Data from: Rev7 and 53BP1/Crb2 prevent RecQ helicase-dependent hyper-resection of DNA double-strand breaks

Poly(ADP ribose) polymerase inhibitors (PARPi) target cancer cells deficient in homology-directed repair of DNA double-strand breaks (DSBs). In preclinical models, PARPi resistance is tied to altered nucleolytic processing (resection) at the 5' ends of a DSB. For example, loss of 53BP1 or Rev7/MAD2L2/FANCV derepresses resection to drive PARPi resistance, although the mechanisms are poorly understood. Long-range resection can be catalyzed by two machineries: the exonuclease Exo1, or the combination of a RecQ helicase and Dna2. Here, we develop a single cell microscopy assay that allows the distinct phases and machineries of resection to be interrogated simultaneously in living S. pombe cells. Using this assay, we find that the 53BP1 orthologue and Rev7 specifically repress long-range resection through the RecQ helicase-dependent pathway, thereby preventing hyper-resection. These results suggest that 'rewiring' of BRCA1-deficient cells to employ an Exo1-independent hyper-resection pathway is a driver of PARPi resistance.

聚(ADP-核糖)聚合酶抑制剂（Poly(ADP ribose) polymerase inhibitors，PARPi）可靶向存在DNA双链断裂（DNA double-strand breaks，DSBs）同源定向修复缺陷的癌细胞。在临床前模型中，PARPi耐药性与DNA双链断裂5'端的核酸酶解加工（末端切除，resection）异常密切相关。例如，53BP1或Rev7/MAD2L2/FANCV的缺失会解除对末端切除的抑制，进而介导PARPi耐药性，但其具体分子机制目前仍不甚明确。长距离末端切除可通过两种机制完成：一是核酸外切酶Exo1，二是RecQ解旋酶与Dna2的协同作用。本研究开发了一种单细胞显微镜成像检测方法，可在活的粟酒裂殖酵母（S. pombe）细胞中同时检测末端切除的不同阶段及参与的分子机制。利用该检测方法，我们发现53BP1同源蛋白与Rev7可通过依赖RecQ解旋酶的通路特异性抑制长距离末端切除，从而避免过度切除事件的发生。上述研究结果提示，BRCA1缺陷细胞通过通路重编程转而采用不依赖Exo1的过度切除通路，是其产生PARPi耐药性的关键驱动因素。