t2aa  (MedChemExpress)

Journal: Nature

Article Title: PAF15–PCNA exhaustion governs the strand-specific control of DNA replication

doi: 10.1038/s41586-025-10011-3

Figure Lengend Snippet: a , QIBC analysis of chromatin-bound (left) Timeless and (right) Claspin in U2OS cells with simultaneous depletion of PAF15 and addition of PCNA inhibition (T2AA, 5 and 10 μM). b , Modelling of PCNA inhibitor T2AA (PDB: 3WGW) onto the AlphaFold 3-predicted structure of PCNA (trimer)-DNA-PAF15 shows a steric clash between PAF15 PIP and T2AA. c , AlphaFold-predicted structural model of three PCNAs in complex with PAF15, Claspin (CLSPN), and dsDNA shows high confidence in the folding of PCNA and DNA and very low confidence in the folding of CLSPN. According to the PAE score of the model, the 304–341 residues of Claspin that contain the PIP motif (residues 311–318) are in the closest proximity of PCNA, but they are within a predicted alignment error of 20–25 Å, which can be situated beyond the interaction distance. d , QIBC analysis of PLA focus sum intensity in pairs of PAF15-POLε1 and PAF15-POLδ1 in U2OS cells treated with control siRNA and siRNA of TIMELESS . S-phase quantification is used in Fig. . e , Immunoprecipitation of FLAG-tagged PAF15 in U2OS parental, U2OS cells constitutively expressing PAF15-Myc-FLAG and U2OS PAF15 KO cells doxycycline inducible PAF15 WT. U2OS cells constitutively expressing PAF15-Myc-FLAG were treated with control, TIMELESS , CLSPN siRNAs and indicated proteins from input and Flag IP lysates were analysed by western blot. f , (Top) Schematic of DNA-fibre protocol, (bottom) Replication fork speed in U2OS parental and U2OS PAF15 KO cells treated with 20 µg/ml doxycycline to induce WT PAF15 expression upon TIMELESS gene depletion. n = 200 fibres per condition. P values were determined by one-way ANOVA with Tukey’s test, n = 2 experiments. g , Survival analysis of parental and PAF15- KO U2OS cells with depletion of Timeless and Claspin. Values in bar graphs denote mean ± s.d. All the P values were determined by one-way ANOVA with Tukey’s test. h , QIBC analysis of Cyclin A2 and EdU in HeLa Kyoto cells with depletion of indicated proteins. 2n: G1, 4n: G2, n > 5,000 cells were analysed per condition. Red arrows indicate S-phase population revival in Timeless or Claspin depletion together with PAF15. All the experimental data are derived from a minimum of 2 independent experiments.

Article Snippet: Reagents were as follows: hydroxyurea (ribonucleotide reductase (RNR) inhibitor; Sigma-Aldrich, H8627; solubilized in H 2 O), ceralasertib (AZD6738) (ATR inhibitor; Selleckchem, S7693), adavosertib (MK-1775) (WEE1 inhibitor; Selleckchem, s1525), palbociclib (PD-0332991) (CDK4/6 inhibitor; Selleckchem, S1116), MG132 (proteasome inhibitor; Selleckchem, s2619), bortezomib (proteasome inhibitor; Selleckchem, S1013), PDD 00017273 (PARG inhibitor; Tocris, 5952), doxycycline (Thermo Fisher Scientific, BP2653-5), T2AA (PCNA inhibitor; Tocris, 4723), nocodozole (Tocris, 1228), 5-chloro-2′-deoxyuridine (CldU; Sigma-Aldrich, c6891), 5-iodo-2′-deoxyuridine (IdU; Sigma-Aldrich, I7125), olaparib (PARP inhibitor; Selleckchem S1060), decitabine (DNMT1i; Tocris, 2624), PHA-767491 hydrochloride (CDC7 inhibitor; Tocris, 3140), 5-Ph-IAA (Merck, SML3574) and 5-ethynyl-2′-deoxyuridine (EdU; Thermo Fisher Scientific, A10044).

