UBQLN4 Represses Homologous Recombination and Is Overexpressed in Aggressive Tumors.

Genomic instability can be a hallmark of both human genetic disease and cancer. We identify a deleterious UBQLN4 mutation in families with an autosomal recessive syndrome reminiscent of genome instability disorders. UBQLN4 deficiency leads to increased sensitivity to genotoxic stress and delayed DNA double-strand break (DSB) repair. The proteasomal shuttle factor UBQLN4 is phosphorylated by ATM and interacts with ubiquitylated MRE11 to mediate early steps of homologous recombination-mediated DSB repair (HRR). Loss of UBQLN4 leads to chromatin retention of MRE11, promoting non-physiological HRR activity in vitro and in vivo. Conversely, UBQLN4 overexpression represses HRR and favors non-homologous end joining. Moreover, we find UBQLN4 overexpressed in aggressive tumors. In line with an HRR defect in these tumors, UBQLN4 overexpression is associated with PARP1 inhibitor sensitivity. UBQLN4 therefore curtails HRR activity through removal of MRE11 from damaged chromatin and thus offers a therapeutic window for PARP1 inhibitor treatment in UBQLN4-overexpressing tumors.

PLK1 regulates CtIP and DNA2 interplay in long-range DNA end resection.

DNA double-strand break (DSB) repair is initiated by DNA end resection. CtIP acts in short-range resection to stimulate MRE11-RAD50-NBS1 (MRN) to endonucleolytically cleave 5'-terminated DNA to bypass protein blocks. CtIP also promotes the DNA2 helicase-nuclease to accelerate long-range resection downstream from MRN. Here, using AlphaFold2, we identified CtIP-F728E-Y736E as a separation-of-function mutant that is still proficient in conjunction with MRN but is not able to stimulate ssDNA degradation by DNA2. Accordingly, CtIP-F728E-Y736E impairs physical interaction with DNA2. Cellular assays revealed that CtIP-F728E-Y736E cells exhibit reduced DSB-dependent chromatin-bound RPA, impaired long-range resection, and increased sensitivity to DSB-inducing drugs. Previously, CtIP was shown to be targeted by PLK1 to inhibit long-range resection, yet the underlying mechanism was unclear. We show that the DNA2-interacting region in CtIP includes the PLK1 target site at S723. The integrity of S723 in CtIP is necessary for the stimulation of DNA2, and phosphorylation of CtIP by PLK1 in vitro is consequently inhibitory, explaining why PLK1 restricts long-range resection. Our data support a model in which CDK-dependent phosphorylation of CtIP activates resection by MRN in S phase, and PLK1-mediated phosphorylation of CtIP disrupts CtIP stimulation of DNA2 to attenuate long-range resection later at G2/M.

Mechanism of BRCA1-BARD1 function in DNA end resection and DNA protection.

DNA double-strand break (DSB) repair by homologous recombination is initiated by DNA end resection, a process involving the controlled degradation of the 5'-terminated strands at DSB sites1,2. The breast cancer suppressor BRCA1-BARD1 not only promotes resection and homologous recombination, but it also protects DNA upon replication stress1,3-9. BRCA1-BARD1 counteracts the anti-resection and pro-non-homologous end-joining factor 53BP1, but whether it functions in resection directly has been unclear10-16. Using purified recombinant proteins, we show here that BRCA1-BARD1 directly promotes long-range DNA end resection pathways catalysed by the EXO1 or DNA2 nucleases. In the DNA2-dependent pathway, BRCA1-BARD1 stimulates DNA unwinding by the Werner or Bloom helicase. Together with MRE11-RAD50-NBS1 and phosphorylated CtIP, BRCA1-BARD1 forms the BRCA1-C complex17,18, which stimulates resection synergistically to an even greater extent. A mutation in phosphorylated CtIP (S327A), which disrupts its binding to the BRCT repeats of BRCA1 and hence the integrity of the BRCA1-C complex19-21, inhibits resection, showing that BRCA1-C is a functionally integrated ensemble. Whereas BRCA1-BARD1 stimulates resection in DSB repair, it paradoxically also protects replication forks from unscheduled degradation upon stress, which involves a homologous recombination-independent function of the recombinase RAD51 (refs. 4-6,8). We show that in the presence of RAD51, BRCA1-BARD1 instead inhibits DNA degradation. On the basis of our data, the presence and local concentration of RAD51 might determine the balance between the pronuclease and the DNA protection functions of BRCA1-BARD1 in various physiological contexts.

