Fig 2: Effect of the p53 isoforms on the subcellular distribution and association of POL? and PCNA.K562 cells were transfected with expression plasmids for p53a, alternative p53 isoforms [A-C p53ß, p53?, D-F ?40p53a, ?133p53a, ?160p53a] or EV in controls (ctrl). Forty-eight hours after transfection, cells were mock- or MMC-treated (3 µM, 45 min, 3 h release) and processed for immunostaining to visualize POL? and PCNA foci as well as their co-localization. At least 100 nuclei were scored in = two independent experiments. Mean values for p53a expressing cells after MMC-treatment were set to 1 [on average 103 POL? (A, D), 133 PCNA (B, E), and 15 POL?-PCNA co-localized (C, F) foci/nucleus]. For graphic presentation, calculation of SEM and statistically significant differences via Dunn’s multiple comparisons test GraphPadPrism8.4 software was used. Representative images of MMC-treated samples are shown in (G). The experiments shown in (A) and (D), (B) and (E) as well as (C) and (F) were performed together (values of ctrl and p53a are identical but separated in different panels for clarity) and with the experiments presented in Fig. 6. *(#)P < 0.05, **(# #)P < 0.01, ***(# # #)P < 0.001, ****(# # # #), P < 0.0001 (scale bar: 5 µm).

Fig 3: p53 isoforms differently interact with PCNA and POL?.K562 cells were transfected with expression plasmids for p53a, alternative p53 isoforms [A p53ß, p53?, B ?40p53a, ?133p53a, ?160p53a] or EV in controls (ctrl). Forty-eight hours after transfection, cells were harvested for immunoprecipitations (IP) after preparation of crosslinked chromatin (A) or without crosslinked chromatin preparation (B). For the pull-downs of PCNA (A) anti-PCNA antibody (mouse, ab29, abcam, Cambridge, UK) or control Mouse IgG (Santa Cruz, Dallas, Texas, USA), for the pull-downs of POL? (A) POL?-antibody (rabbit, A301-304A, Bethyl, Montgomery, USA) or Rabbit IgG were used. For the pull-down of p53 (B) a mix of anti-p53(DO11) (mouse, MCA1704, BioRad Laboratories, München, Germany) and anti-p53(Pab421) (mouse, OP03, Calbiochem, Darmstadt, Germany) or control Mouse IgG were used. Subsequent immunoblotting relied on anti-POL? (rabbit, A301-304A, Bethyl, Montgomery, USA), anti-p53(DO1) (mouse, mAb, 554293, BD, Biosciences, Franklin Lakes, New Jersey, USA) in (A), anti-p53(DO11) (mouse, GTX75258, Genetex, Irvine California, USA), anti-p53 (PAb421, rabbit, ab245685, Abcam Cambridge, UK), anti-PCNA (mouse, ab29, abcam, Cambridge, UK) in (B) and light chain-specific peroxidase-coupled secondary antibody. *, asterisks mark bands possibly stemming from proteolytic cleavage of the p53-isoform. Quantification of band intensities of co-precipitated proteins relative to their input is indicated. Representative Western blots from three to four experiments are shown.

Fig 4: Knockdown of endogenous p53-isoforms induces changes in fiber track lengths.U2OS cells (A) or HSPCs (B) were transfected with nonsense siRNA (sictrl), siRNA targeting all p53-isoforms (sip53) or siRNA targeting ?133p53/?160p53 isoforms (si?133p53). A, B: 24 h after transfection, cells were sequentially incubated with CldU (20 µM) and IdU (200 µM) for 20 min. During IdU-incorporation, cells were either mock-treated or treated with 3 µM MMC. Both CldU- and IdU-tracks were measured while only IdU-tracks in ongoing forks (=257 fibers from two independent biological experiments) are graphically presented for clarity. Knockdown of p53-isoforms was verified by immunoblot staining using anti-p53 (DO-11, MCA1704, Biorad) shown in the right panel of (A, B) each. Quantification of ?133p53a relative to a-Tubulin levels was achieved by Imagelab and is indicated above the blots. The dashed frame in (A) marks an unspecific band stained by DO-11 in U2OS cells that had to be cut out, followed by antibody reincubation and a long exposure for 300 s to permit immunodetection of the ?133p53a-band. Statistically significant differences between groups were calculated by Dunn’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 5: Model for the impact of different p53 isoforms on POL?-dependent DDT.A Hetero-oligomerization or aggregation of p53a with alternative p53 isoforms, particularly with p53a + ?133p53a or p53a + ?160p53a, can antagonize p53a functions in the POL?-dependent DDT. B C-terminal p53 isoforms still interact with PCNA [20, 41] but are compromised in binding to three-stranded DNA junctions like replication forks due to shortened OD [38]. Intact DBD, OD, and RPA-interaction sites [8], but compromised interactions with POL? and PCNA due to lack of the N-terminal end in ?40p53a permit limited replication fork recognition only. ?133p53a or ?160p53a are compromised in DNA-binding due to the N-terminally truncated DBD and in RPA-, POL? and PCNA-binding due to the missing N-terminus. However, all N-terminal p53 isoforms retain the OD, enabling hetero-oligomerization with p53a and therefore dominant-negative interference with p53a functions in performing idling events in complex with POL? and PCNA, giving time for PCNA ubiquitination and FR events by HLTF and ZRANB3 [19]. Black arrows, DNA-binding; blue arrows, PCNA-binding; stippled arrow, compromised interaction.