Fig 2: RAD52 Substantially Promotes a Subset of RMDs; however, BLM and MSH2 Inhibit RMDs Independently of RAD52(A) Immunoblotting analysis for several mESC lines, and transfections of siRNA or complementation vectors, for RAD52, MSH2, BLM, and ACTIN.(B) RMD analysis in WT (EV), Rad52−/− (EV), and Rad52−/− mESCs transfected with a RAD52 expression vector. Frequencies for WT EV are from Figure 2D. n = 6. *p ≤ 0.0481 and †p-unadjusted ≤ 0.0332 for EV versus RAD52 complemented in Rad52−/− or WT versus Rad52−/−.(C) RMD frequencies of Rad52−/− mESCs treated with siBLM or siCTRL. Frequencies from WT siCTRL are from Figure 1C. n = 6. *p ≤ 0.0036 for siCTRL versus siBLM or for WT versus Rad52−/−.(D) RMD analysis for WT, Rad52−/− mESCs, Rad52−/−Msh2−/−, and Rad52−/−Msh2−/− transfected with an MSH2 expression vector. Frequencies for WT EV and Rad52−/− EV are from Figure 2D and (B), respectively. n = 6. *p ≤ 0.0221 for Rad52−/− versus Rad52−/−Msh2−/− or for MSH2 complemented versus EV.

Fig 3: MRE11 foci merged with γH2AX are more effectively formed under MMR deficient background compared to those of WT cells. (A,B) HeLa cells knocked down for MSH2 and the control were treated with HU as shown in the top box, and γH2AX and MRE11 foci were assessed by immunofluorescence microscopy (A). Representative cells are zoomed. Foci numbers were counted (B). Error bars indicate SD. Scale bars, 10 μm.

Fig 4: Replication stress-associated DSBs are persisted under intermediates of HR in MMR-proficient cells. (A–D) WT and Msh2−/− MEFs were treated with HU, as shown in the workflow (A). HR statuses were determined by the intensities of p- and pp-Rad51 signals (A,B). MMR-deficiency dependent decay of p- and pp-Rad51 signals are pointed by red arrowheads (A). Source images of blots are shown in Supplementary Fig. 1. p- and pp-Rad51 signals normalized with β-Actin signal are shown (B). DSB statuses were determined by the merged foci of γH2AX and 53BP1 (C,D). The representative images are shown (C). The numbers of 53BP1 foci in each cell were plotted (n numbers and P values are indicated in graph) (D). Two-tailed Welch's t-test was used for statistical analysis. (E) WT and Msh2−/− MEFs were exogenously growth-accelerated, as shown in the workflow. HR and DSB statuses were determined by the merged foci of Rad51 and γH2AX. Representative cells are zoomed. Error bars represent ±SD. Scale bars, 10 μm.

Fig 5: BLM-Mediated Suppression of RMDs Is Not Epistatic with the Influence of MSH2 (except at the 16-bp Distance) or BRCA2(A) Influence of BLM depletion on RMDs in Msh2−/− mESCs. Shown is BLM and ACTIN immunoblot analysis, and RMD frequencies, of Msh2−/− mESCs treated with siBLM versus siCTRL. The WT siCTRL RMD frequencies are from Figure 1C. n = 6. *p ≤ 0.0222 and †p-unadjusted ≤ 0.0272 for siCTRL versus siBLM in Msh2−/− mESCs or for WT versus Msh2−/−. Shown are fold effects of significant differences, using the mean.(B) Influence of BLM levels on HDR (DR-GFP assay) In Blmtet/tet mESCs treated with DMSO or Dox, as for the RMD assays. n = 6. ns, not significant.(C) Influence of siBRCA2 treatment on HDR in mESCs. n = 6. *p = 0.0002. Immunoblot analysis of BRCA2 and ACTIN using this siRNA is shown. The asterisk (*) indicates nonspecific bands.(D) Immunoblot analysis of BRCA2 depletion in Blmtet/tet mESCs treated with DMSO or Dox.(E) Effects of siBRCA2 on RMDs in Blmtet/tet mESCs treated with Dox (i.e., BLM depletion) or DMSO control. n = 6. *p ≤ 0.0354 and †p-unadjusted ≤ 0.0435 for siCTRL versus siBRCA2. Fold effects as in (A) are shown.