The Ubiquitin E3/E4 Ligase UBE4A Adjusts Protein Ubiquitylation and Accumulation at Sites of DNA Damage, Facilitating Double-Strand Break Repair.

Double-strand breaks (DSBs) are critical DNA lesions that robustly activate the elaborate DNA damage response (DDR) network. We identified a critical player in DDR fine-tuning: the E3/E4 ubiquitin ligase UBE4A. UBE4A's recruitment to sites of DNA damage is dependent on primary E3 ligases in the DDR and promotes enhancement and sustainment of K48- and K63-linked ubiquitin chains at these sites. This step is required for timely recruitment of the RAP80 and BRCA1 proteins and proper organization of RAP80- and BRCA1-associated protein complexes at DSB sites. This pathway is essential for optimal end resection at DSBs, and its abrogation leads to upregulation of the highly mutagenic alternative end-joining repair at the expense of error-free homologous recombination repair. Our data uncover a critical regulatory level in the DSB response and underscore the importance of fine-tuning the complex DDR network for accurate and balanced execution of DSB repair.

EXO1 and DNA2-mediated ssDNA gap expansion is essential for ATR activation and to maintain viability in BRCA1-deficient cells.

DNA replication faces challenges from DNA lesions originated from endogenous or exogenous sources of stress, leading to the accumulation of single-stranded DNA (ssDNA) that triggers the activation of the ATR checkpoint response. To complete genome replication in the presence of damaged DNA, cells employ DNA damage tolerance mechanisms that operate not only at stalled replication forks but also at ssDNA gaps originated by repriming of DNA synthesis downstream of lesions. Here, we demonstrate that human cells accumulate post-replicative ssDNA gaps following replicative stress induction. These gaps, initiated by PrimPol repriming and expanded by the long-range resection factors EXO1 and DNA2, constitute the principal origin of the ssDNA signal responsible for ATR activation upon replication stress, in contrast to stalled forks. Strikingly, the loss of EXO1 or DNA2 results in synthetic lethality when combined with BRCA1 deficiency, but not BRCA2. This phenomenon aligns with the observation that BRCA1 alone contributes to the expansion of ssDNA gaps. Remarkably, BRCA1-deficient cells become addicted to the overexpression of EXO1, DNA2 or BLM. This dependence on long-range resection unveils a new vulnerability of BRCA1-mutant tumors, shedding light on potential therapeutic targets for these cancers.

Centriolar subdistal appendages promote double-strand break repair through homologous recombination.

The centrosome is a cytoplasmic organelle with roles in microtubule organization that has also been proposed to act as a hub for cellular signaling. Some centrosomal components are required for full activation of the DNA damage response. However, whether the centrosome regulates specific DNA repair pathways is not known. Here, we show that centrosome presence is required to fully activate recombination, specifically to completely license its initial step, the so-called DNA end resection. Furthermore, we identify a centriolar structure, the subdistal appendages, and a specific factor, CEP170, as the critical centrosomal component involved in the regulation of recombination and resection. Cells lacking centrosomes or depleted for CEP170 are, consequently, hypersensitive to DNA damaging agents. Moreover, low levels of CEP170 in multiple cancer types correlate with an increase of the mutation burden associated with specific mutational signatures and a better prognosis, suggesting that changes in CEP170 can act as a mutation driver but could also be targeted to improve current oncological treatments.

ALC1/eIF4A1-mediated regulation of CtIP mRNA stability controls DNA end resection.

