Journal: Cell communication and signaling : CCS

Article Title: Poly(ADP-ribosyl)ation of acetyltransferase NAT10 by PARP1 is required for its nucleoplasmic translocation and function in response to DNA damage.

doi: 10.1186/s12964-022-00932-1

Figure Lengend Snippet: Fig. 2 PARP1 PARylates NAT10 at K1016, K1017, and K1020 both in vitro and in vivo. A Four GST-NAT10 deletion mutants (∆1–201, ∆202–488, ∆489–753, and ∆754–1025) and GST control were bacterially purified, and subjected to in vitro PARation assay in the presence of PARP1 and NAD+. The reaction samples were resolved by SDS-PAGE, and analyzed by immunoblotting analyses with anti-PAR and anti-GST antibodies. B In vitro PARylation assays were performed using purified GST-NAT10 deletion fragments in the presence or absence of recombinant PARP1 enzyme, NAD+, and Olaparib. PARylated NAT10 was detected with an anti-PAR antibody. C–E GST-NAT10 deletion fragments were subjected to in vitro PARation assay as described in A. F, G Purified GST-NAT10 754–1025 proteins (WT, K1016A, K1017A, D1018A, and K1020A, K3A) were subjected to in vitro PARation assays in the presence of PARP1 and NAD+. PARylation of NAT10 was detected by immunoblotting with an anti-PAR antibody. In G, K3A represents the combined mutation in all three residues (K1016, K1017, and K1020). H Alignment of the NAT10 protein sequence among different organisms. Asterisk (*) indicates the full conservation of the residues of NAT10 among different species. I, J MCF-7 cells were transfected with HA-NAT10 or HA-NAT10 K3A expression vector. After 48 h of transfection, cells were treated with or without 1 mM MMS for 2 h (I) or 6 Gy IR (J). Thereafter, IP and immunoblotting analyses were conducted with the indicated antibodies

Article Snippet: The purified GST-NAT10 fragment (1 μg) was incubated with 100 ng of recombinant full-length PARP1 (Origene, #TP710053) in a reaction buffer containing 100 mM Tris–HCl (pH 8.0), 10 mM MgCl2, 1 mM DTT, 4 ng/ml sonicated salmon sperm DNA (Invitrogen, #AM9680), and 300 μM nicotinamide adenine dinucleotide (NAD+) at 37 °C for 30 min.

Techniques: In Vitro, In Vivo, Control, Purification, SDS Page, Western Blot, Recombinant, Mutagenesis, Sequencing, Transfection, Expressing, Plasmid Preparation

Journal: Cell communication and signaling : CCS

Article Title: Poly(ADP-ribosyl)ation of acetyltransferase NAT10 by PARP1 is required for its nucleoplasmic translocation and function in response to DNA damage.

doi: 10.1186/s12964-022-00932-1

Figure Lengend Snippet: Fig. 3 PARylation of NAT10 by PARP1 regulates its nucleoplasmic translocation and co-localization with MORC2. A MCF-7 cells were transfected with plasmid DNAs encoding Flag-MORC2, HA-NAT10, or HA-NAT10 K3A. After 48 h of transfection, cells were treated with or without 1 mM MMS for 2 h or 6 Gy IR. IF staining was performed with an anti-Flag (green) or anti-HA (red) antibody. DNA was counterstained with DAPI (blue). Scale bar, 2.5 μm. The quantitative results of cells with NAT10 nucleoplasmic translocation are presented in the right panel. **p < 0.01; NS, no significance. B, C MCF-7 cells were transfected with HA-NAT10 and Flag-MORC2. After 48 h of transfection, cells were pretreated with or without 10 μM Olaparib for 3 h, and then treated with or without 1 mM MMS for another 2 h (B) or 6 Gy IR (C). IF staining was performed with an anti-Flag (green) or anti-HA (red) antibody. DNA was counterstained with DAPI (blue). Scale bar, 2.5 μm. The quantitative results of cells with NAT10 nucleoplasmic translocation are displayed in the right panel. **p < 0.01; ***p < 0.001. D, E PARP1-KO MCF-7 cells were transfected with HA-NAT10 and Flag-MORC2. After 48 h of transfection, cells were treated with or without 1 mM MMS for another 2 h (D) or 6 Gy IR (E). IF staining was performed with an anti-Flag (green) or anti-HA (red) antibody. DNA was counterstained with DAPI (blue). Scale bar, 2.5 μm. The quantitative results of cells with NAT10 nucleoplasmic translocation are displayed in the right panel. **p < 0.01; ***p < 0.001. Arrows indicate the colocalization between MORC2 and NAT10

