Fig 2: Inhibition of fork reversal rescues fork degradation in GRB2 deficient cells.a Overexpression of BRC4 has no further fork degradation effect on GRB2 deficient cells. b BRCA2 knockdown has no further fork degradation effect on GRB2 deficient cells. c Knockdown of HTLF, SMARCAL1 or ZRANB3 is sufficient to alleviate replication fork degradation in GRB2-depleted cells. d Knockdown of RAD51 rescues the observed fork degradation in GRB2-depleted cells. e Overexpression of K133R ATPase deficient mutant RAD51 is sufficient to prevent fork degradation in GRB2-depleted HAP1 cells. f Western blot analysis showing the expression of K133RRAD51 mutant in WT and GRB2 KO HAP1 cells using anti-Flag antibody. Antibody targeting GRB2 was used to distinguish WT from GRB2 KO cells. Beta-Actin serves as a loading control. In (a–e), radiometric analysis of ≥200 fibers, n = 3. The significance was analyzed by two-sided Student’s t test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001; NS, not significant. Error bars showing standard deviations (SD). For (a–f), source data are provided as a Source Data file.

Fig 3: Increased heterochromatin formation induced by SMARCA4 loss promotes ATRi-induced fork reversal and Mre11 recruitment. (A) SMARCA4MUT (A549 and H1299) cells transduced with EV or SMARCA4 were immunostained with anti-HP1β antibody, and the nuclei were counterstained with DAPI. Scale bar: 10 μm. (B) Quantification of the intensities of individual nuclei in (A). Red bars represent mean intensities. Representative results of two independent reproducible experiments are shown. (C) SMARCA4WT (PC9 and H1975) cells were transfected with siCTL or siSMARCA4 for 48 h and immunostained with anti-HP1β antibody. The nuclei were counterstained with DAPI. Scale bar: 10 μm. (D) Quantification of the intensities of total HP1β intensities in individual nuclei in (C). Red bars represent mean intensities. Representative results of two independent reproducible experiments are shown. (E) A549 cells transduced with EV or SMARCA4 were treated with 1 μM ATRi for 2 h and immunostained with anti-H3K9me3 and anti-Mre11 antibody, and the nuclei were counterstained with DAPI. Scale bar: 10 μm. (F) Scatter diagram showing the total intensity of H3K9me3 and Mre11 in (E). Percentages of cells doubly positive for Mre11 and H3K9me3 (red) are indicated. Results are representative of two independent reproducible experiments. (G) A549 cells transduced with EV or SMARCA4 were transfected with the indicated siRNAs. After 48 h, the cells were incubated with 10 μM EdU for 15 min and treated with 1 μM ATRi for 2 h. After EdU staining, PLAs using anti-HP1α and anti-SMARCAL1 antibodies were performed. The nuclei were counterstained with DAPI. Scale bar: 10 μm. (H) Quantification of PLA signal in (G). The results represent the mean ± SD of three independent experiments.

Fig 4: Model for the molecular mechanisms involved in HRASV12- and cyclin E1-induced replication stress and its resolution.DNA replication stress induced by HRASV12 oncogene is driven by excessive reactive oxygen species (ROS) generated by the upregulation NADPH oxidase 1 and 4. This leads to PRDX2-mediated dissociation of the TIMELESS-TIPIN complex from the replisome, resulting in replication fork slowdown. The slow progression of replication forks towards head-on transcription complexes causes a gradual buildup of positive DNA supercoiling (+SC) in the DNA template, which promotes R-loop formation followed by ZRANB3/SMARCAL1-mediated fork reversal. Cyclin E1 overexpression increases intragenic origin firing, enhancing the frequency of head-on transcription-replication conflicts and concomitantly reducing replication fork velocity due to excessive origin firing, ultimately leading to the same outcome as observed with HRASV12. R-loop-mediated fork reversal is counteracted by RECQ1 DNA helicase, which unwinds reversed forks and promotes replication restart via the MUS81-LIG4-ELL pathway involving PRIMPOL repriming. This ensures the completion of DNA replication and proper chromosome segregation in mitosis. Persistent reversed forks impair chromosome segregation, giving rise to anaphase bridges and micronuclei, which can lead to chromosomal rearrangements. RNAPII, RNA polymerase II.