Techniques: Inhibition, Control, Immunoprecipitation, Expressing, Western Blot, Derivative Assay

Journal: mBio

Article Title: Adeno-Associated Virus Monoinfection Induces a DNA Damage Response and DNA Repair That Contributes to Viral DNA Replication

doi: 10.1128/mbio.03528-22

Figure Lengend Snippet: Pharmacological inhibitors targeting both DNA replication DNA polymerase and DNA repair polymerase abolish or significantly reduce AAV2 DNA replication in vitro . (A) Purification of AAV2 Rep68 His protein. AAV2 Rep68 His protein was purified as described in Materials and Methods. The eluted protein from peaked fractions were separated on an SDS-(12%) PAGE gel, along with a protein size ladder (M), and stained with Coomassie brilliant blue. (B) In vitro DNA replication assay. Various inhibitors HAMNO (specifically targeting to RPA complex) at 100 μM, T2AA (to PCNA complex) at 1 μM, aphidicolin (to Pol α/δ/ε) at 10 μg/mL, MK886 (to Pol κ/η) at 200 μM, and PNR7-02 (to Pol η/λ) at 20 μM were added into the reaction of in vitro replication assays. DMSO served as a vehicle control. After DpnI digestion for 1 h at 37°C, the in vitro replicated products (lanes 2 to 7) were resolved on 1% agarose gel, and stained with ethidium bromide (EB) for DpnI digested inputs (lower panel). The dehydrated gel was exposed to a phosphor screen and scanned on a Typhoon FLA 9000 scanner. (B, lane 1), the reaction product without DpnI digestion was loaded as a size maker (~4.7 kb). The arrowhead indicates the in vitro replicated AAV2 genome. (C) Quantification of the relative replication efficiency. The fully in vitro replicated (DpnI-digestion resistant) viral DNA signals at ~4.7 kb were quantified by using ImageQuant Tl (IQTL) 8.2 software (Cytiva). The quantities are presented as relative levels to the DMSO control (B, lane 7). The data were obtained from triple experiments. ****, P < 0.0001.

Article Snippet: We used HAMNO (#S0148, Selleckchem), T2AA (#21921, Cayman Chemical), aphidicolin (#14007, Cayman Chemical), MK886 (#1311, Tocris Bioscience), and PNR7-02 (#2965, Axon Medchem).

Techniques: In Vitro, Purification, Staining, Control, Agarose Gel Electrophoresis, Software

Journal: Frontiers in immunology

Article Title: ROS and DNA repair in spontaneous versus agonist-induced NETosis: Context matters.

doi: 10.3389/fimmu.2022.1033815

Figure Lengend Snippet: FIGURE 1 Oxidative DNA damage and repair drive NETosis: inhibition of early repair steps suppresses spontaneous NETosis while inhibition of late repair steps promotes it. (A) Immunoconfocal imaging confirms that neutrophils undergo low level spontaneous NETosis in media, while PMA, an oxidative burst inducer, promotes robust NETosis. Presence of DPI, an inhibitor of ROS generation, suppresses NETosis in both experimental conditions. DNA (DAPI, blue), MPO (pink). (B) NET DNA release from neutrophils incubated in media, or media with DPI or NAC indicates that ROS inhibitors suppress spontaneous NETosis (SYTOX; *, p<0.05 compared to control). (C) DNA release from neutrophils incubated in media or media with inhibitors (inh) of early stage oxidative DNA damage repair proteins APE (inh 1 or 2), PARP1 (inh 1 or 2) or LIG suggests that inhibition of these proteins suppresses spontaneous NETosis (SYTOX; *, p<0.05 compared to control). (D) DNA release from neutrophils incubated in media, or media with inhibitors of late stage repair proteins PCNA, Pol b or Pol d suggest that inhibition of these proteins promotes spontaneous NETosis (SYTOX; *, p<0.05 compared to control). (E) DNA release from neutrophils incubated with ROS inhibitors (DPI, NAC or FBS) shows that spontaneous NETosis is suppressed by ROS inhibitors regardless of the presence of inhibitors to PCNA, Pol b or Pol d in the media (SYTOX; *, p<0.05 compared to media treated control in each respective cluster). (F) Confocal imaging confirms that ROS inhibitor DPI suppresses spontaneous NETosis promoted by the inhibitors of PCNA, Pol b or Pol d. DNA (DAPI, blue). (G) ROS measurements by DHR123 assays show that neutrophils used in the experiments generate high levels of ROS when stimulated with PMA (+ve control) compared to non-activated neutrophils. Presence of inhibitors to PCNA, Pol b or Pol d does not alter the baseline ROS generation (Plate reader; *, p<0.05 compared to control). Serum was not added to the media unless otherwise stated. DNA repair inhibitors used were: APE inh 1 (CRT0044876), APE inh 2 (APE1 Inhibitor III), PARP1 inh 1 (BSI201), PARP inh 2 (PJ34) or LIG inh (L189), PCNA inh (T2AA), Pol b inh (AM-TS23) or Pol d inh (Aphidicolin). Fluorescence was recorded at 4-hour time points by SYTOX Green plate reader assays (n = 3 for all the experiments). The images are representative of 3 independent experiments, at 4-hour time points. Scale bar on images, 5 mm.