Article Snippet: The purified GST-NAT10 fragment (1 μg) was incubated with 100 ng of recombinant full-length PARP1 (Origene, #TP710053) in a reaction buffer containing 100 mM Tris–HCl (pH 8.0), 10 mM MgCl2, 1 mM DTT, 4 ng/ml sonicated salmon sperm DNA (Invitrogen, #AM9680), and 300 μM nicotinamide adenine dinucleotide (NAD+) at 37 °C for 30 min.

Techniques: Translocation Assay, Transfection, Plasmid Preparation, Staining

Journal: Cell communication and signaling : CCS

Article Title: Poly(ADP-ribosyl)ation of acetyltransferase NAT10 by PARP1 is required for its nucleoplasmic translocation and function in response to DNA damage.

doi: 10.1186/s12964-022-00932-1

Figure Lengend Snippet: Fig. 4 PARylation of NAT10 by PARP1 regulates its interaction with MORC2. A HEK293T cells were transfected with the indicated expression vectors. After 48 h of transfection, cells were treated with or without 1 mM MMS for 2 h and subjected to IP and immunoblotting analyses with the indicated antibodies. B, C BT549 cells were transfected with plasmid DNAs encoding pCDH, HA-NAT10, or HA-NAT10 K3A. After 48 h of transfection, cells were treated with or without 1 mM MMS for 2 h (B) or 6 Gy IR (C). IP and immunoblotting analyses were performed with the indicated antibodies. D–F MCF-7 and BT549 cells were pretreated with or without 10 μM Olaparib for 3 h, and then treated with or without 1 mM MMS for another 2 h. The sequential IP and immunoblotting analyses were performed with the indicated antibodies. G–I MCF-7 and BT549 cells were pretreated with or without 10 μM Olaparib for 3 h, and then treated with or without 6 Gy IR. The sequential IP and immunoblotting analyses were performed with the indicated antibodies

Article Snippet: The purified GST-NAT10 fragment (1 μg) was incubated with 100 ng of recombinant full-length PARP1 (Origene, #TP710053) in a reaction buffer containing 100 mM Tris–HCl (pH 8.0), 10 mM MgCl2, 1 mM DTT, 4 ng/ml sonicated salmon sperm DNA (Invitrogen, #AM9680), and 300 μM nicotinamide adenine dinucleotide (NAD+) at 37 °C for 30 min.

Techniques: Transfection, Expressing, Western Blot, Plasmid Preparation

Journal: Cell communication and signaling : CCS

Article Title: Poly(ADP-ribosyl)ation of acetyltransferase NAT10 by PARP1 is required for its nucleoplasmic translocation and function in response to DNA damage.

doi: 10.1186/s12964-022-00932-1

Figure Lengend Snippet: Fig. 6 DNA damage induces MORC2 K767Ac in a PARP1-dependent manner. A MCF-7 cells were pretreated with or without 10 μM ATM inhibitor (KU-55933), 10 μM ATR inhibitor (VE-821), 10 μM DNA-PKcs inhibitor (NU7441), and 10 μM PARP inhibitor (Olaparib) for 3 h, and then treated with 1 mM MMS for 1 h. IP and immunoblotting analyses were performed with the indicated antibodies. Positive controls for these inhibitors are shown in the input. B HEK293T cells stably expressing pCDH and Flag-MORC2 were pretreated with or without 10 μM Olaparib for 3 h, and then treated with 1 mM MMS for another 2 h or 6 Gy IR. IP and immunoblotting analyses were performed with the indicated antibodies. C MCF-7 and BT549 cells were pretreated with or without 10 μM Olaparib for 3 h, and then treated with 1 mM MMS for another 2 h or 6 Gy IR. IP and immunoblotting analyses were performed with the indicated antibodies. D WT and PARP1-KO MCF-7 cells were treated with or without 1 mM MMS for 2 h or 6 Gy IR. Thereafter, IP and immunoblotting analyses were carried out with the inidicated antibodies. E BT549 cells were transfected with siNC or two independent siRNA targeting PARP1 (siPARP1). After 48 h of transfection, cells were treated with or without 1 mM MMS for 2 h or 6 Gy IR. IP and immunoblotting analyses were subsequently performed with the indicated antibodies

