The SUMO‐targeted ubiquitin ligase RNF4 functions at the crossroads of the SUMO and ubiquitin systems. Here, we report that the deubiquitylation enzyme (DUB) ataxin‐3 counteracts RNF4 activity during the DNA double‐strand break (DSB) response. We find that ataxin‐3 negatively regulates ubiquitylation of the checkpoint mediator MDC1, a known RNF4 substrate. Loss of ataxin‐3 markedly decreases the chromatin dwell time of MDC1 at DSBs, which can be fully reversed by co‐depletion of RNF4. Ataxin‐3 is recruited to DSBs in a SUMOylation‐dependent fashion, and in vitro it directly interacts with and is stimulated by recombinant SUMO, defining a SUMO‐dependent mechanism for DUB activity toward MDC1. Loss of ataxin‐3 results in reduced DNA damage‐induced ubiquitylation due to impaired MDC1‐dependent recruitment of the ubiquitin ligases RNF8 and RNF168, and reduced recruitment of 53BP1 and BRCA1. Finally, ataxin‐3 is required for efficient MDC1‐dependent DSB repair by non‐homologous end‐joining and homologous recombination. Consequently, loss of ataxin‐3 sensitizes cells to ionizing radiation and poly(ADP‐ribose) polymerase inhibitor. We propose that the opposing activities of RNF4 and ataxin‐3 consolidate robust MDC1‐dependent signaling and repair of DSBs.

The pathology of spinocerebellar ataxia type 3, also known as Machado‐Joseph disease, is triggered by aggregation of toxic ataxin‐3 (ATXN3) variants containing expanded polyglutamine repeats. The physiological role of this deubiquitylase, however, remains largely unclear. Our recent work showed that ATX‐3, the nematode orthologue of ATXN3, together with the ubiquitin‐directed segregase CDC‐48, regulates longevity in Caenorhabditis elegans. Here, we demonstrate that the long‐lived cdc‐48.1; atx‐3 double mutant displays reduced viability under prolonged starvation conditions that can be attributed to the loss of catalytically active ATX‐3. Reducing the levels of the autophagy protein BEC‐1 sensitized worms to the effect of ATX‐3 deficiency, suggesting a role of ATX‐3 in autophagy. In support of this conclusion, the depletion of ATXN3 in human cells caused a reduction in autophagosomal degradation of proteins. Surprisingly, reduced degradation in ATXN3‐depleted cells coincided with an increase in the number of autophagosomes while levels of lipidated LC3 remained unaffected. We identified two conserved LIR domains in the catalytic Josephin domain of ATXN3 that directly interacted with the autophagy adaptors LC3C and GABARAP in vitro. While ATXN3 localized to early autophagosomes, it was not subject to lysosomal degradation, suggesting a transient regulatory interaction early in the autophagic pathway. We propose that the deubiquitylase ATX‐3/ATXN3 stimulates autophagic degradation by preventing superfluous initiation of autophagosomes, thereby promoting an efficient autophagic flux important to survive starvation.

Enzymes in the PARP family partake in the regulation of vital cellular signaling pathways by ADP-ribosylating their targets. The roles of these signaling pathways in disease development and the de-regulation of several PARP enzymes in cancer cells have motivated the pursuit of PARP inhibitors for therapeutic applications. In this rapidly expanding research area, availability of simple research tools will help assess the functions of ADP-ribosylation in a wider range of contexts. Here, we generated a mutant Af1521 macrodomain fused to green fluorescent protein (GFP) to generate a high-affinity ADP-ribosyl binding reagent. The resulting tool – which we call MacroGreen – is easily produced by expression in Escherichia coli , and can detect both mono-and poly-ADP-ribosylation of diverse proteins in vitro. Staining with MacroGreen allows detection of ADP-ribosylation at sites of DNA damage by fluorescence microscopy. MacroGreen can also be used to quantify modification of target proteins in overlay assays, and to screen for PARP inhibitors in high-throughput format with excellent assay statistics. We expect that this broadly applicable tool will facilitate ADP-ribosylation related discoveries, including by laboratories that do not specialize in this field.

Lysosomal membrane damage represents a threat to cell viability. As such, cells have evolved sophisticated mechanisms to maintain lysosomal integrity. Small membrane lesions are detected and repaired by the endosomal sorting complex required for transport (ESCRT) machinery while more extensively damaged lysosomes are cleared by a galectin‐dependent selective macroautophagic pathway (lysophagy). In this study, we identify a novel role for the autophagosome‐lysosome tethering factor, TECPR1, in lysosomal membrane repair. Lysosomal damage promotes TECPR1 recruitment to damaged membranes via its N‐terminal dysferlin domain. This recruitment occurs upstream of galectin and precedes the induction of lysophagy. At the damaged membrane, TECPR1 forms an alternative E3‐like conjugation complex with the ATG12‐ATG5 conjugate to regulate ATG16L1‐independent unconventional LC3 lipidation. Abolishment of LC3 lipidation via ATG16L1/TECPR1 double knockout impairs lysosomal recovery following damage.