During repair of DNA double-strand breaks, resection of DNA ends influences how these lesions will be repaired. If resection is activated, the break will be channeled through homologous recombination; if not, it will be simply ligated using the non-homologous end-joining machinery. Regulation of resection relies greatly on modulating CtIP, which can be done by modifying: i) its interaction partners, ii) its post-translational modifications, or iii) its cellular levels, by regulating transcription, splicing and/or protein stability/degradation. Here, we have analyzed the role of ALC1, a chromatin remodeler previously described as an integral part of the DNA damage response, in resection. Strikingly, we found that ALC1 affects resection independently of chromatin remodeling activity or its ability to bind damaged chromatin. In fact, it cooperates with the RNA-helicase eIF4A1 to help stabilize the most abundant splicing form of CtIP mRNA. This function relies on the presence of a specific RNA sequence in the 5' UTR of CtIP. Therefore, we describe an additional layer of regulation of CtIP-at the level of mRNA stability through ALC1 and eIF4A1.

CtIP -mediated alternative mRNA splicing finetunes the DNA damage response.

In order to survive to the exposure of DNA damaging agents, cells activate a complex response that coordinates the cellular metabolism, cell cycle progression and DNA repair. Among many other events, recent evidence has described global changes in mRNA splicing in cells treated with genotoxic agents. Here, we explore further this DNA damage-dependent alternative splicing. Indeed, we show that both the splicing factor SF3B2 and the repair protein CtIP contribute to the global pattern of splicing both in cells treated or not to DNA damaging agents. Additionally, we focus on a specific DNA damage- and CtIP-dependent alternative splicing event of the helicase PIF1 and explore its relevance for the survival of cells upon exposure to ionizing radiation. Indeed, we described how the nuclear, active form of PIF1 is substituted by a splicing variant, named vPIF1, in a fashion that requires both the presence of DNA damage and CtIP. Interestingly, timely expression of vPIF1 is required for optimal survival to exposure to DNA damaging agents, but early expression of this isoform delays early events of the DNA damage response. On the contrary, expression of the full length PIF1 facilitates those early events, but increases the sensitivity to DNA damaging agents if the expression is maintained long-term.

PHF2 regulates homology-directed DNA repair by controlling the resection of DNA double strand breaks.

Post-translational histone modifications and chromatin remodelling play a critical role controlling the integrity of the genome. Here, we identify histone lysine demethylase PHF2 as a novel regulator of the DNA damage response by regulating DNA damage-induced focus formation of 53BP1 and BRCA1, critical factors in the pathway choice for DNA double strand break repair. PHF2 knockdown leads to impaired BRCA1 focus formation and delays the resolution of 53BP1 foci. Moreover, irradiation-induced RPA phosphorylation and focus formation, as well as localization of CtIP, required for DNA end resection, to sites of DNA lesions are affected by depletion of PHF2. These results are indicative of a defective resection of double strand breaks and thereby an impaired homologous recombination upon PHF2 depletion. In accordance with these data, Rad51 focus formation and homology-directed double strand break repair is inhibited in cells depleted for PHF2. Importantly, we demonstrate that PHF2 knockdown decreases CtIP and BRCA1 protein and mRNA levels, an effect that is dependent on the demethylase activity of PHF2. Furthermore, PHF2-depleted cells display genome instability and are mildly sensitive to the inhibition of PARP. Together these results demonstrate that PHF2 promotes DNA repair by homologous recombination by controlling CtIP-dependent resection of double strand breaks.

Isolation of Nontuberculous Mycobacteria from Bovine Raw Lungs Bought in Butchers' Shops.

Nontuberculous Mycobacteria (NTM) can cause opportunistic disease in animals and humans, causing mycobacteriosis. In this study, bovine lungs were collected from butchers' shops and slaughterhouses after food official's inspection from the metropolitan area of Buenos Aires. All samples were cultured and then identified by molecular methods. Twelve isolates of NTM were identified being the most prevalent Mycolicibacterium insubricum. This demonstrates that viable Mycobacteria can pass food inspection and contaminate surfaces and food, making manipulation of raw organs and feeding of animals with raw lungs a potential source of infection for pets and owners.

Nuclear poly(A)-binding protein 1 is an ATM target and essential for DNA double-strand break repair.