Article Snippet: The purified GST-NAT10 fragment (1 μg) was incubated with 100 ng of recombinant full-length PARP1 (Origene, #TP710053) in a reaction buffer containing 100 mM Tris–HCl (pH 8.0), 10 mM MgCl2, 1 mM DTT, 4 ng/ml sonicated salmon sperm DNA (Invitrogen, #AM9680), and 300 μM nicotinamide adenine dinucleotide (NAD+) at 37 °C for 30 min.

Techniques: Western Blot, Stable Transfection, Expressing, Transfection

Journal: Cell communication and signaling : CCS

Article Title: Poly(ADP-ribosyl)ation of acetyltransferase NAT10 by PARP1 is required for its nucleoplasmic translocation and function in response to DNA damage.

doi: 10.1186/s12964-022-00932-1

Figure Lengend Snippet: Fig. 5 PARylation of NAT10 by PARP1 regulates MORC2 acetylation in response to DNA damage. A, B NAT10-KO MCF-7 and BT549 cells were transfected with plasmid DNAs encoding pCDH, HA-NAT10, or HA-NAT10 K3A. After 48 h of transfection, cells were treated with or without 1 mM MMS for 2 h (A) or 6 Gy IR (B), and then subjected to IP and immunoblotting with the indicated antibodies

Article Snippet: The purified GST-NAT10 fragment (1 μg) was incubated with 100 ng of recombinant full-length PARP1 (Origene, #TP710053) in a reaction buffer containing 100 mM Tris–HCl (pH 8.0), 10 mM MgCl2, 1 mM DTT, 4 ng/ml sonicated salmon sperm DNA (Invitrogen, #AM9680), and 300 μM nicotinamide adenine dinucleotide (NAD+) at 37 °C for 30 min.

Techniques: Transfection, Plasmid Preparation, Western Blot

Journal: Cell communication and signaling : CCS

Article Title: Poly(ADP-ribosyl)ation of acetyltransferase NAT10 by PARP1 is required for its nucleoplasmic translocation and function in response to DNA damage.

doi: 10.1186/s12964-022-00932-1

Figure Lengend Snippet: Fig. 7 PARylation of NAT10 by PARP1 is required for cell survival in response to DNA damage. A, B NAT10-KO MCF-7 and BT549 cells stably expressing pCDH, HA-NAT10 WT, or HA-NAT10 K3A were treated with increasing doses of MMS and then subjected to clonogenic survival assays. Representative images of survival colonies are displayed in A and the corresponding quantitative results are shown in B. C, D NAT10-KO MCF-7 and BT549 cells stably expressing pCDH, HA-NAT10 WT, or HA-NAT10 K3A were treated with or without 6 Gy IR, and then subjected to clonogenic survival assays. Representative images of survival colonies are displayed in C and the corresponding quantitative results are shown in D

Article Snippet: The purified GST-NAT10 fragment (1 μg) was incubated with 100 ng of recombinant full-length PARP1 (Origene, #TP710053) in a reaction buffer containing 100 mM Tris–HCl (pH 8.0), 10 mM MgCl2, 1 mM DTT, 4 ng/ml sonicated salmon sperm DNA (Invitrogen, #AM9680), and 300 μM nicotinamide adenine dinucleotide (NAD+) at 37 °C for 30 min.

