Journal: Cell reports

Article Title: Poly(ADP-ribosyl)ation of TIMELESS limits DNA replication stress and promotes stalled fork protection

doi: 10.1016/j.celrep.2024.113845

Figure Lengend Snippet: (A) coIP of endogenous TIM and PARP1 in 293T cells released from G1/S block. (B) Left: PLA of PARP1 with EdU-labeled replication forks in U2OS WT versus PARP1 KO cells. Right: quantification of cells positive for PARP1:EdU PLA foci. Scale bar: 10 μm; **p < 0.01, Student’s t test. (C) Left: The PARP1:TIM PLA in U2OS cells. S phase cells were marked with EdU. Right: percentages of cells positive for PLA foci in EdU positive or negative cells. Scale bar: 10 μm; **p < 0.01, Student’s t test. (D) Schematic of TIM-PAB and the catalytic domain of PARP1. (E) Anti-FLAG coIP of TIM variants with endogenous PARP1 in 293T cells. EV, empty vector. (F) Anti-myc coIP of TIM-myc with FLAG-PARP1 variants. (G) Top: schematic of the Flp-In T-REx system to express siRNA-resistant FLAG-TIM WT or EQ/EQ/TD by doxycycline (dox) mediated derepression of the promoter. Bottom: verification of U2OS Flp-In cell lines reconstituting TIM WT or EQ/EQ/TD in response to increasing doses of dox, following transfection of TIM siRNA. (H) Anti-FLAG coIP of endogenous PARP1 by TIM WT or EQ/EQ/TD from the Flp-In cells. (I) Dot plot of the DNA fiber iodo-deoxyuridine (IdU) track length from Flp-In cells. Red bars indicate the median from at least 150 tracks. ****p < 0.0001, n.s., not significant; two-way ANOVA with post hoc test and false discovery rate (FDR) adjustment. (J) Dot plot of the IdU/chloro-deoxyuridine (CldU) ratios from Flp-In cells treated with 4 mM HU for 4 h. ****p < 0.0001; two-way ANOVA. (B and C) Mean ± SD from 3 biological replicates. (I and J) Representative plot from 3 biological replicates. See also .

Article Snippet: PARP1 autoPARylation assay was carried out in 10 μL reaction volume, by first equilibrating 10 U of recombinant PARP1 (R&D Systems, #4668–100-01) with 2 μM 16-mer dsDNA in 1X reaction buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 4 mM MgCl 2 ) complemented with EDTA-free protease inhibitor cocktail (Thermo Fisher) for 5 min on ice.

Techniques: Blocking Assay, Labeling, Plasmid Preparation, Transfection

Journal: Cell reports

Article Title: Poly(ADP-ribosyl)ation of TIMELESS limits DNA replication stress and promotes stalled fork protection

doi: 10.1016/j.celrep.2024.113845

Figure Lengend Snippet: (A) Schematic of two conserved PBMs within TIM with the relative positions of binding partners TIPIN and PARP1. (B) Top: schematic of the glutathione S-transferase-tagged recombinant TIM fragment containing amino acids with PBM2 WT or K949A/K950A (KA). Bottom: slot blot loaded with corresponding recombinant PBM2 and incubated with biotinylated PAR chains (pADPr). (C) Top: schematic of synthesized PBM1 26 amino acid-peptides, either WT or with 4 mutations in basic residues. Bottom: slot blot of corresponding peptides incubated with pADPr. (D) In vitro auto-PARylation assay with recombinant PARP1, activating dsDNA, and 50 μM NAD + , as well as 10 μM olaparib, where indicated. (E) Anti-FLAG IP of IVTT FLAG-TIM WT reacted with recombinant PARP1 in the presence of 25 μM of biotinylated NAD + . (F) Anti-FLAG IP of IVTT FLAG-TIM reacted with recombinant PARP1 in the presence of 25 μM of biotinylated NAD + . (G) Quantification of (F). Mean ± SD from three biological replicates. *p < 0.05; ***p < 0.001; ****p < 0.0001; one-way ANOVA. (H) Model for the recognition of auto-PARylated PARP1 by TIM (1) and subsequent PARP1-dependent TIM PARylation (2). (I and J) Anti-FLAG IP of FLAG-TIM in denaturing conditions followed by anti-PAR western blot (WB) from 293T cells treated with 1 μM CPT and 10 μM PARGi (PDD00017273) with or without 10 mM olaparib pretreatment for 1 h. (K) coIP of endogenous PARP1 and PARylated PARP1 by anti-FLAG TIM IP in chromatin-enriched fractions of U2OS cells treated with 1 μM CPT and 10 μM PARGi for 1 h. (L) In vivo PARylation of FLAG-TIM in cells transfected with 2 independent siRNAs against HPF1 and treated with CPT/PARGi. See also .

