Background: Ubiquitylation plays important roles in DNA damage signal transduction. However, mechanistic details that drive these ubiquitin-dependent signals at DNA breaks remain to be determined. Results: RNF169 localized at DNA double-strand breaks (DSBs), inhibited DNA damage-induced ubiquitin formation, and attenuated 53BP1 accumulation. Conclusion: RNF169 antagonizes ubiquitin signaling at DNA DSBs. Significance: RNF169 represents a negative regulator of the ubiquitin-dependent DNA damage signaling cascade.

SUMMARY The DNA-dependent pattern recognition receptor, cGAS (cyclic GMP-AMP synthase), mediates communication between the DNA damage and the immune responses. Mitotic chromosome missegregation stimulates cGAS activity; however, it is unclear whether progression through mitosis is required for cancercell-intrinsic activation of anti-tumor immune responses. Moreover, it is unknown whether cell cycle checkpoint disruption can restore responses in cancer cells that are recalcitrant to DNAdamage-induced inflammation. Here, we demonstrate that prolonged cell cycle arrest at the G 2 -mitosis boundary from either excessive DNA damage or CDK1 inhibition prevents inflammatory-stimulated gene expression and immune-mediated destruction of distal tumors. Remarkably, DNAdamage-induced inflammatory signaling is restored in a RIG-I-dependent manner upon concomitant disruption of p53 and the G 2 checkpoint. These findings link aberrant cell progression and p53 loss to an expanded spectrum of damage-associated molecular pattern recognition and have implications for the design of rational approaches to augment anti-tumor immune responses.

Loading of p53-binding protein 1 (53BP1) and receptor-associated protein 80 (RAP80) at DNA double-strand breaks (DSBs) drives cell cycle checkpoint activation but is counterproductive to high-fidelity DNA repair. ring finger protein 169 (RNF169) maintains the balance by limiting the deposition of DNA damage mediator proteins at the damaged chromatin. We report here that this attribute is accomplished, in part, by a predicted nuclear localization signal (NLS) that not only shuttles RNF169 into the nucleus but also promotes its stability by mediating a direct interaction with the ubiquitin-specific protease USP7. Guided by the crystal structure of USP7 in complex with the RNF169 NLS, we uncoupled USP7 binding from its nuclear import function and showed that perturbing the USP7-RNF169 complex destabilized RNF169, compromised high-fidelity DSB repair, and hypersensitized cells to poly (ADP-ribose) polymerase inhibition. Finally, expression of USP7 and RNF169 positively correlated in breast cancer specimens. Collectively, our findings uncover an NLS-mediated bipartite mechanism that supports the nuclear function of a DSB response protein.ells respond to DNA double-strand breaks (DSBs) by mounting a series of signal transduction events that culminate in cell arrest and DNA repair (1). Signal transduction entails the coordinated assembly of a cohort of DNA damage mediator proteins, including p53-binding protein 1 (53BP1) and receptor-associated protein 80 (RAP80), at the damaged chromatin, which, in turn, enforces checkpoint control and cell tolerance to DNA damage (2, 3). Paradoxically, although DSB loading of 53BP1 and RAP80 underlies robust activation of DNA damage responses (DDRs), they operate at the expense of high-fidelity DNA repair, because their productive accumulation at DSB-flanking chromatin blocks DNA end resection and suppresses RAD51-dependent homologous recombination (HR) repair (4-11). Exactly how cells achieve a balance between robust DSB signal transduction and HR repair remains to be defined, but several lines of evidence converge on the idea that limiting the extent of DSB ubiquitylation may selectively promote HR repair (12-15), highlighting the need for cell-intrinsic mechanisms to restrain chromatin responses arising from DSBs (16).Intriguingly, one such cell-intrinsic strategy involves ring finger protein 169 (RNF169), a paralog of the RIDDLE (radiosensitivity, immunodeficiency, dysmorphic features, and learning difficulties) syndrome protein RNF168 (17,18). In stark contrast to RNF168, which plays positive roles in amplifying ubiquitin-dependent DSB signals (18,19), RNF169 is endowed with antagonistic properties in the DSB signal transduction pathway. Indeed, RNF169 competes for RNF168-catalyzed ubiquitin adducts and displaces 53BP1 and RAP80 from DSBs (14,20,21). Despite its putative role as an integral component of the DSB signal transduction cascade, how RNF169 is regulated mechanistically and how it contributes to the multipartite self-restraining mechanisms in DNA damage surveillance and...

Dysregulation of the DYRK1A protein kinase has been associated with human disease. On the one hand, its overexpression in trisomy 21 has been linked to certain pathological traits of Down syndrome, while on the other, inactivating mutations in just one allele are responsible for a distinct yet rare clinical syndrome, DYRK1A haploinsufficiency. Moreover, altered expression of this kinase may also provoke other human pathologies, including cancer and diabetes. Although a few DYRK1A substrates have been described, its upstream regulators and downstream targets are still poorly understood, an information that could shed light on the functions of DYRK1A in the cell. Here, we carried out a proteomic screen using antibody-based affinity purification coupled to mass spectrometry to identify proteins that directly or indirectly bind to endogenous DYRK1A. We show that the use of a cell line not expressing DYRK1A, generated by CRISPR/Cas9 technology, was needed in order to discriminate between true positives and non-specific interactions. Most of the proteins identified in the screen are novel candidate DYRK1A interactors linked to a variety of activities in the cell. The in-depth characterization of DYRK1A’s functional interaction with one of them, the E3 ubiquitin ligase RNF169, revealed a role for this kinase in the DNA damage response. We found that RNF169 is a DYRK1A substrate and we identified several of its phosphorylation sites. In particular, one of these sites appears to modify the ability of RNF169 to displace 53BP1 from sites of DNA damage. Indeed, DYRK1A depletion increases cell sensitivity to ionizing irradiation. Therefore, our unbiased proteomic screen has revealed a novel activity of DYRK1A, expanding the complex role of this kinase in controlling cell homeostasis.