Techniques: Stable Transfection, Expressing

Journal: Cell communication and signaling : CCS

Article Title: Poly(ADP-ribosyl)ation of acetyltransferase NAT10 by PARP1 is required for its nucleoplasmic translocation and function in response to DNA damage.

doi: 10.1186/s12964-022-00932-1

Figure Lengend Snippet: Fig. 8 The proposed working model. Activated PARP1 after DNA damage catalyzes the PARylation of NAT10, which is required for the translocation of NAT10 from the nucleolus to the nucleoplasm. NAT10 relocalization increases its co-localization and interaction with its substrate, MORC2, thereby enhancing MORC2 K767Ac in response to DNA damage

Article Snippet: The purified GST-NAT10 fragment (1 μg) was incubated with 100 ng of recombinant full-length PARP1 (Origene, #TP710053) in a reaction buffer containing 100 mM Tris–HCl (pH 8.0), 10 mM MgCl2, 1 mM DTT, 4 ng/ml sonicated salmon sperm DNA (Invitrogen, #AM9680), and 300 μM nicotinamide adenine dinucleotide (NAD+) at 37 °C for 30 min.

Techniques: Translocation Assay

Journal: Molecular cell

Article Title: SPARCLE , a p53-induced lncRNA, controls apoptosis after genotoxic stress by promoting PARP-1 cleavage

doi: 10.1016/j.molcel.2022.01.001

Figure Lengend Snippet: (A) PARP-1 domains. FI-FIII are Zinc finger domains. Arrow shows the caspase-3/7 cleavage site.

Article Snippet: In vitro transcription 2 μg of linearized plasmid encoding full-length SPARCLE ( SPARCLE 3K ), the first 75 nt ( SPARCLE 75 ), 178 nt ( SPARCLE 178 ) or 275 nt ( SPARCLE 275 ) of SPARCLE sequence or the reverse sequence of SPARCLE 275 ( ELCRAPS ) were in vitro transcribed using the MEGAScript T7 transcription kit (AM1334, ThermoFisher Scientific) following the manufacturer’s directions, but with increased incubation time (6 hr). . PARP-1 in vitro cleavage 50 ng of human recombinant full-length PARP-1 protein (11040-H08B, Sino Biological) were incubated at 37°C for 10 min with 0.5 units of human recombinant cleaved Caspase 3 protein (ab52101, Abcam) and with the indicated amount of in vitro transcribed SPARCLE 75 , SPARCLE 178 , SPARCLE 275 or ELCRAPS .

Techniques:

Journal: Molecular cell

Article Title: SPARCLE , a p53-induced lncRNA, controls apoptosis after genotoxic stress by promoting PARP-1 cleavage

doi: 10.1016/j.molcel.2022.01.001

Figure Lengend Snippet: Key Resources Table

Article Snippet: In vitro transcription 2 μg of linearized plasmid encoding full-length SPARCLE ( SPARCLE 3K ), the first 75 nt ( SPARCLE 75 ), 178 nt ( SPARCLE 178 ) or 275 nt ( SPARCLE 275 ) of SPARCLE sequence or the reverse sequence of SPARCLE 275 ( ELCRAPS ) were in vitro transcribed using the MEGAScript T7 transcription kit (AM1334, ThermoFisher Scientific) following the manufacturer’s directions, but with increased incubation time (6 hr). . PARP-1 in vitro cleavage 50 ng of human recombinant full-length PARP-1 protein (11040-H08B, Sino Biological) were incubated at 37°C for 10 min with 0.5 units of human recombinant cleaved Caspase 3 protein (ab52101, Abcam) and with the indicated amount of in vitro transcribed SPARCLE 75 , SPARCLE 178 , SPARCLE 275 or ELCRAPS .