Article Snippet: PARP1 autoPARylation assay was carried out in 10 μL reaction volume, by first equilibrating 10 U of recombinant PARP1 (R&D Systems, #4668–100-01) with 2 μM 16-mer dsDNA in 1X reaction buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 4 mM MgCl 2 ) complemented with EDTA-free protease inhibitor cocktail (Thermo Fisher) for 5 min on ice.

Techniques: Binding Assay, Recombinant, Dot Blot, Incubation, Synthesized, In Vitro, Western Blot, In Vivo, Transfection

Journal: Cell reports

Article Title: Poly(ADP-ribosyl)ation of TIMELESS limits DNA replication stress and promotes stalled fork protection

doi: 10.1016/j.celrep.2024.113845

Figure Lengend Snippet: (A) Left: TIM-myc and FLAG-PARP1 were transiently expressed in U2OS or PARP1 KO cells and analyzed by WB. Where indicated, 10 mM MG132 was treated for 4 h. (B) Quantification of TIM with each MG132 condition normalized to its corresponding untreated condition. *p < 0.05; **p < 0.01; Student’s t test. (C) WB analysis of FLAG-TIM WT or PBM1/2 levels in U2OS or PARP1 KO cells. (D) Expression of TIM variants in U2OS cells versus IVTT expression. (E) Anti-FLAG IP of FLAG-TIM in denaturing conditions followed by anti-PAR WB in 293T cells treated with 10 μM PARGi with or without 1 μM CPT for 1 h. (F) Ubiquitination assay of TIM-myc WT and PBM1/2 overexpressed in 293T cells with His-Ub and treated with 1 μM CPT and 10 μM MG132. (G) Analysis of pRPA32 S4/S8 in U2OS cells transfected with increasing amounts of FLAG-TIM WT or PBM1/2. (H) Structure depicting the MCM PI of TIM that includes anchor and DBM, and their conservation throughout multiple species. Adapted from PDB: 7PFO and created using Chimera. (I) coIP of FLAG-TIM PBM1/2 or PBM1/2 ∆PI (amino acid D273–281 and amino acid ∆316–322) by endogenous MCM6 in 293T cells. (J) Analysis of pRPA32 S4/S8 in U2OS cells induced by FLAG-TIM WT, PBM1/2, or PBM1/2 ∆PI. (K) The PLA between TIM and PARP1. A total of 10 μM olaparib was treated for 24 h, and 10 μM EdU was added for the last 30 min before fixation. Scale bar: 10 μm ****p < 0.0001; two-way ANOVA. (B and K) Mean ± SD from 3 biological replicates. See also .

Article Snippet: PARP1 autoPARylation assay was carried out in 10 μL reaction volume, by first equilibrating 10 U of recombinant PARP1 (R&D Systems, #4668–100-01) with 2 μM 16-mer dsDNA in 1X reaction buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 4 mM MgCl 2 ) complemented with EDTA-free protease inhibitor cocktail (Thermo Fisher) for 5 min on ice.

Techniques: Expressing, Ubiquitin Proteomics, Transfection

Journal: Cell reports

Article Title: Poly(ADP-ribosyl)ation of TIMELESS limits DNA replication stress and promotes stalled fork protection

doi: 10.1016/j.celrep.2024.113845

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: PARP1 autoPARylation assay was carried out in 10 μL reaction volume, by first equilibrating 10 U of recombinant PARP1 (R&D Systems, #4668–100-01) with 2 μM 16-mer dsDNA in 1X reaction buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 4 mM MgCl 2 ) complemented with EDTA-free protease inhibitor cocktail (Thermo Fisher) for 5 min on ice.

