USP7 and VCP define the SUMO/Ubiquitin landscape at the DNA replication fork

The AAA+ ATPase VCP regulates the extraction of SUMO and ubiquitin-modified DNA replication factors from chromatin. We have previously described that active DNA synthesis is associated with a SUMO-high/ubiquitin-low environment governed by the deubiquitylase USP7. Here, we unveil a functional cooperation between USP7 and VCP in DNA replication, which is conserved from Caenorhabditis elegans to mammals. The role of VCP in chromatin is defined by its cofactor FAF1, which facilitates the extraction of SUMOylated and ubiquitylated proteins that accumulate after the block of DNA replication in the absence of USP7. The inactivation of USP7 and FAF1 is synthetically lethal both in C. elegans and mammalian cells. In addition, USP7 and VCP inhibitors display synergistic toxicity supporting a functional link between deubiquitylation and extraction of chromatin-bound proteins. Our results suggest that USP7 and VCPFAF1 facilitate DNA replication by controlling the balance of SUMO/Ubiquitin-modified DNA replication factors on chromatinMINECO (BFU2014-55168-JIN; RTI2018-093485-B-I00) and a Ramo´ n y Cajal Fellowship from MINECO (RYC-2016-20705), co-funded by European Regional Development Funds (FEDER) to E.L.; by grants from the Spanish Ministry of Science, Innovation and Universities (RTI2018-102204-B-I00, co-financed with European FEDER funds) and the European Research Council (ERC-617840) to O.F.-C.; fellowships from Fundacion Ramón Areces-UAM and La Caixa Foundation to P.V. (LCF/BQ/ES18/11670008

Interrogating DNA replication stress and DNA damage responses via phenotypic and functional CRISPR/Cas9 screens

DNA double-strand breaks are believed to be one of the most cytotoxic DNA lesions, posing a constant threat for genome integrity and cell survival. Consequently, various DNA repair processes and pathways have evolved to deal with different DNA lesions and ensure genome stability. The tumour suppressor ATM is one of the apical kinases in the DNA damage response (DDR), occupying a key position in the DNA damage response network to regulate signalling responses and repair processes upon DNA damage. Disruption of the ATM gene sensitises cells to DNA damage-inducing chemotherapeutics. To identify genetic suppressors of this hypersensitivity, we performed a genome-wide CRISPR/Cas9 screen in ATM-deficient cells treated with the topoisomerase I poison topotecan. This dissertation describes two distinct resistance mechanisms in Atm-/- cells towards TOP1 inhibitors, resulting from the inactivation of the non-homologous end-joining (NHEJ) machinery or alternatively the absence of the BRCA1-A complex. Accordingly, I establish that the catalytic activity of DNA ligase 4 (LIG4) is crucial for topotecan toxicity in Atm-/- cells and mutating the catalytic site of Lig4 reinstates cellular survival of ATM-deficient cells in the presence of topotecan. In contrast, the absence of the BRCA1-A complex increases DNA end-resection kinetics, a pivotal initial step during homologous recombination-mediated repair of DNA double strand breaks. Moreover, I show that the accurate repair of topotecan-induced DNA damage occurs via homologous recombination, and in this context ATM prevents toxic end- joining of broken replication forks. Inactivation of NHEJ or increased end-resection in the absence of BRCA1-A supresses toxic end-joining, thus enabling cellular survival in the presence of topotecan. Additionally I describe that the end-resection defect of BRCA1- deficient cells can be counteracted by the inactivation of genes encoding the SHIELDIN complex, which causes resistance of BRCA1-mutant cells to PARP inhibitors. Lastly, I conceptualised and performed functional genome-wide CRISPR/Cas9 screens, utilising the chromatinisation of RPA as a read-out for DNA repair processes, to identify novel factors involved in the DNA damage response. Based on these screens, I provide evidence for the deubiquitinating enzyme USP37 regulating RPA loading and DNA repair during DNA replication

Interfaces between cellular responses to DNA damage and cancer immunotherapy.

The DNA damage response (DDR) fulfils essential roles to preserve genome integrity. Targeting the DDR in tumors has had remarkable success over the last decade, exemplified by the licensing of PARP inhibitors for cancer therapy. Recent studies suggest that the application of DDR inhibitors impacts on cellular innate and adaptive immune responses, wherein key DNA repair factors have roles in limiting chronic inflammatory signaling. Antitumor immunity plays an emerging part in cancer therapy, and extensive efforts have led to the development of immune checkpoint inhibitors overcoming immune suppressive signals in tumors. Here, we review the current understanding of the molecular mechanisms underlying DNA damage-triggered immune responses, including cytosolic DNA sensing via the cGAS/STING pathway. We highlight the implications of DDR components for therapeutic outcomes of immune checkpoint inhibitors or their use as biomarkers. Finally, we discuss the rationale for novel combinations of DDR inhibitors with antagonists of immune checkpoints and current hindrances limiting their broader therapeutic applications