Techniques: Recombinant, Binding Assay, Modification, Protease Inhibitor, Immunoprecipitation, Hybridization, Staining, Labeling, TUNEL Assay, Caspase-Glo Assay, PCR Cloning, Luciferase, Reporter Assay, Purification, Gel Extraction, Plasmid Preparation, Western Blot, Microscopy, Negative Control, Software, Imaging

Journal: Journal of Biological Chemistry

Article Title: Transcriptional Repression by Binding of Poly(ADP-ribose) Polymerase to Promoter Sequences

doi: 10.1074/jbc.m108551200

Figure Lengend Snippet: FIG. 1. Binding of recombinant PARP to three-way DNA junc- tions. A, schematic representation of heteroduplex DNA with an un- paired region at the apex of hairpin. B, AFM images of three-way DNA junctions containing a 50-bp hairpin (visible as the protrusion from the bend near the center of the molecule). C–E, representative AFM images of PARP-DNA complexes. End-bound (yellow arrows) and internally bound (white arrows) PARP molecules are indicated. Images show a 400- 400-nm surface area. The color scale ranges from 0.0 to 4.0 nm (from dark to bright). F, the effects of NAD (0.1 mM) and 3-aminoben- zamide (3AB) (1 mM) on the interaction of PARP with DNA ends and hairpins. PARP binding to DNA was calculated as the percentage of occurrence of the PARP-DNA complexes to the total number of hetero- duplexes scored. Only unobstructed protein-DNA complexes were quan- tified. The total numbers of DNAs counted in each experiment ranged from 420 to 540 molecules.

Article Snippet: PARP Binding Reactions—A recombinant full-length human PARP (R&D Systems) was used in DNA binding reactions at a 4:1 molar ratio (protein to DNA) under the ionic conditions required for optimal PARP activity (4, 21).

Techniques: Binding Assay, Recombinant

Journal: Journal of Biological Chemistry

Article Title: Transcriptional Repression by Binding of Poly(ADP-ribose) Polymerase to Promoter Sequences

doi: 10.1074/jbc.m108551200

Figure Lengend Snippet: FIG. 2. Interaction of PARP protein with the 1.1-kb 5-region of the PARP gene. A, binding of PARP to topologically relaxed pPR- PARP plasmid containing the PARP promoter region (from 899 to 156). B, binding of PARP to negatively supercoiled ( 0.050) pPR-PARP plasmid. C, AFM images of the PARP protein-promoter complexes. Bound PARP molecules were cross-linked to plasmid DNA with a superhelical density, of 0.050, and the promoter-containing fragment (1.1 kb) was isolated for AFM examination. Representative images A and B show a 700- 700-nm surface area, and image C shows an enlarged surface area (340 183 nm). Arrows (B and C) point to the PARP-DNA complex.

Article Snippet: PARP Binding Reactions—A recombinant full-length human PARP (R&D Systems) was used in DNA binding reactions at a 4:1 molar ratio (protein to DNA) under the ionic conditions required for optimal PARP activity (4, 21).

Techniques: Binding Assay, Plasmid Preparation, Isolation

Journal: Journal of Biological Chemistry

Article Title: Transcriptional Repression by Binding of Poly(ADP-ribose) Polymerase to Promoter Sequences

doi: 10.1074/jbc.m108551200

Figure Lengend Snippet: FIG. 3. Detection of P1 nuclease-sensitive sites in the PARP promoter. A, schematic representation of the human PARP promoter (from 899 to 1). The position of dyad symmetry elements (DSE) in the promoter sequence and the hairpin free energies calculated by the MFOLD program are indicated in the boxed area. Putative P1 nuclease- sensitive sites are shown with arrows. B, pPR-PARP topoisomers with superhelical density () ranging from 0 to 0.111 were treated with P1 nuclease. The promoter-containing fragment (1.1 kb) was isolated and analyzed by alkaline agarose gel electrophoresis. The products of P1 nuclease digestion are denoted on the right. Topoisomer fractions 0–7 numbered at the bottom had the average of 0, 0.019, 0.031, 0.050, 0.065, 0.080, 0.094, and 0.111, respectively.

Article Snippet: PARP Binding Reactions—A recombinant full-length human PARP (R&D Systems) was used in DNA binding reactions at a 4:1 molar ratio (protein to DNA) under the ionic conditions required for optimal PARP activity (4, 21).

