Repair of DNA double strand breaks is critical to genomic stability and the prevention of developmental disorders and cancer. A central pathway for this repair is homologous recombination (HR). Most knowledge of HR is derived from work in prokaryotic and eukaryotic model organisms. We performed a genome-wide siRNA-based screen in human cells. Among positive regulators of HR we identified networks of DNA damage response and pre-mRNA processing proteins, and among negative regulators we identified a phosphatase network. Three candidate proteins localized to DNA lesions including RBMX, a heterogeneous nuclear ribonucleoprotein that has a role in alternative splicing. RBMX accumulated at DNA lesions via multiple domains in a poly(ADP-ribose) polymerase 1-dependent manner and promoted HR by facilitating proper BRCA2 expression. Our screen also revealed that off-target depletion of Rad51 is a common source of RNAi false-positives, sounding a cautionary note for siRNA screens and RNAi-based studies of HR.

Summary Microtubule inhibitors are important cancer drugs that induce mitotic arrest by activating the spindle assembly checkpoint (SAC), which in turn inhibits the ubiquitin ligase activity of the Anaphase-Promoting Complex (APC). Here we report a small molecule, Tosyl-L-Arginine Methyl Ester (TAME), which binds to the APC and prevents its activation by Cdc20 and Cdh1. A prodrug of TAME arrests cells in metaphase without perturbing the spindle, but nonetheless the arrest is dependent on the SAC. Metaphase arrest induced by a proteasome inhibitor is also SAC-dependent, suggesting that APC-dependent proteolysis is required to inactivate the SAC. We propose that mutual antagonism between the APC and the SAC yields a positive feedback loop that amplifies the ability of TAME to induce mitotic arrest.

Summary Protein machines are multi-subunit protein complexes that orchestrate highly regulated biochemical tasks. An example is the Anaphase-Promoting Complex/Cyclosome (APC/C), a thirteen-subunit ubiquitin ligase that initiates the metaphase-anaphase transition and mitotic exit by targeting proteins such as securin and cyclin B1 for ubiquitin-dependent destruction by the proteasome1,2. Because blocking mitotic exit is an effective approach for inducing tumor cell death3,4, the APC/C represents a potential novel target for cancer therapy. APC/C activation in mitosis requires binding of Cdc205, which forms a co-receptor with the APC/C to recognize substrates containing a Destruction box (D-box)6-14. Here we demonstrate that we can synergistically inhibit APC/C-dependent proteolysis and mitotic exit by simultaneously disrupting two protein-protein interactions within the APC/C-Cdc20-substrate ternary complex. We identified a small molecule, called apcin (APC inhibitor), which binds to Cdc20 and competitively inhibits the ubiquitylation of D-box-containing substrates. Analysis of the crystal structure of the apcin-Cdc20 complex suggests that apcin occupies the D-box-binding pocket on the side face of the WD40-domain. The ability of apcin to block mitotic exit is synergistically amplified by co-addition of tosyl-L-arginine methyl ester (TAME), a small molecule that blocks the APC/C-Cdc20 interaction15,16. This work suggests that simultaneous disruption of multiple, weak protein-protein interactions is an effective approach for inactivating a protein machine.

Biliary epithelial cells (BECs) form bile ducts in the liver and are facultative liver stem cells that establish a ductular reaction (DR) to support liver regeneration following injury. Liver damage induces periportal LGR5+ putative liver stem cells that can form BEClike organoids, suggesting that RSPO-LGR4/5-mediated WNT/b-catenin activity is important for a DR. We addressed the roles of this and other signaling pathways in a DR by performing a focused CRISPRbased loss-of-function screen in BEC-like organoids, followed by in vivo validation and single-cell RNA sequencing. We found that BECs lack and do not require LGR4/5-mediated WNT/b-catenin signaling during a DR, whereas YAP and mTORC1 signaling are required for this process. Upregulation of AXIN2 and LGR5 is required in hepatocytes to enable their regenerative capacity in response to injury. Together, these data highlight heterogeneity within the BEC pool, delineate signaling pathways involved in a DR, and clarify the identity and roles of injury-induced periportal LGR5+ cells.