Solvents and chemicals including DMSO, glutaraldehyde, temozolomide, and talazoparib were obtained from Sigma-Aldrich (St. Louis, MO, United States). DO1 p53 (Cat# sc-126) monoclonal and PARP1 (Cat# sc-7150) rabbit polyclonal antibodies were purchased from Santa Cruz (United States). MCM2 (Cat# 12079s) mouse antibody was purchased from Cell Signaling Technology. An Eppendorf 5,415 refrigerated centrifuge was used for preparation of all extracts.

Human breast cancer cell line MDA-MB-468 was purchased from ATCC (www.atcc.org) and the HCT116 colon cancer cell line that is p53−/− was a gift from Bert Vogelstein and were made as described (Bunz et al., 1998; Bunz et al., 1999). Cell lines were regularly authenticated via short tandem repeat technology (Genetica DNA Laboratories). Cells were routinely checked for mycoplasma contamination by PCR assay (ATCC). Fresh cells were thawed when the passaging period was around 30. Cells were maintained at 5% CO₂ in a 37°C humidified incubator. HCT116 p53−/− and MDA-MB-468 cells were cultured in McCoy’s 5A (Gibco) and DMEM media (Corning) respectively, with 50 U/ml penicillin, 50 μg/ml streptomycin (Mediatech), 5 ug/ml plasmocin (InvivoGen) and supplemented with 10% FBS (Gemini). PARP-dependent recruitment of proteins to chromatin was assayed by exposure of HCT116 cells to 1 mM Temozolomide (Sigma-Aldrich; 100 mM stock solution in DMSO) and 10 μM Talazoparib (Selleckchem; 20 mM stock solution in DMSO) combination treatment for 4 h at 37°C followed by chromatin isolation as described (Qiu et al., 2017).

To generate clones expressing p53 with the desired mutations in the oligomerization domain, we used the NEBasechanger (https://nebasechanger.neb.com) platform to design primers to introduce specific point mutations within the OD of pCMV-FLAG-wtp53 and pCMV-FLAG-p53R273H plasmids which express wtp53 or R273H mtp53 (Hamard et al., 2012). For R337C, the primer pair was F: TGG​GCG​TGA​GtG​CTT​CGA​GAT and R: CGG​ATC​TGA​AGG​GTG​AAA​TAT​TCT​C (annealing temperature 66°C). For A347D, the primer pair was F: CTG​AAT​GAG​GaC​TTG​GAA​CTC and R: CTC​TCG​GAA​CAT​CTC​GAA​G (annealing temperature 62°C). Lastly, for L344P the primer pair was F: TTC​CGA​GAG​CcG​AAT​GAG​GCC and R: CATCTCGAAGCGCTCACG (annealing temperature 65°C). A PCR reaction was set up with CMV-wtp53 or CMV-R273H plasmid template and the Q5 Hotstart high-fidelity 2X master mix (NEB) at the specific annealing temperature of each primer pair. After confirming PCR amplicon with agarose gel electrophoresis, a kinase-ligase-Dpn1 (NEB) reaction was done before transforming DH5α competent cells. DNA was isolated from cultured transformants (grown in LB+50 μg/ml Amp) using the Qiagen miniprep/midiprep kit, sequenced (Genewiz) using a p53 Exon 8 F primer 5′ ACA​GCA​CAT​GAC​GGA​GGT​TGT, and plasmids harboring mutations were confirmed using the Benchling™ platform. Sequences were compared to TP53 cDNA from the GRCh38 homo sapien reference genome using the benchling platform’s external databases. Plasmids with the desired OD mutations were transfected into HCT116 p53−/− cells using either the lipofectamine™ (Invitrogen) or the electroporation-based Neon Transfection System™ (ThermoFisher) as directed by the manufacturers. For the nucleofection, the transfection protocol as previously described for MDA-MB-468 cells was followed with little modification (Ellison et al., 2021). The conditions for introduction of plasmids into HCT116 p53−/− cells (7 μg/1,000,000 cells) using the Neon were pulsation 1x for 20 ms at 1530 V followed by culturing in McCoy’s 5A media +10% FBS without antibiotics.

