. n = 3, **** P < 0.0001, ** P < 0.01, Mann–Whitney. ( D ) Dot plot of the DNA fiber IdU track lengths from Flp-In cells reconstituted with Flag-tagged TIM WT or EQ/EQ/TD via siRNA-mediated TIM knockdown and doxycycline (dox)-dependent expression of siRNA-resistant cDNA. Where indicated, cells were treated with 20 U/ml S1 nuclease for 30 min. Red bar: median, n = 3, **** P < 0.0001, ** P < 0.001, ns: not significant, Mann–Whitney. ( E ) Representative images of poly(ADP-ribose) or pADPr signals in U2OS cells knocked down of TIM using two independent siRNA. Where indicated, cells were treated with 10 μM PARGi for 30 min before fixation. Scale bar: 10 μm. ( F ) Quantification of pADPr immunofluorescence signals in either EdU negative or EdU positive cells. At least 250 cells were analyzed in each condition. Red bar: median, n = 3, **** P < 0.0001, ns: not significant, Mann–Whitney. ( G ) WB analysis of cellular pADPr levels in U2OS cells transfected with siRNA TIM (versus Ctrl) in the presence or absence of 10 μM PARG inhibitor (PARGi). ( H ) WB analysis of DNA damage in U2OS cells transfected with siRNA TIM (versus Ctrl) for 72 h.

Article Snippet: U2OS Flp-In T-REx TIM WT and EQ/EQ/TD cell lines were generated by co-transfecting pcDNA5/FRT/TO Flag-TIM and the pOG44 plasmid encoding the Flp recombinase (Invitrogen) into the host U2OS cell line stably expressing the Tet-repressor (T-REx) and carrying a single FRT locus, followed by hygromycin selection and recovery of stably transfected cells after 4 weeks.

Techniques: Knockdown, Western Blot, Negative Control, Positive Control, MANN-WHITNEY, Expressing, Immunofluorescence, Transfection

Journal: Nucleic Acids Research

Article Title: The TIMELESS and PARP1 interaction suppresses replication-associated DNA gap accumulation

doi: 10.1093/nar/gkae445

Figure Lengend Snippet: Physical disruption of the TIM–PARP1 interaction impairs PARP1-dependent OF processing. ( A ) Schematic depicting a strategy to disrupt the TIM–PARP1 interaction in an inducible manner. The C-terminal PARP1-binding region (PAB) of TIM fused to a destabilization domain (DD) is short-lived via rapid proteasomal degradation but stabilized by a synthetic ligand Shield-1 (Shld1), thus competing with endogenous TIM to disrupt its interaction with PARP1. ( B ) WB to show the induction of myc-tagged DD-PAB wildtype (WT) or EQ/EQ/TD mutant in cells treated with 1 μM Shld1 for the indicated times. ( C ) Representative images of the TIM–PARP1 PLA foci from U2OS DD-PAB WT or EQ/EQ/TD mutant cells following treatment of 1 μM Shld1 for the indicated times. Scale bar: 10 μm. ( D ) Quantification of cells positive for the TIM–PARP1 PLA foci. n = 3, mean ± SD, *** P < 0.001, ns: not significant, Student's t -test. ( E ) Dot plot of the DNA fiber IdU track lengths of DD-TIM-PAB WT or EQ/EQ/TD mutant cells following treatment of 1 μM Shld1 for the indicated times. Red bar: median, n = 2, **** P < 0.0001, ns: not significant, Mann–Whitney. ( F ) Dot plot representing the quantification of S phase-specific pADPr intensity from EdU-positive DD-TIM-PAB WT or EQ/EQ/TD mutant cells treated with 1 μM Shld1 for the indicated times. Red bar: median, n = 2, **** P < 0.0001, * P < 0.05, ns: not significant, Mann–Whitney. ( G ) WB to visualize cellular pADPr levels in DD-TIM-PAB WT or EQ/EQ/TD mutant cells treated with 10 μM FEN1i for 4 h and/or 1 μM Shld1 for 8 h. 10 μM PARGi was treated for 20 min before harvest. ( H ) Representative images of pADPr signals in DD-TIM-PAB WT or EQ/EQ/TD mutant cells transfected with siRNA LIG1 for 66 h and/or treated with 1 μM Shld1 for 8 h. 10 μM PARGi was treated for 20 min before fixation. (I) Dot plot representing S phase-specific XRCC1 foci intensity in EdU-positive DD-TIM-PAB WT or EQ/EQ/TD mutant cells transfected with siRNA LIG1 for 66 h and/or treated with 1 μM Shld1 for 8 h. Red bar: median, n = 2, **** P < 0.0001, * P < 0.05, ns: not significant, Mann–Whitney.

Article Snippet: U2OS Flp-In T-REx TIM WT and EQ/EQ/TD cell lines were generated by co-transfecting pcDNA5/FRT/TO Flag-TIM and the pOG44 plasmid encoding the Flp recombinase (Invitrogen) into the host U2OS cell line stably expressing the Tet-repressor (T-REx) and carrying a single FRT locus, followed by hygromycin selection and recovery of stably transfected cells after 4 weeks.