Techniques: Sequencing, Isolation, Agarose Gel Electrophoresis

Journal: Journal of Biological Chemistry

Article Title: Transcriptional Repression by Binding of Poly(ADP-ribose) Polymerase to Promoter Sequences

doi: 10.1074/jbc.m108551200

Figure Lengend Snippet: FIG. 4. PARP protein binds to the 5-flanking region of the human PARP gene in vivo. Formaldehyde-cross-linked chromatin from asynchronously growing Ewing’s sarcoma cells (cell line A4573) was immunoprecipitated using anti-PARP polyclonal antibody. A no- antibody immunoprecipitation was performed for a negative control (None). The input sample contains total chromatin before selection by immunoprecipitation. Top panel, immunoprecipitated DNA was ana- lyzed by PCR using primers specific for the human PARP promoter. A 240-bp PCR fragment amplified from the PARP promoter sequence is shown. Bottom panel, immunoblotting analysis of PARP protein in cross-linked chromatin. IP, immunoprecipitation.

Article Snippet: PARP Binding Reactions—A recombinant full-length human PARP (R&D Systems) was used in DNA binding reactions at a 4:1 molar ratio (protein to DNA) under the ionic conditions required for optimal PARP activity (4, 21).

Techniques: In Vivo, Immunoprecipitation, Negative Control, Selection, Amplification, Sequencing, Western Blot, Chromatin Immunoprecipitation

Journal: Journal of Biological Chemistry

Article Title: Transcriptional Repression by Binding of Poly(ADP-ribose) Polymerase to Promoter Sequences

doi: 10.1074/jbc.m108551200

Figure Lengend Snippet: FIG. 5. PARP protein is a transcriptional repressor. A, PARP promoter transcriptional activity in wild type (PARP/) and PARP/

Article Snippet: PARP Binding Reactions—A recombinant full-length human PARP (R&D Systems) was used in DNA binding reactions at a 4:1 molar ratio (protein to DNA) under the ionic conditions required for optimal PARP activity (4, 21).

Techniques: Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Transcriptional Repression by Binding of Poly(ADP-ribose) Polymerase to Promoter Sequences

doi: 10.1074/jbc.m108551200

Figure Lengend Snippet: FIG. 6. A model for PARP-mediated regulation of transcrip- tion. I, in undamaged cells, unmodified PARP molecules bind to the DNA secondary structures within the gene promoter (denoted by a striped box). Such macromolecular interactions between PARP protein and a promoter region constitute a repressor function for PARP in transcription. II, in response to DNA damage, PARP binding to the DNA ends triggers its catalytic activity. Subsequent poly(ADP-ribosyl) ation of free and bound PARP in the presence of intracellular NAD

Article Snippet: PARP Binding Reactions—A recombinant full-length human PARP (R&D Systems) was used in DNA binding reactions at a 4:1 molar ratio (protein to DNA) under the ionic conditions required for optimal PARP activity (4, 21).

Techniques: Binding Assay, Activity Assay

Journal:

Article Title: Poly(ADP-ribosyl)ation of p53 In Vitro and In Vivo Modulates Binding to its DNA Consensus Sequence 1

doi:

Figure Lengend Snippet: Immunoblot analysis of recombinant p53 and PARP-1 proteins; catalytic activities of wild-type and mutant PARP-1. (A) Immunoblot analysis with anti-p53 of purified GST-p53(1–393) (0.1 µg) and of an osteosarcoma cell extract (30 µg of protein). (B) Immunoblot analysis with anti-PARP of purified wild-type (wt) and catalytically inactive mutant (mut) PARP-1. The positions of molecular size standards (in kDa) and of the proteins (arrows) are indicated. (C) Relative catalytic activities of wild-type and mutant PARP-1 were determined by incubation of PARP-1 (0.1 µg) for 1 minute at 25°C in a reaction mixture (50 µl) containing 50 mM Tris-HCl (pH 7.8), 25 mM MgCl2, 1 mM dithiothreitol, 4 µg of activated DNA, 100 µM NAD, and 1 µl [32P]NAD (2 mCi/mmol), as described in Materials and Methods section. Data are expressed as nanomoles of [32P]NAD incorporated per minute per milligram of protein, and are means of triplicate determinations from a representative experiment.