Article Snippet: ROS inhibitors (NOX inh Diphenyleneiodonium chloride, 1 mM DPI, Sigma), ROS scavengers (N-Acetyl-L-cysteine, 3 mMNAC, Sigma) and fetal bovine serum (1% (v/v) FBS, ThermoFisher Scientific) were added to the cells 1 hour before adding DNA repair inhibitors: Pol d inh (Aphidicolin, 50 mM, Sigma), Pol b inh (AM-TS23, 25 mM, Tocris), PCNA : Polymerase interaction inh or PCNA inh (T2AA, 25 mM, Tocris), APE inh 1 (CRT0044876, 125 mM, Sigma), APE inh 2 (APE1 Inhibitor III, 50 mM, EMD-Millipore), PARP1 inh 1 (BSI201, 100 mM, Sigma), PARP inh 2 (PJ34, 50 mM, EMD-Millipore), LIG inh (L189, 100 mM, Tocris).

Techniques: Inhibition, Imaging, Incubation, Control, Fluorescence

Journal: Frontiers in immunology

Article Title: ROS and DNA repair in spontaneous versus agonist-induced NETosis: Context matters.

doi: 10.3389/fimmu.2022.1033815

Figure Lengend Snippet: FIGURE 2 Confocal images showing that inhibiting the late stage oxidative DNA damage repair pathways induce spontaneous NETosis, and a diagram summarizing the regulation of spontaneous NETosis by various DNA repair proteins. (A, B) Neutrophils were treated with PCNA inhibitor (T2AA), immunostained and imaged by confocal microscopy. MPO (pink) colocalizing to DNA (DAPI blue) shows that PCNA inhibition promotes spontaneous NETosis (A). PCNA (red) is cytoplasmically distributed in the neutrophils but the inhibition of late stage DNA repair by inhibition of PCNA: polymerase binding leads to NETosis with PCNA present throughout the NET DNA (DAPI, blue) (B). Images for panels are representative of 3 independent experiments. Scale bar, 10 mm. (C) Diagram highlighting the proteins involved in the oxidative DNA damage repair pathways and their effects on spontaneous NETosis. Non-bulky adducts are repaired by BER. Baseline NETosis is suppressed by early DNA repair events that lead up to the chromatin decondensation/nick formation by APE1. Inhibition of late events of DNA repair promotes spontaneous NETosis. Proteins whose inhibition induces ( , blue arrow) or suppresses ( , red arrow) spontaneous NETosis are indicated. Overall, inhibition of early

Article Snippet: ROS inhibitors (NOX inh Diphenyleneiodonium chloride, 1 mM DPI, Sigma), ROS scavengers (N-Acetyl-L-cysteine, 3 mMNAC, Sigma) and fetal bovine serum (1% (v/v) FBS, ThermoFisher Scientific) were added to the cells 1 hour before adding DNA repair inhibitors: Pol d inh (Aphidicolin, 50 mM, Sigma), Pol b inh (AM-TS23, 25 mM, Tocris), PCNA : Polymerase interaction inh or PCNA inh (T2AA, 25 mM, Tocris), APE inh 1 (CRT0044876, 125 mM, Sigma), APE inh 2 (APE1 Inhibitor III, 50 mM, EMD-Millipore), PARP1 inh 1 (BSI201, 100 mM, Sigma), PARP inh 2 (PJ34, 50 mM, EMD-Millipore), LIG inh (L189, 100 mM, Tocris).

Techniques: Confocal Microscopy, Inhibition, Binding Assay