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.

A new role for Rrm3 in repair of replication-born DNA breakage by sister chromatid recombination.

Replication forks stall at different DNA obstacles such as those originated by transcription. Fork stalling can lead to DNA double-strand breaks (DSBs) that will be preferentially repaired by homologous recombination when the sister chromatid is available. The Rrm3 helicase is a replisome component that promotes replication upon fork stalling, accumulates at highly transcribed regions and prevents not only transcription-induced replication fork stalling but also transcription-associated hyper-recombination. This led us to explore the possible role of Rrm3 in the repair of DSBs when originating at the passage of the replication fork. Using a mini-HO system that induces mainly single-stranded DNA breaks, we show that rrm3Δ cells are defective in DSB repair. The defect is clearly seen in sister chromatid recombination, the major repair pathway of replication-born DSBs. Our results indicate that Rrm3 recruitment to replication-born DSBs is crucial for viability, uncovering a new role for Rrm3 in the repair of broken replication forks.

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.

Histone H3K56 acetylation, Rad52, and non-DNA repair factors control double-strand break repair choice with the sister chromatid.

DNA double-strand breaks (DSBs) are harmful lesions that arise mainly during replication. The choice of the sister chromatid as the preferential repair template is critical for genome integrity, but the mechanisms that guarantee this choice are unknown. Here we identify new genes with a specific role in assuring the sister chromatid as the preferred repair template. Physical analyses of sister chromatid recombination (SCR) in 28 selected mutants that increase Rad52 foci and inter-homolog recombination uncovered 8 new genes required for SCR. These include the SUMO/Ub-SUMO protease Wss1, the stress-response proteins Bud27 and Pdr10, the ADA histone acetyl-transferase complex proteins Ahc1 and Ada2, as well as the Hst3 and Hst4 histone deacetylase and the Rtt109 histone acetyl-transferase genes, whose target is histone H3 Lysine 56 (H3K56). Importantly, we use mutations in H3K56 residue to A, R, and Q to reveal that H3K56 acetylation/deacetylation is critical to promote SCR as the major repair mechanism for replication-born DSBs. The same phenotype is observed for a particular class of rad52 alleles, represented by rad52-C180A, with a DSB repair defect but a spontaneous hyper-recombination phenotype. We propose that specific Rad52 residues, as well as the histone H3 acetylation/deacetylation state of chromatin and other specific factors, play an important role in identifying the sister as the choice template for the repair of replication-born DSBs. Our work demonstrates the existence of specific functions to guarantee SCR as the main repair event for replication-born DSBs that can occur by two pathways, one Rad51-dependent and the other Pol32-dependent. A dysfunction can lead to genome instability as manifested by high levels of homolog recombination and DSB accumulation.

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.

N7-methylguanosine methylation of tRNAs regulates survival to stress in cancer.

Tumour progression and therapy tolerance are highly regulated and complex processes largely dependent on the plasticity of cancer cells and their capacity to respond to stress. The higher plasticity of cancer cells highlights the need for identifying targetable molecular pathways that challenge cancer cell survival. Here, we show that N7-guanosine methylation (m7G) of tRNAs, mediated by METTL1, regulates survival to stress conditions in cancer cells. Mechanistically, we find that m7G in tRNAs protects them from stress-induced cleavage and processing into 5' tRNA fragments. Our analyses reveal that the loss of tRNA m7G methylation activates stress response pathways, sensitising cancer cells to stress. Furthermore, we find that the loss of METTL1 reduces tumour growth and increases cytotoxic stress in vivo. Our study uncovers the role of m7G methylation of tRNAs in stress responses and highlights the potential of targeting METTL1 to sensitise cancer cells to chemotherapy.

The interface between transcription and mRNP export: from THO to THSC/TREX-2.

Eukaryotic gene expression is a multilayer process covering transcription to post-translational protein modifications. As the nascent pre-mRNA emerges from the RNA polymerase II (RNAPII), it is packed in a messenger ribonucleoparticle (mRNP) whose optimal configuration is critical for the normal pre-mRNA processing and mRNA export, mRNA integrity as well as for transcription elongation efficiency. The interplay between transcription and mRNP formation feeds forward and backward and involves a number of conserved factors, from THO to THSC/TREX-2, which in addition have a unique impact on transcription-dependent genome instability. Here we review our actual knowledge of the role that these factors play at the interface between transcription and mRNA export in the model organism Saccharomyces cerevisiae.

The Effect of Atypical Nucleic Acids Structures in DNA Double Strand Break Repair: A Tale of R-loops and G-Quadruplexes.

