The breast cancer susceptibility proteins BRCA1 and BRCA2 have emerged as key stabilizing factors for the maintenance of replication fork integrity following replication stress. In their absence, stalled replication forks are extensively degraded by the MRE11 nuclease, leading to chemotherapeutic sensitivity. Here we report that BRCA proteins prevent nucleolytic degradation by protecting replication forks that have undergone fork reversal upon drug treatment. The unprotected regressed arms of reversed forks are the entry point for MRE11 in BRCA-deficient cells. The CtIP protein initiates MRE11-dependent degradation, which is extended by the EXO1 nuclease. Next, we show that the initial limited resection of the regressed arms establishes the substrate for MUS81 in BRCA2-deficient cells. In turn, MUS81 cleavage of regressed forks with a ssDNA tail promotes POLD3-dependent fork rescue. We propose that targeting this pathway may represent a new strategy to modulate BRCA2-deficient cancer cell response to chemotherapeutics that cause fork degradation.

During the process of cross presentation, viral or tumor-derived antigens are presented to CD8+ T cells by the Batf3-dependent CD8α+/XCR1+ classical dendritic cell (cDC1). We designed a functional CRISPR screen for novel regulators of cross presentation, and identified the BEACH-domain containing protein WDFY4 as essential for cross-presentation of cell-associated antigens by cDC1. WDFY4 was not, however, required for MHC class II presentation or for cross-presentation by monocyte-derived DCs. In contrast to Batf3−/− mice, Wdfy4−/− mice have normal lymphoid and non-lymphoid cDC1 populations that produce IL-12 and protect against Toxoplasma gondii infection. However similar to Batf3−/− mice, Wdfy4−/− mice fail to prime virus- specific CD8+ T cells in vivo or induce tumor rejection, revealing a critical role for cross-presentation in anti-viral and anti-tumor immunity.

DNA repair is essential to prevent the cytotoxic or mutagenic effects of various types of DNA lesions, which are sensed by distinct pathways to recruit repair factors specific to the damage type. Although biochemical mechanisms for repairing several forms of genomic insults are well understood, the upstream signalling pathways that trigger repair are established for only certain types of damage, such as double-stranded breaks and interstrand crosslinks. Understanding the upstream signalling events that mediate recognition and repair of DNA alkylation damage is particularly important, since alkylation chemotherapy is one of the most widely used systemic modalities for cancer treatment and because environmental chemicals may trigger DNA alkylation. Here we demonstrate that human cells have a previously unrecognized signalling mechanism for sensing damage induced by alkylation. We find that the alkylation repair complex ASCC (activating signal cointegrator complex) relocalizes to distinct nuclear foci specifically upon exposure of cells to alkylating agents. These foci associate with alkylated nucleotides, and coincide spatially with elongating RNA polymerase II and splicing components. Proper recruitment of the repair complex requires recognition of K63-linked polyubiquitin by the CUE (coupling of ubiquitin conjugation to ER degradation) domain of the subunit ASCC2. Loss of this subunit impedes alkylation adduct repair kinetics and increases sensitivity to alkylating agents, but not other forms of DNA damage. We identify RING finger protein 113A (RNF113A) as the E3 ligase responsible for upstream ubiquitin signalling in the ASCC pathway. Cells from patients with X-linked trichothiodystrophy, which harbour a mutation in RNF113A, are defective in ASCC foci formation and are hypersensitive to alkylating agents. Together, our work reveals a previously unrecognized ubiquitin-dependent pathway induced specifically to repair alkylation damage, shedding light on the molecular mechanism of X-linked trichothiodystrophy.

Repair of DNA alkylation damage is critical for genomic stability and involves multiple conserved enzymatic pathways. Alkylation damage resistance, which is critical in cancer chemotherapy, depends on the overexpression of alkylation repair proteins. However, the mechanisms responsible for this upregulation are unknown. Here, we show that an OTU domain deubiquitinase, OTUD4, is a positive regulator of ALKBH2 and ALKBH3, two DNA demethylases critical for alkylation repair. Remarkably, we find that OTUD4 catalytic activity is completely dispensable for this function. Rather, OTUD4 is a scaffold for USP7 and USP9X, two deubiquitinases that act directly on the AlkB proteins. Moreover, we show that loss of OTUD4, USP7, or USP9X in tumor cells makes them significantly more sensitive to alkylating agents. Taken together, this work reveals a novel, noncanonical mechanism by which an OTU family deubiquitinase regulates its substrates, and provides multiple new targets for alkylation chemotherapy sensitization of tumors.

No abstract