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DNA double strand breaks (DSBs) are the most cytotoxic DNA lesions and, if not repaired, lead to chromosomal rearrangement, genomic instability and cell death. Cells have evolved a complex network of DNA repair and signalling molecules which promptly detect and repair DSBs, commonly known as the DNA damage response (DDR). The DDR is orchestrated by various post-translational modifications such as phosphorylation, methylation, ubiquitination or SUMOylation. As DSBs are located in complex chromatin structures, the repair of DSBs is engineered at two levels: (i) at sites of broken DNA and (ii) at chromatin structures that surround DNA lesions. Thus, DNA repair and chromatin remodelling machineries must work together to efficiently repair DSBs. Here, we summarize the current knowledge of the ubiquitin-dependent molecular unfoldase/segregase p97 (VCP in vertebrates and Cdc48 in worms and lower eukaryotes) in DSB repair. We identify p97 as an essential factor that regulates DSB repair. p97-dependent extraction of ubiquitinated substrates mediates spatio-temporal protein turnover at and around the sites of DSBs, thus orchestrating chromatin remodelling and DSB repair. As p97 is a druggable target, p97 inhibition in the context of DDR has great potential for cancer therapy, as shown for other DDR components such as PARP, ATR and CHK1. This article is part of the themed issue ‘Chromatin modifiers and remodellers in DNA repair and signalling’.

Eukaryotic topoisomerase 1 (TOP1) regulates DNA topology to ensure efficient DNA replication and transcription. TOP1 is also a major driver of endogenous genome instability, particularly when its catalytic intermediate—a covalent TOP1-DNA adduct known as a TOP1 cleavage complex (TOP1cc)—is stabilised. TOP1ccs are highly cytotoxic and a failure to resolve them underlies the pathology of neurological disorders but is also exploited in cancer therapy where TOP1ccs are the target of widely used frontline anti-cancer drugs. A critical enzyme for TOP1cc resolution is the tyrosyl-DNA phosphodiesterase (TDP1), which hydrolyses the bond that links a tyrosine in the active site of TOP1 to a 3’ phosphate group on a single-stranded (ss)DNA break. However, TDP1 can only process small peptide fragments from ssDNA ends, raising the question of how the ~90 kDa TOP1 protein is processed upstream of TDP1. Here we find that TEX264 fulfils this role by forming a complex with the p97 ATPase and the SPRTN metalloprotease. We show that TEX264 recognises both unmodified and SUMO1-modifed TOP1 and initiates TOP1cc repair by recruiting p97 and SPRTN. TEX264 localises to the nuclear periphery, associates with DNA replication forks, and counteracts TOP1ccs during DNA replication. Altogether, our study elucidates the existence of a specialised repair complex required for upstream proteolysis of TOP1ccs and their subsequent resolution. Eukaryotic topoisomerase 1 (TOP1) regulates DNA topology to ensure efficient DNA replication and transcription. Here, the authors reveal insights into the molecular resolution of topoisomerase 1-DNA adducts by TEX264, p97 and SPRTN.

The human MRE11/RAD50/NBS1 (MRN) complex plays a crucial role in sensing and repairing DNA DSB. MRE11 possesses dual 3′−5′ exonuclease and endonuclease activity and forms the core of the multifunctional MRN complex. We previously identified a C-terminally truncated form of MRE11 (TR-MRE11) associated with post-translational MRE11 degradation. Here we identified SPRTN as the essential protease for the formation of TR-MRE11 and characterised the role of this MRE11 form in its DNA damage response (DDR). Using tandem mass spectrometry and site-directed mutagenesis, the SPRTN-dependent cleavage site for MRE11 was identified between 559 and 580 amino acids. Despite the intact interaction of TR-MRE11 with its constitutive core complex proteins RAD50 and NBS1, both nuclease activities of truncated MRE11 were dramatically reduced due to its deficient binding to DNA. Furthermore, lack of the MRE11 C-terminal decreased HR repair efficiency, very likely due to abolished recruitment of TR-MRE11 to the sites of DNA damage, which consequently led to increased cellular radiosensitivity. The presence of this DNA repair-defective TR-MRE11 could explain our previous finding that the high MRE11 protein expression by immunohistochemistry correlates with improved survival following radical radiotherapy in bladder cancer patients.

The SPRTN metalloprotease is essential for DNA-protein crosslink (DPC) repair and DNA replication in vertebrate cells. Cells deficient in SPRTN protease exhibit DPC-induced replication stress and genome instability, manifesting as premature ageing and liver cancer. Here, we provide a body of evidence suggesting that SPRTN activates the ATR-CHK1 phosphorylation signalling cascade during physiological DNA replication by proteolysis-dependent eviction of CHK1 from replicative chromatin. During this process, SPRTN proteolyses the C-terminal/inhibitory part of CHK1, liberating N-terminal CHK1 kinase active fragments. Simultaneously, CHK1 full length and its N-terminal fragments phosphorylate SPRTN at the C-terminal regulatory domain, which stimulates SPRTN recruitment to chromatin to promote unperturbed DNA replication fork progression and DPC repair. Our data suggest that a SPRTN-CHK1 cross-activation loop plays a part in DNA replication and protection from DNA replication stress. Finally, our results with purified components of this pathway further support the proposed model of a SPRTN-CHK1 cross-activation loop. Cells deficient in SPRTN protease activity exhibit severe DNA-protein crosslink induced replication stress and genome instability. Here the author reveal a functional link between the SPRTN protease and the CHK1 kinase during physiological DNA replication.