This assay was carried out as described previously for MDA-MB-468 cells and purified R273H mtp53 (Ellison et al., 2021; Xiao et al., 2021). Cells were washed with cold PBS, harvested by scraping in cold PBS, and centrifuging at 1,400 g (1,100 rpm) for 7 min. Harvested cells were lysed with phosphate lysis buffer (1x PBS, 10% glycerol, 10 mMEDTA, 0.5% NP-40, 0.1M KCl, 1 mM PMSF, 8.5 μg/ml aprotinin, 2 μg/ml leupeptin, and phosphatase inhibitor cocktail). A total of 50 μg of protein lysates were treated without, or with, glutaraldehyde at a final concentration of 0.005%. The lysates were then incubated at room temperature for 20 min on a shaker. The crosslinking was stopped by the addition of 1/6 of the volume with 6X protein sample buffer (6X SDS Laemmli sample buffer, 0.2 M DTT) and heated at 95°C for 10 min 25 μg was resolved on an 8% SDS-PAGE gel and Western blot analysis was performed with DO1 p53 monoclonal antibody.

Total RNA was isolated from cell cultures as dictated by the experimental design using the Qiagen RNeasy™ kit as directed by the manufacturers (www.qiagen.com/HB-0435). The Applied Biosystems High-Capacity cDNA Reverse Transcription kit was used to generate cDNA from 5 μg of total RNA from each cell sample. The relative abundance of the TP53, p21, MDM2, RRM2 and CDC7 mRNA within each cell was measured by quantitative-PCR using the Applied Biosystems TaqMan real-time PCR with FAM dye-labeled probes and GAPDH as an endogenous control (Thermofisher Scientific TP53 ID# 01034249_m1, p21 cat# 4331182, MDM2 cat# 4351372 RRM2 cat# 01072069_g1, CDC7 ID# 00177487_m1, and GAPDH ID# 02786624_g1) and the Applied Biosystems QuantStudio seven Flex instrument.

Cells were harvested by low speed centrifugation at 1,400 g (1,100 rpm) for 7 min at 4°C. Cells were washed three times with ice-cold PBS and resuspended in RIPA buffer (0.1% SDS, 1% IGEPAL NP-40, 0.5% Deoxycholate, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 50 mM Tris-Cl pH 8.0, 1 mM PMSF, 8.5 μg/ml Aprotinin, 2 μg/ml Leupeptin and phosphatase inhibitor cocktail). The cell suspension was incubated on ice for 30 min to lyse the cells, with gentle vortexing every 5 min, after which, lysates were subject to sonication 3x for 30 s pulses/30 s rest on ice at 98% amplitude and then centrifuged at 15,700 g (13,200 rpm) for 30 min at 4°C. The protein concentrations of clarified cell extracts were determined using the Bradford assay (Bio-Rad), and 50 μg of extracts were analyzed for specific proteins by electro-transfer onto Polyvinylidene fluoride membrane (Amersham-GE Biosciences) following SDS-PAGE. The membrane was blocked with 5% non-fat milk (Bio-Rad) in either 1X PBS-0.1% Tween-20 or 1X TBS-0.1% Tween-20 followed by an overnight incubation with primary antibody at 4°C. The membrane was washed 3x with either 1X PBS-0.1% Tween-20 or 1X TBS-0.1% Tween-20 and incubated with Cy5-and Cy3-linked secondary antibodies (Amersham Biosciences) for 1h at room temperature. The signal was detected with the Typhoon FLA 7000 laser scanner (GE Healthcare). Primary antibodies used were; 1) anti-p53 DO1 p53 (Santa Cruz Cat# sc-126), 2) anti-PARP1 (Santa Cruz Cat# sc-7150), 3) anti-MCM2 1E7 (Cell Signalling Technology Cat# 12079s).