Techniques: Disruption, Binding Assay, Mutagenesis, MANN-WHITNEY, Transfection

Journal: Nucleic Acids Research

Article Title: The TIMELESS and PARP1 interaction suppresses replication-associated DNA gap accumulation

doi: 10.1093/nar/gkae445

Figure Lengend Snippet: The TIM–PARP1 interaction is necessary for engaging PARP1 to ssDNA gaps behind replication forks. ( A ) HCT116-mAID cells were treated with 10 μM FEN1i for 6 h and/or 1 μM 5-Ph-IAA for 6 h. Following 125 μM EdU pulse and click reaction to conjugate EdU-labeled forks with biotin, replication fork-associated proteins were isolated by iPOND and analyzed by WB. ( B ) Quantification of the numbers of PARP1-EdU PLA foci from cells containing more than two PLA foci. U2OS DD-PAB WT cells were treated with 1 μM Shld1 for 24 h and 10 μM FEN1i for 6 h followed by a brief pulse with 125 μM EdU for 12 min and chase with 1 mM thymidine for another 10 min to label DNA behind replication forks. 10 μM PARGi was treated during the last 20 min of FEN1i incubation and during the EdU pulse. Dashed lines indicate Q1, median, and Q3. n = 2, **** P < 0.0001, Mann–Whitney. ( C ) Representative images of the PARP1-EdU PLA foci. Scale bar: 10 μm. ( D ) Quantification of the numbers of XRCC1-EdU PLA foci. Cells were treated as (B). Dashed lines indicate Q1, median, and Q3. n = 2, **** P < 0.0001, Mann–Whitney. ( E ) Representative images of the XRCC1-EdU PLA foci. Scale bar: 10 μm.

Article Snippet: U2OS Flp-In T-REx TIM WT and EQ/EQ/TD cell lines were generated by co-transfecting pcDNA5/FRT/TO Flag-TIM and the pOG44 plasmid encoding the Flp recombinase (Invitrogen) into the host U2OS cell line stably expressing the Tet-repressor (T-REx) and carrying a single FRT locus, followed by hygromycin selection and recovery of stably transfected cells after 4 weeks.

Techniques: Labeling, Isolation, Incubation, MANN-WHITNEY

Journal: Nucleic Acids Research

Article Title: The TIMELESS and PARP1 interaction suppresses replication-associated DNA gap accumulation

doi: 10.1093/nar/gkae445

Figure Lengend Snippet: Disruption of the TIM–PARP1 interaction triggers synergistic replication fork instability in cells deficient in the canonical OF processing pathway. ( A ) Quantification of cellular viability measured by ATP-dependent luminescence. TIM-mAID cells were treated with DMSO or 10 μM FEN1i for the first 24 h followed by 1 μM 5-Ph-IAA (versus control) for 3 more days. n = 3, mean ± SD, **** P < 0.0001, *** P < 0.001, Student's t -test. ( B ) Quantification of clonogenic survival of TIM-mAID cells treated with DMSO or 10 μM FEN1i for the first 24 h followed by 1 μM 5-Ph-IAA (versus control) for 11 more days. n = 3, mean ± SD, **** P < 0.0001, Student's t -test. ( C ) Representative images of colony formation in (B). ( D ) Quantification of cellular viability measured by ATP-dependent luminescence. U2OS DD-PAB WT cells treated with DMSO or 10 μM FEN1i for the first 24 h followed by 1 μM Shld1 (versus control) for 3 more days. n = 3, mean ± SD, **** P < 0.0001, Student's t -test. (E) Quantification of clonogenic survival of U2OS DD-PAB WT cells treated with DMSO or 10 μM FEN1i for the first 24 h followed by 1 μM Shld1 (versus control) for 11 more days. n = 3, mean ± SD, **** P < 0.0001, * P < 0.05, Student's t -test. ( F ) Representative images of colony formation in (E). ( G ) TIM-mAID cells were treated with 1 μM 5-Ph-IAA for 48 h and/or 10 μM FEN1i for the first 24 h, and DNA damage and cell death were analyzed by WB. ( H ) As (G) except where indicated cells were treated with 50 μM mirin for 12 h before harvest. ( I ) U2OS DD-PAB WT cells were first treated with 10 μM FEN1i for 24 h and then replenished with media containing 1 μM Shld1 for additional 48 h. Cell lysates were analyzed by WB.

Article Snippet: U2OS Flp-In T-REx TIM WT and EQ/EQ/TD cell lines were generated by co-transfecting pcDNA5/FRT/TO Flag-TIM and the pOG44 plasmid encoding the Flp recombinase (Invitrogen) into the host U2OS cell line stably expressing the Tet-repressor (T-REx) and carrying a single FRT locus, followed by hygromycin selection and recovery of stably transfected cells after 4 weeks.

Techniques: Disruption, Control