The G-protein coupled receptor (GPCR) gene family represents one of the largest families in the mammalian genome. The flexibility of signalling and widespread tissue distribution of these receptors has allowed GPCRs to be employed in the physiological regulation of nearly all biological functions. This, coupled with the fact that it is possible to chemically produce highly specific ligands to these receptors have made GPCRs attractive targets for pharmacological intervention in a wide variety of disease states. When targeting GPCRs in therapeutic drug design it is traditional, and eminently sensible, to focus on ligands that will provide agonism, antagonism or allosteric modulation. However, as more is understood of the mechanisms that regulate GPCRs, and in particular the dynamic covalent modifications that might endow tissue specific functions, then these regulatory processes may provide alternative targets for GPCR drug discovery. In this review we consider three of the covalent modifications which are considered to regulate the function of GPCRs namely; receptor phosphorylation, palmitoylation and ubiquitination. In particular, we will describe the mechanisms of modification, the functional consequences and the relationship between these three covalent modification events

DNA double strand breaks (DSBs) are the most cytotoxic DNA lesions and, if not repaired, lead to chromosomal rearrangement, genomic instability and cell death. Cells have evolved a complex network of DNA repair and signalling molecules which promptly detect and repair DSBs, commonly known as the DNA damage response (DDR). The DDR is orchestrated by various post-translational modifications such as phosphorylation, methylation, ubiquitination or SUMOylation. As DSBs are located in complex chromatin structures, the repair of DSBs is engineered at two levels: (i) at sites of broken DNA and (ii) at chromatin structures that surround DNA lesions. Thus, DNA repair and chromatin remodelling machineries must work together to efficiently repair DSBs. Here, we summarize the current knowledge of the ubiquitin-dependent molecular unfoldase/segregase p97 (VCP in vertebrates and Cdc48 in worms and lower eukaryotes) in DSB repair. We identify p97 as an essential factor that regulates DSB repair. p97-dependent extraction of ubiquitinated substrates mediates spatio-temporal protein turnover at and around the sites of DSBs, thus orchestrating chromatin remodelling and DSB repair. As p97 is a druggable target, p97 inhibition in the context of DDR has great potential for cancer therapy, as shown for other DDR components such as PARP, ATR and CHK1. This article is part of the themed issue 'Chromatin modifiers and remodellers in DNA repair and signalling'.

We investigated the possibility of Ca2+ signaling in cyanobacteria (blue-green algae) by measuring intracellular free Ca2+ levels ([Ca2+]i) in a recombinant strain of the nitrogen fixing cyanobacterium Anabaena strain sp. PCC7120, which constitutively expresses the Ca2+-binding photoprotein apoaequorin. The homeostasis of intracellular Ca2+ in response to increasing external Ca2+ has been studied in this strain. The resting level of free Ca2+ in Anabaena was found to be between 100 and 200 nM. Additions of increasing concentrations of external Ca2+ gave a transient burst of [Ca2+]i followed by a very quick decline, reaching a plateau within seconds that brought the level of [Ca2+]i back to the resting value. These results indicate that Anabaena strain sp. PCC7120 is able to regulate its internal Ca2+ levels. We also monitored Ca2+ transients in our recombinant strain in response to heat and cold shock. The cell's response to both stresses was dependent on the way they were induced. The use of inhibitors suggests that heat shock mobilizes cytosolic Ca2+ from both intracellular and extracellular sources, while the Ca2+ source for cold shock signaling is mostly extracellular.

We investigated the possibility of Ca(2+) signaling in cyanobacteria (blue-green algae) by measuring intracellular free Ca(2+) levels ([Ca(2+)](i)) in a recombinant strain of the nitrogen fixing cyanobacterium Anabaena strain sp. PCC7120, which constitutively expresses the Ca(2+)-binding photoprotein apoaequorin. The homeostasis of intracellular Ca(2+) in response to increasing external Ca(2+) has been studied in this strain. The resting level of free Ca(2+) in Anabaena was found to be between 100 and 200 nM. Additions of increasing concentrations of external Ca(2+) gave a transient burst of [Ca(2+)](i) followed by a very quick decline, reaching a plateau within seconds that brought the level of [Ca(2+)](i) back to the resting value. These results indicate that Anabaena strain sp. PCC7120 is able to regulate its internal Ca(2+) levels. We also monitored Ca(2+) transients in our recombinant strain in response to heat and cold shock. The cell's response to both stresses was dependent on the way they were induced. The use of inhibitors suggests that heat shock mobilizes cytosolic Ca(2+) from both intracellular and extracellular sources, while the Ca(2+) source for cold shock signaling is mostly extracellular.

DNA-protein crosslinks (DPCs) are a specific type of DNA lesions where proteins are covalently attached to DNA. Unrepaired DPCs lead to genomic instability, cancer, neurodegeneration and accelerated ageing. DPC proteolysis was recently discovered as a specialised pathway for DPC repair. The DNA-dependent SPRTN protease and 26S proteasome emerged as two independent proteolytic systems for DPC repair. DPCs are also repaired by homologous recombination (HR), a canonical DNA repair pathway. While studying the role of ubiquitin and SUMO in DPC repair, we identified mutually exclusive signalling mechanisms associated with DPC repair pathway choice. DPC modification by SUMO-1 favours SPRTN proteolysis as the preferred pathway for DPC repair. DPC SUMOylation counteracts DPC ubiquitination, which promotes DNA breaks and the switch to HR. We propose that modification of DPCs by SUMO-1 promotes SPRTN proteolysis, which is essential for DPC removal to prevent DNA replication defects, chromosomal recombination and genomic instability.