Techniques: Control, Virus, Recombinant, TNKS1 Histone Ribosylation Assay, Transfection, Protease Inhibitor, In Situ, DNA Extraction, Mutagenesis, Cell Viability Assay, Staining, Negative Control, Software

Journal: Communications biology

Article Title: Poly ADP-ribosylation of SET8 leads to aberrant H4K20 methylation in mammalian nuclear genome.

doi: 10.1038/s42003-022-04241-8

Figure Lengend Snippet: Fig. 6 PARP1 regulates H4K20me1 and H4K20me3 distribution in the genome. a Spearman correlation of H4K20me1 and H4K20me3 regions in PARP1 knockdown HeLa cells and its control. b Genome-wide metagene plot showing H4K20me1 profile (ChIP-seq) in control and PARP1 knockdown HeLa cells. c Genome-wide metagene plot showing H4K20me3 profile (ChIP-seq) in control and PARP1 knockdown HeLa cells. d Peak width profile of H4K20me1 by binning the peaks into different lengths in PARP1 knockdown cells and its control. e Peak width profile of H4K20me3 by binning the peaks into different lengths in PARP1 knockdown cells and its control. f Representative IGV genomic tracks showing H4K20me1 and RNAseq profile in PARP1 knockdown cells and its control.

Article Snippet: SET8 recombinant enzyme (New England Biolabs # M0428S) was incubated with recombinant active PARP1 (Trevigen # 4668-100-01) with or without EcoRI hairpin oligo or NAD+ in histone methyltransferase buffer and 6 μM of radiolabeled [3H] AdoMet (Perkin Elmer Life Science # NET155V001MC).

Techniques: Knockdown, Control, Genome Wide, ChIP-sequencing

Journal: Molecular cancer

Article Title: BTApep-TAT peptide inhibits ADP-ribosylation of BORIS to induce DNA damage in cancer.

doi: 10.1186/s12943-022-01621-w

Figure Lengend Snippet: Fig. 5 BTApep-TAT inhibited the ADP ribosylation at residues 198–228 of BORIS and inhibited the interaction of BORIS with Ku70. (A) Truncations of variable lengths of amino acids between AA 2 and 227 in the N-terminus of BORIS were constructed and transfected into H1299 cells. ADP ribosylation of the short BORIS protein, which contains residues 228–663 of BORIS, was reduced compared with that of BORIS 198–663. (B) Schematic representation of the truncated BORIS proteins and the details of the homology of BORIS in Mus musculus (Mus.), Rattus norvegicus (Rattus.) and Homo sapiens (Homo.). Conserved amino acids are shown in red boxes. Five conserved glutamic acid residues were mutated to alanine residues in the BORIS-5EA mutant. (C) The 5EA quintuple mutant of BORIS tagged with myc (5EA) or BORIS-myc (WT) was transfected into H1299 cells. The cell lysates were used to evaluate the interactions of ADP-ribosylated and myc-tagged proteins. (D) ADP-ribosylation of the 5EA quintuple mutant and other mutants (which contained substitutions of glutamic acid residues with alanine residues in the 29–33 or 165–185 regions) were compared by precipitation with an anti-ADP-ribose antibody and visualization by dot blot or Western blot assays with a myc antibody. (E) The BORIS-N198-228 peptide was subjected to PARP1-mediated PARylation in vitro, which was activated by recombinant PARP1 and ssDNA. After the reaction, the peptides and PARP1 were separated by centrifugal filtration and analyzed by the dot blot assay. (F) DNA damage induced by 30 Gy X-ray irradiation was examined in BORIS-WT- and BORIS-5EA-transfected H1299 cells by TUNEL assay. (G) The comparison of BER activity of the mutants is presented as a histogram. (H) BORIS-myc and Ku70-HA were co-transfected into H1299 cells, and the cells were incubated with BTApep-TA or His-TAT to investigate the interaction between BORIS and Ku70. (I) BORIS-myc/BORIS-5EA-myc and Ku70-HA were co-transfected into H1299 cells, and the interaction between BORIS and Ku70 was examined by immunoprecipitation

Article Snippet: Peptides (1–4 μg) incubated with different samples were added to 50 μL PARP1 reaction buffer (50 mM Tris– HCl at pH 7.4, 2 mM MgCl2, 200 μM NAD+), which contained 0.2 μg of recombinant PARP1 (Trevigen, 4668–100-01) and 2.5 μg of ssDNA (Sigma, D8899), and reaction was carried out at 37 °C for 30 min. Low molecular weight peptide and PARP1 protein in the reactions were separated by a 30 kDa cutoff centrifugal filter (Millipore, UFC803096).

