PMC:6145447 / 12023-14368 JSONTXT

{"project":"2_test","denotations":[{"id":"29925947-22941618-75110979","span":{"begin":275,"end":277},"obj":"22941618"},{"id":"29925947-22941618-75110979","span":{"begin":275,"end":277},"obj":"22941618"},{"id":"29925947-15235593-75110980","span":{"begin":507,"end":509},"obj":"15235593"},{"id":"29925947-15235593-75110980","span":{"begin":507,"end":509},"obj":"15235593"},{"id":"29925947-17220302-75110981","span":{"begin":1012,"end":1014},"obj":"17220302"},{"id":"29925947-17220302-75110981","span":{"begin":1012,"end":1014},"obj":"17220302"}],"text":"WASP and Arp2/3 mediate HDR, not NHEJ\nTo determine how DSB clustering impacts DNA repair pathways, we utilized a panel of U2OS cell lines that monitor the repair of I-SceI-induced DSBs by HDR, NHEJ, single-strand annealing (SSA), and microhomology-mediated end joining (MMEJ)26. Arp2/3 inactivation by CK-666 or CK-548 reduced the ability of cells to repair I-SceI-induced DSBs by HDR or SSA by 40 percent (Fig. 5a, b and Extended Data Fig. 7a, e). Similarly, wiskostatin, a small molecule inhibitor of WASP27, reduced HDR and SSA efficiency (Fig. 5a, b). Comparable HDR defects in cells depleted of WASP by RNA interference were also observed (Extended Data Fig. 7b–d). Notably, inhibition of WASP or Arp2/3 did not compromise MMEJ or NHEJ, neither of which require significant resection (Fig. 5c, d and Extended Data Fig. 7f, g). Finally, the application of CK-689 did not reduce the repair of I-SceI-induced DSBs by HDR, SSA or NHEJ (Extended Data Fig. 7h).\nAlthough the Arp2/3 complex is found in the nucleus14, it also polymerizes actin in the cytoplasm. To strengthen the idea that the HDR defect was a direct consequence of inhibiting nuclear actin polymerization, we increased nuclear actin levels by overexpressing actin fused to a nuclear localization sequence (WT-actin-NLS) (Fig. 5e). Whereas expression of WT-actin-NLS did not affect HDR, overexpression of an actin variant that interrupts filament formation (R62D-actin-NLS) significantly inhibited HDR to levels comparable with Arp2/3 inhibition (Fig. 5f). Treatment of cells expressing R62D-actin-NLS with CK-666 did not further reduce HDR, suggesting that nuclear actin and Arp2/3 functioned together. Moreover, WT-actin, R62D-actin, and WT-actin-NLS tagged with mCherry did not affect DSB repair, whereas expression of R62D-actin-NLS significantly inhibited HDR (Extended Data Fig. 8a, b). This strongly suggests that inhibiting actin polymerization in the cytoplasm does not influence DNA repair in the nucleus. Similar to CK-666, expression of R62D-actin-NLS did not reduce NHEJ-based repair (Fig. 5g). Finally, neither siRNA directed against the actin filament nucleator formin-2 nor treatment with the formin inhibitor SMIFH2 inhibited HDR efficiency (Extended Data Fig. 8c–f). This observation thus demonstrates the specific requirement for Arp2/3 complex activity in HDR."}