Article Snippet: Recombinant p53: Expression and Purification of Recombinant Wild-Type and Inactive Mutant PARP-1 Proteins Purified recombinant p53, an 80-kDa GST fusion protein of full-length human p53 (amino acids 1–393) expressed in Escherichia coli and purified by Ni resin column chromatography, and GST were obtained from Santa Cruz Biotechnology.

Techniques: Western Blot, Recombinant, Mutagenesis, Purification, Incubation

Journal:

Article Title: Poly(ADP-ribosyl)ation of p53 In Vitro and In Vivo Modulates Binding to its DNA Consensus Sequence 1

doi:

Figure Lengend Snippet: Poly(ADP-ribosyl)ation of GST-p53(1–393) and GST by wild-type PARP-1 in vitro. (A) Recombinant wild-type or mutant PARP-1 (0.1 µg) was incubated for 30 minutes at 37°C in the presence of 100 µM NAD and in the absence or presence of GST-p53(1–393) (1 µg), as indicated. The reaction mixtures were then subjected to immunoblot analysis with anti-PAR (left panel), after which the blot was stripped of antibodies and reprobed with anti-p53 (right panel). Arrows: p53-PAR, poly(ADP-ribosyl)ated p53 fusion protein; GST-p53, GST-p53(1–393). (B) Recombinant wild-type or mutant PARP-1 (0.1 µg) was incubated for the indicated times at 37°C in the presence of 1 µM [32P]NAD and in the absence or presence of GST-p53(1–393) (1 µg). The reaction mixtures were then subjected to SDS-PAGE and autoradiography. Arrows indicate the positions of poly(ADP-ribosyl)ated PARP-1 and p53 fusion protein. (C) GST-p53(1–393) was incubated in the absence or presence of wild-type or mutant PARP-1 as in (A). The reaction mixtures were immunodepleted of PARP-1 and then subjected to immunoprecipitation with anti-p53 or with control IgG, as indicated. The resulting immunocomplexes were subjected to immunoblot analysis with anti-PAR, after which the blot was stripped of antibodies and reprobed with anti-p53 and then with anti-PARP, as indicated. (D) Purified GST (1 µg) was incubated for 30 minutes at 37°C in the presence of 100 µM NAD and in the absence or presence of wild-type or mutant PARP-1 (0.1 µg) and 1 mM 3-aminobenzamide (3-AB), as indicated. The reaction mixtures were then subjected to immunoblot analysis with anti-PAR (left panel), after which the blot was stripped of antibodies and reprobed with anti-GST (right panel).

Article Snippet: Recombinant p53: Expression and Purification of Recombinant Wild-Type and Inactive Mutant PARP-1 Proteins Purified recombinant p53, an 80-kDa GST fusion protein of full-length human p53 (amino acids 1–393) expressed in Escherichia coli and purified by Ni resin column chromatography, and GST were obtained from Santa Cruz Biotechnology.

Techniques: In Vitro, Recombinant, Mutagenesis, Incubation, Western Blot, SDS Page, Autoradiography, Immunoprecipitation, Control, Purification

Journal:

Article Title: Poly(ADP-ribosyl)ation of p53 In Vitro and In Vivo Modulates Binding to its DNA Consensus Sequence 1

doi:

Figure Lengend Snippet: Effect of poly(ADP-ribosyl)ation of GST-p53(1–393) by PARP-1 in vitro on binding of p53 to its DNA consensus sequence. (A) After incubation for 30 minutes at 37°C, poly(ADP-ribosyl)ation reaction mixtures containing NAD and the indicated combinations of GST-p53(1–393) and wild-type or mutant PARP-1 were immunodepleted of PARP-1 and then subjected to EMSSA by incubation first with anti-p53 and then with a 32P-labeled 30-bp oligonucleotide probe corresponding to the consensus p53-binding sequence (or with a control probe corresponding to the NF-κB-binding sequence). (B) EMSSA analysis was performed as in (A), with the exception that the incubation with the 32P-labeled p53-specific probe was performed in the absence or presence of x10 or x50 excesses of the same unlabeled probe. The positions of free DNA probe and the antibody-p53-DNA complex are indicated.