The fine tuning of the DNA double strand break repair pathway choice relies on different regulatory layers that respond to environmental and local cues. Among them, the presence of non-canonical nucleic acids structures seems to create challenges for the repair of nearby DNA double strand breaks. In this review, we focus on the recently published effects of G-quadruplexes and R-loops on DNA end resection and homologous recombination. Finally, we hypothesized a connection between those two atypical DNA structures in inhibiting the DNA end resection step of HR.

The Emerging Role of RNA Modifications in DNA Double-Strand Break Repair.

The correct repair of DNA double-strand breaks is essential for maintaining the stability of the genome, thus ensuring the survival and fitness of any living organism. Indeed, the repair of these lesions is a complicated affair, in which several pathways compete for the DNA ends in a complex balance. Thus, the fine-tuning of the DNA double-strand break repair pathway choice relies on the different regulatory layers that respond to environmental cues. Among those different tiers of regulation, RNA modifications have just emerged as a promising field.

ADAR-mediated RNA editing of DNA:RNA hybrids is required for DNA double strand break repair.

The maintenance of genomic stability requires the coordination of multiple cellular tasks upon the appearance of DNA lesions. RNA editing, the post-transcriptional sequence alteration of RNA, has a profound effect on cell homeostasis, but its implication in the response to DNA damage was not previously explored. Here we show that, in response to DNA breaks, an overall change of the Adenosine-to-Inosine RNA editing is observed, a phenomenon we call the RNA Editing DAmage Response (REDAR). REDAR relies on the checkpoint kinase ATR and the recombination factor CtIP. Moreover, depletion of the RNA editing enzyme ADAR2 renders cells hypersensitive to genotoxic agents, increases genomic instability and hampers homologous recombination by impairing DNA resection. Such a role of ADAR2 in DNA repair goes beyond the recoding of specific transcripts, but depends on ADAR2 editing DNA:RNA hybrids to ease their dissolution.

Preparation of a radiobiology beam line at the 18 MeV proton cyclotron facility at CNA.

Proton therapy has gained interest in recent years due to its excellent clinical outcomes. However, the lack of accurate biological data, especially in the Bragg peak region of clinical beams, makes it difficult to implement biophysically optimized treatment plans in clinical practice. In this context, low energy proton accelerator facilities provide the perfect environment to collect good radiobiological data, as they can produce high LET beams with narrow energy distributions. This study presents the radiobiology beam line that has been designed at the 18 MeV proton cyclotron facility at the National Centre of Accelerators (CNA, Seville, Spain), to perform irradiations of mono-layer cell cultures. To ensure that all the cells receive the same dose with a suitable dose rate, low beam intensities and broad and homogeneous beam profiles are necessary. To do so, at the CNA an unfocused beam has been used, broadened with a 500 µm thick aluminium scattering foil. Homogeneous dose profiles, with deviations lower than 10% have been obtained over a circular surface of 35 mm diameter for an incident average energy of 12.8 MeV. Further, a Monte Carlo simulation of the beam line has been developed with Geant4, and benchmarked towards experimental measurements, with differences generally below 1%. Once validated, the code has been used, together with an ionization chamber, for dosimetry studies, to characterize the beam and monitor the dose. Finally, cultures of Human Bone Osteosarcoma cells (U2OS) have been successfully irradiated at the radiobiology beam line, investigating the effects of radiation in terms of DNA damage induction.

Tho1, a novel hnRNP, and Sub2 provide alternative pathways for mRNP biogenesis in yeast THO mutants.

THO is a protein complex that functions in cotranscriptional mRNP formation. Yeast THO1 and SUB2 (Saccharomyces cerevisiae) were identified as multicopy suppressors of the expression defects of the hpr1Delta mutant of THO. Here we show that multicopy THO1 suppresses the mRNA accumulation and export defects and the hyperrecombination phenotype of THO mutants but not those of sub2Delta, thp1Delta, or spt4Delta. Similarly, Sub2 overexpression suppresses the RNA export defect of hpr1Delta. Tho1 is a conserved RNA binding nuclear protein that specifically binds to transcribed chromatin in a THO- and RNA-dependent manner and genetically interacts with the shuttling hnRNP Nab2. The ability of Tho1 to suppress hpr1Delta resides in its C-terminal half, which contains the RNA binding activity and is located after a SAP/SAF (scaffold-associated protein/scaffold-associated factor) domain. Altogether, these results suggest that Tho1 is an hnRNP that, similarly to Sub2, assembles onto the nascent mRNA during transcription and participates in mRNP biogenesis and export. Overexpression of Tho1 or Sub2 may provide alternative ways for mRNP formation and export in the absence of a functional THO complex.

The role of RNA and RNA-related proteins in the regulation of DNA double strand break repair pathway choice.