Chromatin-associated degradation is defined by UBXN-3/FAF1 to safeguard DNA replication fork progression

The coordinated activity of DNA replication factors is a highly dynamic process that involves ubiquitin-dependent regulation. In this context, the ubiquitin-directed ATPase CDC-48/p97 recently emerged as a key regulator of chromatin-associated degradation in several of the DNA metabolic pathways that assure genome integrity. However, the spatiotemporal control of distinct CDC-48/p97 substrates in the chromatin environment remained unclear. Here, we report that progression of the DNA replication fork is coordinated by UBXN-3/FAF1. UBXN-3/FAF1 binds to the licensing factor CDT-1 and additional ubiquitylated proteins, thus promoting CDC-48/p97-dependent turnover and disassembly of DNA replication factor complexes. Consequently, inactivation of UBXN-3/FAF1 stabilizes CDT-1 and CDC-45/GINS on chromatin, causing severe defects in replication fork dynamics accompanied by pronounced replication stress and eventually resulting in genome instability. Our work identifies a critical substrate selection module of CDC-48/p97 required for chromatinassociated protein degradation in both Caenorhabditis elegans and humans, which is relevant to oncogenesis and aging

USP7 and VCPFAF1 define the SUMO/Ubiquitin landscape at the DNA replication fork

The AAA(+) ATPase VCP regulates the extraction of SUMO and ubiquitin-modified DNA replication factors from chromatin. We have previously described that active DNA synthesis is associated with a SUMO-high/ubiquitin-low environment governed by the deubiquitylase USP7. Here, we unveil a functional cooperation between USP7 and VCP in DNA replication, which is conserved from Caenorhabditis elegans to mammals. The role of VCP in chromatin is defined by its cofactor FAF1, which facilitates the extraction of SUMOylated and ubiquitylated proteins that accumulate after the block of DNA replication in the absence of USP7. The inactivation of USP7 and FAF1 is synthetically lethal both in C. elegans and mammalian cells. In addition, USP7 and VCP inhibitors display synergistic toxicity supporting a functional link between deubiquitylation and extraction of chromatin-bound proteins. Our results suggest that USP7 and VCPFAF1 facilitate DNA replication by controlling the balance of SUMO/Ubiquitin-modified DNA replication factors on chromatin

ATM orchestrates the DNA-damage response to counter toxic non-homologous end-joining at broken replication forks.

Mutations in the ATM tumor suppressor gene confer hypersensitivity to DNA-damaging chemotherapeutic agents. To explore genetic resistance mechanisms, we performed genome-wide CRISPR-Cas9 screens in cells treated with the DNA topoisomerase I inhibitor topotecan. Thus, we here establish that inactivating terminal components of the non-homologous end-joining (NHEJ) machinery or of the BRCA1-A complex specifically confer topotecan resistance to ATM-deficient cells. We show that hypersensitivity of ATM-mutant cells to topotecan or the poly-(ADP-ribose) polymerase (PARP) inhibitor olaparib reflects delayed engagement of homologous recombination at DNA-replication-fork associated single-ended double-strand breaks (DSBs), allowing some to be subject to toxic NHEJ. Preventing DSB ligation by NHEJ, or enhancing homologous recombination by BRCA1-A complex disruption, suppresses this toxicity, highlighting a crucial role for ATM in preventing toxic LIG4-mediated chromosome fusions. Notably, suppressor mutations in ATM-mutant backgrounds are different to those in BRCA1-mutant scenarios, suggesting new opportunities for patient stratification and additional therapeutic vulnerabilities for clinical exploitation

Shieldin complex promotes DNA end-joining and counters homologous recombination in BRCA1-null cells.

BRCA1 deficiencies cause breast, ovarian, prostate and other cancers, and render tumours hypersensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. To understand the resistance mechanisms, we conducted whole-genome CRISPR-Cas9 synthetic-viability/resistance screens in BRCA1-deficient breast cancer cells treated with PARP inhibitors. We identified two previously uncharacterized proteins, C20orf196 and FAM35A, whose inactivation confers strong PARP-inhibitor resistance. Mechanistically, we show that C20orf196 and FAM35A form a complex, 'Shieldin' (SHLD1/2), with FAM35A interacting with single-stranded DNA through its C-terminal oligonucleotide/oligosaccharide-binding fold region. We establish that Shieldin acts as the downstream effector of 53BP1/RIF1/MAD2L2 to promote DNA double-strand break (DSB) end-joining by restricting DSB resection and to counteract homologous recombination by antagonizing BRCA2/RAD51 loading in BRCA1-deficient cells. Notably, Shieldin inactivation further sensitizes BRCA1-deficient cells to cisplatin, suggesting how defining the SHLD1/2 status of BRCA1-deficient tumours might aid patient stratification and yield new treatment opportunities. Highlighting this potential, we document reduced SHLD1/2 expression in human breast cancers displaying intrinsic or acquired PARP-inhibitor resistance