The DNA damage response (DDR) is an extensive signaling network that is robustly mobilized by DNA double-strand breaks (DSBs). The primary transducer of the DSB response is the protein kinase, ataxia-telangiectasia, mutated (ATM). Here, we establish nuclear poly(A)-binding protein 1 (PABPN1) as a novel target of ATM and a crucial player in the DSB response. PABPN1 usually functions in regulation of RNA processing and stability. We establish that PABPN1 is recruited to the DDR as a critical regulator of DSB repair. A portion of PABPN1 relocalizes to DSB sites and is phosphorylated on Ser95 in an ATM-dependent manner. PABPN1 depletion sensitizes cells to DSB-inducing agents and prolongs the DSB-induced G2/M cell-cycle arrest, and DSB repair is hampered by PABPN1 depletion or elimination of its phosphorylation site. PABPN1 is required for optimal DSB repair via both nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR), and specifically is essential for efficient DNA-end resection, an initial, key step in HRR. Using mass spectrometry analysis, we capture DNA damage-induced interactions of phospho-PABPN1, including well-established DDR players as well as other RNA metabolizing proteins. Our results uncover a novel ATM-dependent axis in the rapidly growing interface between RNA metabolism and the DDR.

Zoonosis and veterinary waste disposal in rural practice.

The objectives of this study were to estimate: (a) the frequency of zoonoses in large animal veterinarians from rural areas of the province of Buenos Aires, Argentina, and (b) to describe the use and disposal of personal protective equipment (PPE) and selective veterinary clinical waste. A cross-sectional study was carried out on large animal veterinary practitioners in the Province of Buenos Aires (n=106). One third (29.2%) of them had been diagnosed with a zoonosis by laboratory-methods, being brucellosis the most frequent (22.6%). The more years passed since their graduation, the greater the chances of becoming ill (p<0.001). Gloves were the most adopted PPE; however, other elements had little or no use at all. Older and experienced professionals used PPE less frequently than young inexperienced practitioners. Some PPE was frequently reused and the final disposal of veterinary waste was often inappropriate. A change in behavior is an urgent need to preserve not only the veterinarians' health but also their families' wellbeing and to ensure proper disposal of potentially hazardous waste.

Detection of Viable Mycobacterium bovis in Lungs and Livers Sold in Butchers' Shops in Buenos Aires, Argentina.

Although Mycobacterium bovis is the major etiological agent of tuberculosis in bovines, it can infect other mammalians. Previously reported cases of tuberculosis caused by M. bovis in cats from the Autonomous City of Buenos Aires (CABA) led to the conclusion that the main source of infection for these felines was the ingestion of raw bovine lungs. Thus, for this study, we collected samples of bovine viscera from butchers' shops of the Greater Buenos Aires (GBA) and the CABA to assess presence and viability of these mycobacteria in bovine lungs (including the lymph nodes) and livers. We analyzed 216 different samples and obtained 5 isolates of M. bovis (4 from lungs and 1 from liver) by culture analysis. We also confirmed the presence of different isolates by polymerase chain reaction, spoligotyping, and MIRU-VNTR assays. The results obtained in this work emphasizes the need of social education for food hygiene, and to change the habit of feeding pets with raw viscera, which carries the risk of epizootic and zoonotic transmission. Moreover, control and eradication programs of bovine tuberculosis should be strengthened and improved.

Neddylation inhibits CtIP-mediated resection and regulates DNA double strand break repair pathway choice.

DNA double strand breaks are the most cytotoxic lesions that can occur on the DNA. They can be repaired by different mechanisms and optimal survival requires a tight control between them. Here we uncover protein deneddylation as a major controller of repair pathway choice. Neddylation inhibition changes the normal repair profile toward an increase on homologous recombination. Indeed, RNF111/UBE2M-mediated neddylation acts as an inhibitor of BRCA1 and CtIP-mediated DNA end resection, a key process in repair pathway choice. By controlling the length of ssDNA produced during DNA resection, protein neddylation not only affects the choice between NHEJ and homologous recombination but also controls the balance between different recombination subpathways. Thus, protein neddylation status has a great impact in the way cells respond to DNA breaks.

The COP9 signalosome is vital for timely repair of DNA double-strand breaks.