Techniques: Construct, Transfection, Mutagenesis, Dot Blot, Western Blot, In Vitro, Recombinant, Filtration, Irradiation, TUNEL Assay, Comparison, Activity Assay, Incubation, Immunoprecipitation

Journal: International Journal of Molecular Sciences

Article Title: PARP Inhibitor Decreases Akt Phosphorylation and Induces Centrosome Amplification and Chromosomal Aneuploidy in CHO-K1 Cells

doi: 10.3390/ijms23073484

Figure Lengend Snippet: 3AB, a PARP inhibitor, induced aneuploidy of chromosomes. ( A ) Photograph of metaphase of cells treated with or without 7 mM 3AB for 72 h. ( B ) Histogram of cells (%) with a defined number of chromosomes. At least 100 metaphases were analyzed. The number of cells with chromosome number outside of the range of 20 ± 2 was judged as abnormal. Significance was analyzed with Fisher’s exact test.

Article Snippet: Each reaction mixture of 100 μL contained 50 mM Tris-HCl, pH 8.0, 10 mM MgCl 2 , 1 mM DTT, 100 μg/mL BSA, 0.1 mM [ 32 P]NAD + (0.2 μCi/μmol), 20 μg/mL of sonicated salmon sperm DNA, 20 μg/mL of calf thymus histone and 3 mU of human PARP1 (Trevigen, PARP-HSA) in the presence or absence of indicated amounts of AKT inhibitors.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: PARP Inhibitor Decreases Akt Phosphorylation and Induces Centrosome Amplification and Chromosomal Aneuploidy in CHO-K1 Cells

doi: 10.3390/ijms23073484

Figure Lengend Snippet: The PARP inhibitor 3AB reduced phosphorylated Akt levels. ( A ) The cell proliferation signaling proteins p38 MAPK, ERK 1/2 and Akt, together with their phosphorylated proteins, were detected by immunoblot analysis after CHO-K1 cells were treated with 7 mM 3AB for 48 h. α-Tubulin is shown as a loading control. ( B ) The relative intensities of Western blots from 3 experiments are summarized. Significance was assessed against the control. * p < 0.05. ** p < 0.01.

Article Snippet: Each reaction mixture of 100 μL contained 50 mM Tris-HCl, pH 8.0, 10 mM MgCl 2 , 1 mM DTT, 100 μg/mL BSA, 0.1 mM [ 32 P]NAD + (0.2 μCi/μmol), 20 μg/mL of sonicated salmon sperm DNA, 20 μg/mL of calf thymus histone and 3 mU of human PARP1 (Trevigen, PARP-HSA) in the presence or absence of indicated amounts of AKT inhibitors.

Techniques: Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: PARP Inhibitor Decreases Akt Phosphorylation and Induces Centrosome Amplification and Chromosomal Aneuploidy in CHO-K1 Cells

doi: 10.3390/ijms23073484

Figure Lengend Snippet: Akt inhibitors did not inhibit PARP1 enzyme activity. Relative PARP1 activity was determined in the presence of defined concentrations of 3AB, API-2 and 10-NCP. H 2 O was used as a negative control.

Article Snippet: Each reaction mixture of 100 μL contained 50 mM Tris-HCl, pH 8.0, 10 mM MgCl 2 , 1 mM DTT, 100 μg/mL BSA, 0.1 mM [ 32 P]NAD + (0.2 μCi/μmol), 20 μg/mL of sonicated salmon sperm DNA, 20 μg/mL of calf thymus histone and 3 mU of human PARP1 (Trevigen, PARP-HSA) in the presence or absence of indicated amounts of AKT inhibitors.

Techniques: Activity Assay, Negative Control