Autologous K63 deubiquitylation within the BRCA1-A complex licenses DNA damage recognition.

The BRCA1-A complex contains matching lysine-63 ubiquitin (K63-Ub) binding and deubiquitylating activities. How these functionalities are coordinated to effectively respond to DNA damage remains unknown. We generated Brcc36 deubiquitylating enzyme (DUB) inactive mice to address this gap in knowledge in a physiologic system. DUB inactivation impaired BRCA1-A complex damage localization and repair activities while causing early lethality when combined with Brca2 mutation. Damage response dysfunction in DUB-inactive cells corresponded to increased K63-Ub on RAP80 and BRCC36. Chemical cross-linking coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and cryogenic-electron microscopy (cryo-EM) analyses of isolated BRCA1-A complexes demonstrated the RAP80 ubiquitin interaction motifs are occupied by ubiquitin exclusively in the DUB-inactive complex, linking auto-inhibition by internal K63-Ub chains to loss of damage site ubiquitin recognition. These findings identify RAP80 and BRCC36 as autologous DUB substrates in the BRCA1-A complex, thus explaining the evolution of matching ubiquitin-binding and hydrolysis activities within a single macromolecular assembly.

Unpaved roads: How the DNA damage response navigates endogenous genotoxins.

Accurate DNA repair is essential for cellular and organismal homeostasis, and DNA repair defects result in genetic diseases and cancer predisposition. Several environmental factors, such as ultraviolet light, damage DNA, but many other molecules with DNA damaging potential are byproducts of normal cellular processes. In this review, we highlight some of the prominent sources of endogenous DNA damage as well as their mechanisms of repair, with a special focus on repair by the homologous recombination and Fanconi anemia pathways. We also discuss how modulating DNA damage caused by endogenous factors may augment current approaches used to treat BRCA-deficient cancers. Finally, we describe how synthetic lethal interactions may be exploited to exacerbate DNA repair deficiencies and cause selective toxicity in additional types of cancers.

CHK1 phosphorylates PRIMPOL to promote replication stress tolerance.

Replication-coupled DNA repair and damage tolerance mechanisms overcome replication stress challenges and complete DNA synthesis. These pathways include fork reversal, translesion synthesis, and repriming by specialized polymerases such as PRIMPOL. Here, we investigated how these pathways are used and regulated in response to varying replication stresses. Blocking lagging-strand priming using a POLα inhibitor slows both leading- and lagging-strand synthesis due in part to RAD51-, HLTF-, and ZRANB3-mediated, but SMARCAL1-independent, fork reversal. ATR is activated, but CHK1 signaling is dampened compared to stalling both the leading and lagging strands with hydroxyurea. Increasing CHK1 activation by overexpressing CLASPIN in POLα-inhibited cells promotes replication elongation through PRIMPOL-dependent repriming. CHK1 phosphorylates PRIMPOL to promote repriming irrespective of the type of replication stress, and this phosphorylation is important for cellular resistance to DNA damage. However, PRIMPOL activation comes at the expense of single-strand gap formation, and constitutive PRIMPOL activity results in reduced cell fitness.

ATR activation is regulated by dimerization of ATR activating proteins.

The checkpoint kinase ATR regulates DNA repair, cell cycle progression, and other DNA damage and replication stress responses. ATR signaling is stimulated by an ATR activating protein, and in metazoan cells, there are at least two ATR activators: TOPBP1 and ETAA1. Current evidence indicates TOPBP1 and ETAA1 activate ATR via the same biochemical mechanism, but several aspects of this mechanism remain undefined. For example, ATR and its obligate binding partner ATR interacting protein (ATRIP) form a tetrameric complex consisting of two ATR and two ATRIP molecules, but whether TOPBP1 or ETAA1 dimerization is similarly required for ATR function is unclear. Here, we show that fusion of the TOPBP1 and ETAA1 ATR activation domains (AADs) to dimeric tags makes them more potent activators of ATR in vitro. Furthermore, induced dimerization of both AADs using chemical dimerization of a modified FKBP tag enhances ATR kinase activation and signaling in cells. ETAA1 forms oligomeric complexes mediated by regions of the protein that are predicted to be intrinsically disordered. Induced dimerization of a "mini-ETAA1" protein that contains the AAD and Replication Protein A (RPA) interaction motifs enhances ATR signaling, rescues cellular hypersensitivity to DNA damaging agents, and suppresses micronuclei formation in ETAA1-deficient cells. Together, our results indicate that TOPBP1 and ETAA1 dimerization is important for optimal ATR signaling and genome stability.

Common motifs in ETAA1 and TOPBP1 required for ATR kinase activation.

DNA damage response Ser/Thr kinases, including ataxia telangiectasia-mutated (ATM) and Rad3-related (ATR), control cell cycle progression, DNA repair, and apoptosis. ATR is activated by ETAA1 activator of ATR kinase (ETAA1) or DNA topoisomerase II binding protein 1 (TOPBP1). Both ETAA1 and TOPBP1 contain experimentally defined ATR activation domains (AADs) that are mostly unstructured and have minimal sequence similarity. A tryptophan residue in both AADs is required for ATR activation, but the other features of these domains and the mechanism by which they activate ATR are unknown. In this study, using bioinformatic analyses, kinase assays, co-immunoprecipitation, and immunofluorescence measures of signaling, we more specifically defined the TOPBP1 and ETAA1 AADs and identified additional features of the AADs needed for ATR activation. We found that both ETAA1 and TOPBP1 contain a predicted coiled-coil motif that is required for ATR activation in vitro and in cells. Mutation of the predicted coiled coils does not alter AAD oligomerization but does impair binding of the AADs to ATR. These results suggest that TOPBP1 and ETAA1 activate ATR using similar motifs and mechanisms.

Tissue-specific variation in nonsense mutant transcript level and drug-induced read-through efficiency in the Cln1(R151X) mouse model of INCL.

About 10% of inherited diseases are caused by nonsense mutations [Trends Mol Med 18 (2012) 688], and nonsense suppression drug therapy promoting translation through premature stop codons is an emerging therapeutic approach. Infantile neuronal ceroid lipofuscinosis (INCL), a childhood neurodegenerative disease, results from mutations in the CLN1 gene encoding the lysosomal enzyme, palmitoyl-protein thioesterase 1 (PPT1) [Biochim Biophys Acta 1832 (2013) 1806, Hum Mutat (2012) 63, Biochim Biophys Acta 1832 (2013) 1881]. The nonsense mutation p.R151X is the most common disease-causing CLN1 mutation Hum Mutat (2012) 63. In the novel Cln1(R151X) mouse model of INCL, we found large, tissue-specific variations in Cln1(R151X) mRNA level and PPT1 residual enzyme activity. These tissue-specific differences strongly influenced the read-through efficiency of ataluren (PTC124), a well-known nonsense suppression drug. A two-day treatment with ataluren (10 mg/kg) increased PPT1 enzyme activity in the liver and muscle, but not in any other tissue examined. Our study identifies a new challenge/hurdle for read-through drug therapy: variable efficiency of read-through therapy in the different tissues/organs because of tissue-specific variations in nonsense mutant transcript levels.