Supplementary Materialszcaa006_Supplemental_Document. ATR inhibitors can be used for effective manipulation of DNA end resection capacity and DNA repair outcomes in cancer cells. INTRODUCTION DNA replication is a major source of DNA double-strand breaks (DSBs), which arise as replication forks encounter nicks on DNA or collide with obstacles such as DNACprotein or DNACDNA cross-links, actively transcribed genes and hard-to-replicate sequences (1). The ability of cells to sense and repair replication-induced lesions heavily relies on the = gene has been removed by CRISPR-Cas9, and both alleles of were tagged with an mAID epitope to conditionally induce TOPBP1 degradation upon auxin treatment (45,46) (Figure ?(Figure1F).1F). TOPBP1 auxin-dependent degradation resulted in destabilized BRCA1, BLM and?CTIP?(Figure 1G), similar to the effect observed with ATRi treatment. The abundance of resection factors was restored after auxin washout, indicating that loss of resection capacity is transient and is caused by the temporary and reversible suppression of ATR signaling (Figure ?(Figure1H).1H). Importantly, auxin-induced TOPBP1 depletion did not alter the cell cycle distribution (Figure ?(Figure1I).1I). Taken together, these results show that ATR signaling plays Acrivastine a key role in maintaining the abundance of crucial pro-resection factors. Since genotoxins are not used in the described experiments, the findings suggest that the maintenance of resection factor abundance relies on intrinsic ATR activation. Furthermore, since acute treatment (up to 24 hours) with ATRi does not result in similar depletion COL4A1 of resection factors, the activity of ATR must be inhibited over multiple cell division cycles for the altered abundances to become noticeable. Open in a separate window Figure 1. Chemical and genetic ablation of ATR signaling depletes the abundance of key resection factors. (A) U-2OS cells were cultured for 5 days in medium containing DMSO or the indicated concentrations of ATRi VE-821 and analyzed by immunoblotting. (B) Quantification of blots in (A). (C) U-2OS cells were treated as in (A) but with the ATRi AZD6738. (D) Quantification of blots in (C). (E) IdU incorporation analysis of U-2OS cells treated as in (C). (F) Strategy for abrogating ATR activators using the HCT116-= 4). (C) DNA end resection analysis in U-2OS-SEC 72 h after transfection of siRNA against BRCA1. Results are the same as shown in (F) (= 2). (D) DNA end resection analysis in U-2OS-SEC treated with 5 M VE-821 (ATRi) Acrivastine or 0.5 M UCN-01 (CHK1i) 8 h after sgRNA transfection. Cas9-eGFP expression was induced 24 h before sgRNA transfection. Mean SD (= 2); * 0.05. (E) Immunoblot analysis of cells treated as in (D). (F) DNA end resection analysis in U-2OS-SEC 72 h after transfection of the indicated siRNA. Mean SD (= 2); * 0.05, ** 0.01. (G) Immunoblot analysis of cells treated as in (F). (H) DNA end resection analysis in U-2OS-SEC-shSCR and U-2OS-SEC-sh53BP1 cells treated for 5 days with the indicated VE-821 concentrations. After ATRi pre-treatment, DSB was induced by co-transfecting sgRNA and purified Cas9. Mean SD (= 3); ** 0.01. (I) Immunoblot analysis of cells treated as in (H). (J) A schematic model showing how long-term ATRi treatment leads to the efficient Acrivastine depletion of HR proteins by avoiding the synthesis of fresh elements. Because BRCA1 great Acrivastine quantity is strongly suffering from long-term ATR inhibition (Shape?1A-?-D),D), we asked if the impairment of resection was predominantly due to the increased loss of BRCA1s function in counteracting the anti-resection element 53BP1. Since 53BP1 inactivation restores resection and HR in BRCA1-lacking tumors (48C50), we asked whether lack of 53BP1 could restore resection in cells treated chronically with ATRi. In keeping with earlier works, we discovered that 53BP1 Acrivastine depletion by siRNA rescues resection in cells depleted for BRCA1 considerably, as assessed by ddPCR at Cas9-induced breaks (Shape ?(Shape2F2F and?G). Additional evaluation in U-2Operating-system cells stably expressing inducible shRNA against 53BP1 and put through a 5-day time pre-treatment with VE-821 exposed that 53BP1 inactivation will not speed up resection acceleration upon long-term ATRi treatment (Shape ?(Shape2H2H and?We). Therefore, lack of resection capability in cells treated chronically with ATRi isn’t solely because of lack of BRCA1 but is probable a rsulting consequence the increased loss of multiple.