Localization of mtp53 proteins to chromosomes was assessed using a version of the Chromatin Fractionation Assay as described (Ellison et al., 2021). Cells were harvested 24 h post-transfection as dictated by experimental conditions by scraping, pelleted, and washed with ice-cold PBS three times. The pellet was resuspended in Buffer A (10 mM HEPES, 10 mM KCl, 1.5 mM MgCl₂, 300 mM Sucrose, 1 mM DTT, 10% Glycerol, 0.1 mM PMSF, 1 μg/ml Leupeptin, 1 μg/ml Pepstatin A, and 2 μg/ml Aprotinin) with 0.1% Triton X-100 using 3X the pellet volume. The resuspended pellet was centrifuged at 1,500 g (4,000 rpm) for 5 min after incubating on ice for 5 min. The resulting pellet containing nuclei was saved and the supernatant spun at 15,700 g (13,200 rpm) for 5 min; the supernatant from this centrifugation step was saved as S1. The nuclei from each sample were washed twice in Buffer A+ 0.15% Triton X-100 then lysed in Buffer B (3 mM EDTA, 0.2 mM EGTA, 1 mM DTT, 0.1 mM PMSF, 1 μg/ml Leupeptin, 1 μg/ml Pepstatin A, and 2 μg/ml Aprotinin) on ice for 30 min. The chromatin for each sample was separated from the nuclear lysate (S2) by centrifugation for 4 min at 1,500 g (4,000 rpm) at 4°C, washed with Buffer B, and then collected by centrifugation as described above. The chromatin pellet was resuspended in Buffer B and sonicated on ice to shear genomic DNA, the resulting solution was saved as the chromatin fraction. The protein concentrations were measured using the Bradford assay (Bio-Rad). ImageJ quantification is shown in Supplementary Figure S3A. The ImageJ software was used to determine the western blot band intensity with signals normalized to their respective Lamin control. The relative expression of p53 in the HCT116−/− transfected cells was determined by using the level of R273H protein in the no drug treatment as the reference sample. The relative expression of MCM2 and PARP1, was determine by using the protein levels in the empty vector transfection sample with no drug treatment as the reference samples.

The method was derived from a DNA repair tight tethering detergent assay (Iwabuchi et al., 2003). Transfected HCT116 p53−/− cells were harvested and then stored at −80°C. For preparation of the soluble and insoluble fractions, the frozen cell pellets were lysed for 1 h at 4°C in TNE (pH 8.0) buffer (50 mM Tris, pH8.0,120 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 100 g/ml phenylmethylsulphonyl fluoride, 5 g/ml aprotinin, 5 g/ml Pepstatin, 2 g/ml leupeptin, 50 mM NaF, 2 mM Na₃VO₄·2H₂O (Sodium Orthovanadate)). Approximately 3x the cell pellet volume of TNE buffer was used for lysis. The lysate was divided into the soluble and insoluble fractions by centrifugation at 15,700 g (13,200 rpm) for 10 min at 4°C. The supernatant-soluble fraction was transferred to a new tube. The remaining pellet, or insoluble fraction was resuspended in PBS containing 2% SDS (∼5x insoluble cell pellet volume) and then sonicated 3 times for 30 s pulses/30 s rest on ice at 98% amplitude (QSonica, LLC Q700). The protein concentration for the soluble fraction was measured using the Bradford assay, while the protein concentration of the insoluble fraction was obtained by absorbance at 280 nm reading from Nanodrop Spectrophotometer. Samples were analyzed by western blotting as described above. ImageJ quantification is shown in Supplementary Figure S3B and determined as described above for chromatin fractionation samples.