The DNA damage response is vigorously activated by DNA double-strand breaks (DSBs). The chief mobilizer of the DSB response is the ATM protein kinase. We discovered that the COP9 signalosome (CSN) is a crucial player in the DSB response and an ATM target. CSN is a protein complex that regulates the activity of cullin ring ubiquitin ligase (CRL) complexes by removing the ubiquitin-like protein, NEDD8, from their cullin scaffold. We find that the CSN is physically recruited to DSB sites in a neddylation-dependent manner, and is required for timely repair of DSBs, affecting the balance between the two major DSB repair pathways-nonhomologous end-joining and homologous recombination repair (HRR). The CSN is essential for the processivity of deep end-resection-the initial step in HRR. Cullin 4a (CUL4A) is recruited to DSB sites in a CSN- and neddylation-dependent manner, suggesting that CSN partners with CRL4 in this pathway. Furthermore, we found that ATM-mediated phosphorylation of CSN subunit 3 on S410 is critical for proper DSB repair, and that loss of this phosphorylation site alone is sufficient to cause a DDR deficiency phenotype in the mouse. This novel branch of the DSB response thus significantly affects genome stability.

Competing roles of DNA end resection and non-homologous end joining functions in the repair of replication-born double-strand breaks by sister-chromatid recombination.

While regulating the choice between homologous recombination and non-homologous end joining (NHEJ) as mechanisms of double-strand break (DSB) repair is exerted at several steps, the key step is DNA end resection, which in Saccharomyces cerevisiae is controlled by the MRX complex and the Sgs1 DNA helicase or the Sae2 and Exo1 nucleases. To assay the role of DNA resection in sister-chromatid recombination (SCR) as the major repair mechanism of spontaneous DSBs, we used a circular minichromosome system for the repair of replication-born DSBs by SCR in yeast. We provide evidence that MRX, particularly its Mre11 nuclease activity, and Sae2 are required for SCR-mediated repair of DSBs. The phenotype of nuclease-deficient MRX mutants is suppressed by ablation of Yku70 or overexpression of Exo1, suggesting a competition between NHEJ and resection factors for DNA ends arising during replication. In addition, we observe partially redundant roles for Sgs1 and Exo1 in SCR, with a more prominent role for Sgs1. Using human U2OS cells, we also show that the competitive nature of these reactions is likely evolutionarily conserved. These results further our understanding of the role of DNA resection in repair of replication-born DSBs revealing unanticipated differences between these events and repair of enzymatically induced DSBs.

CDK targets Sae2 to control DNA-end resection and homologous recombination.

DNA double-strand breaks (DSBs) are repaired by two principal mechanisms: non-homologous end-joining (NHEJ) and homologous recombination (HR). HR is the most accurate DSB repair mechanism but is generally restricted to the S and G2 phases of the cell cycle, when DNA has been replicated and a sister chromatid is available as a repair template. By contrast, NHEJ operates throughout the cell cycle but assumes most importance in G1 (refs 4, 6). The choice between repair pathways is governed by cyclin-dependent protein kinases (CDKs), with a major site of control being at the level of DSB resection, an event that is necessary for HR but not NHEJ, and which takes place most effectively in S and G2 (refs 2, 5). Here we establish that cell-cycle control of DSB resection in Saccharomyces cerevisiae results from the phosphorylation by CDK of an evolutionarily conserved motif in the Sae2 protein. We show that mutating Ser 267 of Sae2 to a non-phosphorylatable residue causes phenotypes comparable to those of a sae2Delta null mutant, including hypersensitivity to camptothecin, defective sporulation, reduced hairpin-induced recombination, severely impaired DNA-end processing and faulty assembly and disassembly of HR factors. Furthermore, a Sae2 mutation that mimics constitutive Ser 267 phosphorylation complements these phenotypes and overcomes the necessity of CDK activity for DSB resection. The Sae2 mutations also cause cell-cycle-stage specific hypersensitivity to DNA damage and affect the balance between HR and NHEJ. These findings therefore provide a mechanistic basis for cell-cycle control of DSB repair and highlight the importance of regulating DSB resection.

CtIP Mutations Cause Seckel and Jawad Syndromes.

