Almudena Serrano-Benitez

DNA double-strand breaks (DSBs) both pose threats to genome integrity and are commonly used for genome editing applications. Structural features of DSB ends play key roles in determining DNA repair pathway usage and outcomes during genome editing, but the cellular factors involved in these processes are only partially known. Through genome-wide CRISPRi screening, we identify ERCC6L2 as critical for repairing Cas12a-induced staggered DSBs but irrelevant for Cas9-induced blunt DSBs. We show that ERCC6L2 acts as a protection factor for staggered DSBs with either 5′ or 3′ polarity, preventing large deletions and translocations stemming from DNA damage induced by Cas12a, TALENs, or dual Cas9 nicks. Furthermore, ERCC6L2 loss hyper-sensitizes cells to multiple staggered DSBs induced by promiscuous Cas12a activity or etoposide-induced TOP2 trapping. By combining genetics and biochemical reconstitution, we find that ERCC6L2 counteracts MRE11-RAD50-NBS1 (MRN)-mediated resection by binding and melting staggered DNA ends, thereby promoting accurate end joining. Our data reveal a protective role of ERCC6L2 in staggered-end DSB repair, which suggests the molecular underpinnings of pathology in patients with ERCC6L2 mutations and cautions against using overhang-inducing genome editing tools for their treatment.

The catalytic cycle of topoisomerase 2 (TOP2) enzymes proceeds via a transient DNA double-strand break (DSB) intermediate termed the TOP2 cleavage complex (TOP2cc), in which the TOP2 protein is covalently bound to DNA. Anticancer agents such as etoposide operate by stabilizing TOP2ccs, ultimately generating genotoxic TOP2-DNA protein cross-links that require processing and repair. Here, we identify RAD54 like 2 (RAD54L2) as a factor promoting TOP2cc resolution. We demonstrate that RAD54L2 acts through a novel mechanism together with zinc finger protein associated with tyrosyl-DNA phosphodiesterase 2 (TDP2) and TOP2 (ZATT/ZNF451) and independent of TDP2. Our work suggests a model wherein RAD54L2 recognizes sumoylated TOP2 and, using its ATPase activity, promotes TOP2cc resolution and prevents DSB exposure. These findings suggest RAD54L2-mediated TOP2cc resolution as a potential mechanism for cancer therapy resistance and highlight RAD54L2 as an attractive candidate for drug discovery.