The protocol for the proximity ligation assay using the Sigma Aldrich Duolink Kit™ (Cat # DUO92008) was as described previously (Xiao et al., 2020). Cells were seeded in a 12-well glass-bottomed plate at 1 × 10⁵ cells per well in complete McCoy 5A media supplemented with 10% FBS without antibiotics. After 24 h, 1.6 ug of plasmid DNA was added to each well via the lipofectamine transfection system as described by the manufacturer. After 24 h post-transfection, the media was removed, and the cells were washed with cold PBS 3 times. The cells were then fixed in 4% formaldehyde for 15 min and permeabilized in 0.5% Triton X-100 for 10 min at room temperature. The Duolink In-situ Red Kit (Cat # DUO92101) was used for the PLA. Blocking buffer was added to each well and incubated in a humidified chamber at 37°C for 30 min. After aspirating the blocking buffer, primary antibodies were added to each well and incubated in a humidified chamber overnight at room temperature. Wash buffer A (cat # DUO82049) was used to wash the cells 3 times for 5 min each. The PLA Plus/Minus secondary antibody probes were added to the wells and incubated in a humidified chamber for 60 min at 37°C. After that, the cells were washed 2 times with buffer A for 2 min each. The ligation step was performed for 30 min incubation at 37°C, washed 2 times with buffer A and the amplification was done for 100 min at 37°C. The wells were washed with buffer B for 10 min and incubated with a FITC-labelled secondary antibody to detect p53. After washing 3 times, mounting media containing DAPI was added to each well, rocked for 15 min, and images were taken using the Nikon A1 confocal microscope. Images obtained were processed with the Nikon NIS Element software, ImageJ and Cellprofiler. The foci/cell were plotted, and statistical analysis was performed in GraphPad Prism 9.

Statistical analyses were performed using GraphPad Prism 9. Results are expressed as mean +SEM. Statistical significance for hypothesis testing was performed by a one-way ANOVA with multiple comparison. The following format was used to assign significance based on p-value: **** represents a p-value ≤ 0.0001 and ns represent non-significant.

We used site-directed mutagenesis to introduce oligomerization mutations to change p53 amino acid residues R337C, A347D, or L344P in plasmids for exogenous expression of either wtp53 or mtp53 R273H. When the Li-Fraumeni OD-specific associated point mutations were introduced individually, or as dual mutants within mtp53 R273H, they destabilized tetramer formation (Figures 1A,B). The exogenously expressed wtp53 and mtp53 R273H proteins predominantly formed dimers and tetramers when 0.005% glutaraldehyde was added (Figures 1A,B, lanes 1, 2), whereas both single A347D and dual R273H-A347D polypeptides shifted to form predominantly dimers (Figures 1A,B, lanes 5, 6). The Li-Fraumeni mutations R337C and L344P, as well as the dual mutants R273H-R337C and R273H-L344P, were unable to form dimers or tetramers (Figures 1A,B, lanes 3–8). We also confirmed that endogenous mtp53 in many different human breast cancer cell lines formed tetramers (Supplementary Figure S1). These data indicate that mtp53 can exist as tetramers in human cells, and that we successfully disrupted mtp53 R273H oligomerization in the R273H R337C, A347D, or L344P dual mutants.

The p53 protein activates the transcription of the cyclin-dependent kinase p21 and the p53 inhibitor MDM2 (Levine, 1997; Wu and Levine, 1997). We wanted to confirm that destabilization of wtp53 oligomerization blocked transactivation. We expressed the R337C, A347D, or L344P single or dual mtp53 R273H in HCT116 p53−/− cells and observed significant TP53 message and protein expression in all the transfection experiments (Figures 2A,B, compare empty vector (EV) to expression constructs as indicated). As expected, upregulation of the endogenous targets p21 and MDM2 occurred in the presence of wtp53 expression (Figures 2C,D), and the LFS mutations blocked the ability of p53 to transactivate p21 and MDM2 (Figures 2C,D). Equally as predictable, the mtp53 R273H mtp53 expression was unable to activate expression of p21 or MDM2, and dual mutants were no different (Figures 2C,D). The mtp53 R273H has been shown to activate transcriptional targets CDC7 and RRM2 (Kollareddy et al., 2015; Datta et al., 2017). As such, we examined the amount of mRNA and protein expression levels of these targets. We examined whether mtp53 R273H, or the mtp53 R273H dual mutants, significantly upregulated the expression of either CDC7 or RRM2 (Supplementary Figure S2). However, we did not detect upregulation for these genes. This could be due to low transfection efficiency (30% efficiency), or other variables that may have influenced mtp53 mediated transcription effects.