Article Snippet: Recombinant p53: Expression and Purification of Recombinant Wild-Type and Inactive Mutant PARP-1 Proteins Purified recombinant p53, an 80-kDa GST fusion protein of full-length human p53 (amino acids 1–393) expressed in Escherichia coli and purified by Ni resin column chromatography, and GST were obtained from Santa Cruz Biotechnology.

Techniques: In Vitro, Binding Assay, Sequencing, Incubation, Mutagenesis, Labeling, Control

Journal:

Article Title: Poly(ADP-ribosyl)ation of p53 In Vitro and In Vivo Modulates Binding to its DNA Consensus Sequence 1

doi:

Figure Lengend Snippet: Effect of poly(ADP-ribosyl)ation of p53 during early apoptosis in osteosarcoma cells on p53 binding to its DNA consensus sequence in vitro. (A) Cell extracts from osteosarcoma cells before (day 1), after (day 5), and at (day 3) the peak of poly(ADP-ribosyl)ation during the early stage of apoptosis were subjected to immunoblot analysis with antibodies to Bax and Fas (lower panel) and to immunoprecipitation with anti-p53. After immunoprecipitation with anti-p53, the resulting immunocomplexes were then subjected to immunoblot analysis with anti-p53 (upper panel) and anti-PAR (middle panel). (B) Osteosarcoma cell extracts prepared before (D1), after (D5), and at the peak of poly(ADP-ribosyl)ation (D3) were subjected to gel supershift assays with anti-p53 and the 32P-labeled 30-bp oligonucleotide containing the consensus p53-binding sequence. Control EMSSAs were also performed in the absence of cell extract, or with an unrelated control oligonucleotide (NF-κB consensus sequence) to confirm specificity of binding.

Article Snippet: Recombinant p53: Expression and Purification of Recombinant Wild-Type and Inactive Mutant PARP-1 Proteins Purified recombinant p53, an 80-kDa GST fusion protein of full-length human p53 (amino acids 1–393) expressed in Escherichia coli and purified by Ni resin column chromatography, and GST were obtained from Santa Cruz Biotechnology.

Techniques: Binding Assay, Sequencing, In Vitro, Western Blot, Immunoprecipitation, Labeling, Control

Journal:

Article Title: Poly(ADP-ribosyl)ation of p53 In Vitro and In Vivo Modulates Binding to its DNA Consensus Sequence 1

doi:

Figure Lengend Snippet: PARP-1 activation and cleavage in an “amplification loop” toward caspase activation and downstream apoptotic events. Stimulation of the Fas receptor induces the release of mitochondrial factors, such as AIF, that translocates to the nucleus and induces caspase-independent chromatin cleavage into large 50-kb fragments. These large DNA breaks activate PARP, rapidly decreasing NAD and ATP levels, which contributes to both receptor and mitochondrial pathways of apoptosis. Partial depletion of ATP further upregulates Fas, Fas ligand, and FADD; binding of Fas to Fas ligand recruits FADD, resulting in subsequent activation or amplification of the caspase cascade leading to apoptosis. Subsequent cleavage of PARP by caspase-3 prevents depletion of NAD and ATP below a critical level as well as releases certain nuclear proteins, such as Ca2+ -Mg2+-dependent endonucleases and p53, from poly(ADP-ribosyl)ation-induced inhibition.

Article Snippet: Recombinant p53: Expression and Purification of Recombinant Wild-Type and Inactive Mutant PARP-1 Proteins Purified recombinant p53, an 80-kDa GST fusion protein of full-length human p53 (amino acids 1–393) expressed in Escherichia coli and purified by Ni resin column chromatography, and GST were obtained from Santa Cruz Biotechnology.

Techniques: Activation Assay, Amplification, Binding Assay, Inhibition