Seckel syndrome is a recessively inherited dwarfism disorder characterized by microcephaly and a unique head profile. Genetically, it constitutes a heterogeneous condition, with several loci mapped (SCKL1-5) but only three disease genes identified: the ATR, CENPJ, and CEP152 genes that control cellular responses to DNA damage. We previously mapped a Seckel syndrome locus to chromosome 18p11.31-q11.2 (SCKL2). Here, we report two mutations in the CtIP (RBBP8) gene within this locus that result in expression of C-terminally truncated forms of CtIP. We propose that these mutations are the molecular cause of the disease observed in the previously described SCKL2 family and in an additional unrelated family diagnosed with a similar form of congenital microcephaly termed Jawad syndrome. While an exonic frameshift mutation was found in the Jawad family, the SCKL2 family carries a splicing mutation that yields a dominant-negative form of CtIP. Further characterization of cell lines derived from the SCKL2 family revealed defective DNA damage induced formation of single-stranded DNA, a critical co-factor for ATR activation. Accordingly, SCKL2 cells present a lowered apoptopic threshold and hypersensitivity to DNA damage. Notably, over-expression of a comparable truncated CtIP variant in non-Seckel cells recapitulates SCKL2 cellular phenotypes in a dose-dependent manner. This work thus identifies CtIP as a disease gene for Seckel and Jawad syndromes and defines a new type of genetic disease mechanism in which a dominant negative mutation yields a recessively inherited disorder.

BRCA1/BARD1 ubiquitinates PCNA in unperturbed conditions to promote continuous DNA synthesis.

Deficiencies in the BRCA1 tumor suppressor gene are the main cause of hereditary breast and ovarian cancer. BRCA1 is involved in the Homologous Recombination DNA repair pathway and, together with BARD1, forms a heterodimer with ubiquitin E3 activity. The relevance of the BRCA1/BARD1 ubiquitin E3 activity for tumor suppression and DNA repair remains controversial. Here, we observe that the BRCA1/BARD1 ubiquitin E3 activity is not required for Homologous Recombination or resistance to Olaparib. Using TULIP2 methodology, which enables the direct identification of E3-specific ubiquitination substrates, we identify substrates for BRCA1/BARD1. We find that PCNA is ubiquitinated by BRCA1/BARD1 in unperturbed conditions independently of RAD18. PCNA ubiquitination by BRCA1/BARD1 avoids the formation of ssDNA gaps during DNA replication and promotes continuous DNA synthesis. These results provide additional insight about the importance of BRCA1/BARD1 E3 activity in Homologous Recombination.

Standardization through education of molecular pathology: a spotlight on the European Masters in Molecular Pathology.

Despite advancements in precision medicine, many cancer patients globally, particularly those in resource-constrained environments, face significant challenges in accessing high-quality molecular testing and targeted therapies. The considerable heterogeneity in molecular testing highlights the urgent need to harmonize practices across Europe and beyond, establishing a more standardized and consistent approach in MP laboratories. Professionals, especially molecular pathologists, must move beyond traditional education to cope with this heterogeneity. This perspective addresses critical issues in molecular pathology (MP), such as limited access to high-quality molecular testing, leading to disparities in cancer treatment, and the consequences of inconsistent practices. Recognizing the necessity for a standardized framework for education to address these issues, educational programs play a pivotal role in updating professionals' skills to achieve standardization in MP. European experts from the Steering Committee, the Pathology Section of the European Union of Medical Specialists, and the European Society of Pathology have proposed creating a comprehensive Master's degree program called the "European Masters in Molecular Pathology" (EMMP). This program emerges as a strategic response to the demand for a specialized and standardized framework for education in MP, catering to professionals who concurrently work and study. The program's design aligns with evidence-based education methods, ensuring effective learning and engagement while integrating computational pathology to analyze complex molecular data, enhance diagnostic accuracy, and improve treatment outcomes. EMMP's structured curriculum, strategic partnerships, and regular updates underscore its significance in standardizing MP practices. Exploring future developments, this perspective delves into technology integration and interdisciplinary collaboration, anticipating ongoing advances and harmonization. Challenges and future directions in MP education are discussed, emphasizing the necessity for dynamic curriculum updates, seamless technology integration, and interdisciplinary cooperation. This perspective underscores EMMP's pivotal role in preparing pathologists for this dynamic field, advocating continuous advancements in education and training to uphold excellence in MP practices and maintain the highest patient care standards.