Endogenous mtp53 R273H, and other mtp53 proteins, tightly tether to chromatin (Polotskaia et al., 2015; Qiu et al., 2017). We tested whether the tetramerization state of mtp53 was crucial for the mtp53 chromatin association. We transiently transfected in mtp53 R273H without, and with, the OD mutations. To further examine how dual mutants influenced chromatin interaction, we treated cells with a combination of the alkylating agent temozolomide (Temo) and the PARP1 trapping drug talazoparib (Tal) to induce replication stress and trap PARP1 on the chromatin (Qiu et al., 2017; Xiao et al., 2020). We then isolated chromatin by either chromatin fractionation, or the more stringent detergent insoluble fractionation assay, as methods to assess protein-DNA tethering. We observed mtp53 R273H and mtp53 R273H-dual oligomerization mutants tightly tethered to chromatin (Figures 3A,B, with imageJ quantification shown in Supplementary Figures S3A,B). The replication stress did not significantly alter the ability of mtp53 to interact with chromatin regardless of the OD domain status (Figures 3A,B; Supplementary Figures S3A,B). We therefore conclude that oligomerization of mtp53 R273H is not required for mtp53 chromatin association. Moreover, both PARP and MCM2 interacted well with the chromatin in both unstressed and stressed conditions. The p53 protein that cannot form tetramers does not activate gene transcription. However, the possibility exists that monomers and dimers are able to maintain non-specific chromatin interactions. We compared wtp53, with and without OD mutations, for their general chromatin interactions by transfecting HCT116 p53 −/− cells with plasmids expressing wtp53 or the single OD mutants. Western blot analysis of the chromatin fractionated samples demonstrated that the different oligomerization forms tethered well to the chromatin. As such, mutation in the OD of p53 disrupts transcription factor function but does not disrupt the ability of the protein to generally interact with chromatin (Supplementary Figure S3C).

We tested if oligomerization influenced the interaction of mtp53 R273H with either MCM2 or PARP1 by using the proximity ligation assay (PLA), which was previously used to demonstrate their endogenous interactions (Xiao et al., 2020). We used transfected HCT116 p53−/− cells in order to compare the interactions with similar levels of exogenously expressed protein for wtp53 and R273H mtp53 single and dual mutants. The expression of GFP was used as a marker for cells expressing p53 and importantly PLA foci were only detected in p53 expressing cells. Previously we had detected a low number of PLA foci for wtp53 in an endogenously expressing cell line. This was due to the fact that endogenous wtp53 protein is maintained at low levels in MCF7 cancer cells due to degradation by the E3 ubiqutin ligase MDM2 (Xiao et al., 2020). Interestingly, when the expression level was the same we detected similar interaction levels between MCM2 with both wtp53 and mtp53 R273H (Figures 4A,B). In contrast, the dual mtp53 R273H-OD mutants demonstrated slightly more MCM2-mtp53 associated foci (Figures 4A,B). The interaction between mtp53 and PARP1 was also maintained after introducing mutations to destabilize tetramer formation, with no observable increase for the dual mutants (Figures 4C,D). This suggests that R273H hotspot mtp53 may interact with replicating DNA in forms that are not tetrameric. We also assessed the single-OD mutants and observed interactions between the single-OD p53 mutants and MCM2 as well as PARP1 (Supplementary Figure S4). This further supports the possibility that non-tetramerized p53 may function in alternative, non-transcription related, pathways that mediate GOF activity.

Generation of R273H mtp53 deletions of a small portion of the C-terminus (R273HΔ381-388) and a larger deletion removing some of the OD and all of the C-terminus (R273HΔ347-393) in MDA-MB-468 cells causes replication stress (Ellison et al., 2021). The larger deletion R273HΔ347-393 dual mutant causes drastic inhibition of cell proliferation. As such, we wondered if the entire deletion of the C-terminal non-specific DNA binding domain R273HΔ347-393 inhibited the interaction of mtp53 R273H with the replication machinery. We tested the interaction between mtp53 and MCM2 using the PLA method and observed that R273HΔ347-393, but not the smaller deletion R273HΔ347-393, exhibited decreased interaction with the MCM2 replication helicase (Figures 5A,B).