DNA end resection is a critical step in the repair of DNA double strand breaks. It controls the way the lesion is going to be repaired, thus its regulation has a great importance in maintaining genomic stability. In this review, we focus in recent discoveries in the field that point to a modulation of resection by RNA molecules and RNA-related proteins. Moreover, we aim to reconcile contradictory reports on the positive or negative effect of DNA:RNA hybrids in the resection process.

Controlling the balance between chromosome break repair pathways.

Broken chromosomes are among the most complex and more difficult to repair DNA lesions. The loss of the continuity of the DNA molecule presents a challenge to the cells, thus the repair of DNA double strand breaks might lead to genomic alterations. Indeed, to minimize this threat to genomic integrity, different DNA repair pathways can act on a broken chromosome. The balance between them is tightly controlled, and it heavily depends on global and local cellular cues. In this chapter, we review our current understanding on the repair of DNA double strand breaks and focus in the regulation of the balance between alternative pathways. Most of this modulation takes place at the level of DNA end resection. Here, we focus mostly on the local signals that control the repair pathway choice, as the global cues have been extensively reviewed recently. We described epigenetic marks that either facilitate or inhibit DNA resection and homologous recombination, from histone marks and chromatin remodelers to non-coding RNA and RNA-related factors.

EXOSC10 is required for RPA assembly and controlled DNA end resection at DNA double-strand breaks.

The exosome is a ribonucleolytic complex that plays important roles in RNA metabolism. Here we show that the exosome is necessary for the repair of DNA double-strand breaks (DSBs) in human cells and that RNA clearance is an essential step in homologous recombination. Transcription of DSB-flanking sequences results in the production of damage-induced long non-coding RNAs (dilncRNAs) that engage in DNA-RNA hybrid formation. Depletion of EXOSC10, an exosome catalytic subunit, leads to increased dilncRNA and DNA-RNA hybrid levels. Moreover, the targeting of the ssDNA-binding protein RPA to sites of DNA damage is impaired whereas DNA end resection is hyper-stimulated in EXOSC10-depleted cells. The DNA end resection deregulation is abolished by transcription inhibitors, and RNase H1 overexpression restores the RPA recruitment defect caused by EXOSC10 depletion, which suggests that RNA clearance of newly synthesized dilncRNAs is required for RPA recruitment, controlled DNA end resection and assembly of the homologous recombination machinery.

Development and Validation of a New High-Performance Liquid Chromatography Method for the Simultaneous Quantification of Coenzyme Q10, Phosphatidylserine, and Vitamin C from a Cutting-Edge Liposomal Vehiculization.

A high-performance liquid chromatography (HPLC) method was developed to simultaneously quantify three widely used active substances such as coenzyme Q10, phosphatidylserine, and vitamin C. This new method optimizes current timing and costs in the analyses of these three active substances. Additionally, since the analyzed compounds were encapsulated on a cutting-edge liposomal formulation, further processing was necessary to be developed prior to HPLC analyses. The technique was studied and adequately validated in accordance with the guidelines of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) regarding selectivity, linearity, accuracy, precision, and robustness. After data treatment of results, linear regressions for all active substances showed an optimal linearity with a correlation coefficient of >0.999 in the concentration range between 70 to 130% of the liposomal formulation and less than a 3% relative standard deviation (RSD) in accuracy and precision.

Multiple roles of the splicing complex SF3B in DNA end resection and homologous recombination.

The appropriate repair of DNA double strand breaks is critical for genome maintenance. Thus, several cellular pathways collaborate to orchestrate a coordinated response. These include the repair of the breaks, which could be achieved by different mechanisms. A key protein involved in the regulation of the repair of broken chromosomes is CtIP. Here, we have found new partners of CtIP involved in the regulation of DNA break repair through affecting DNA end resection. We focus on the splicing complex SF3B and show that its depletion impairs DNA end resection and hampers homologous recombination. Functionally, SF3B controls CtIP function at, as least, two levels: by affecting CtIP mRNA levels and controlling CtIP recruitment to DNA breaks, in a way that requires ATM-mediated phosphorylation of SF3B2 at serine 289. Indeed, overexpression of CtIP rescues the resection defect caused by SF3B downregulation. Strikingly, other SF3B depletion phenotypes, such as impaired homologous recombination or cellular sensitivity to DNA damaging agents, are independent of CtIP levels, suggesting a more general role of SF3B in controlling the response to chromosome breaks.

The Helicase PIF1 Facilitates Resection over Sequences Prone to Forming G4 Structures.

DNA breaks are complex lesions that can be repaired either by non-homologous end joining (NHEJ) or by homologous recombination (HR). The decision between these two routes of DNA repair is a key point of the DNA damage response (DDR) that is controlled by DNA resection. The core machinery catalyzing the resection process is well established. However, little is known about the additional requirements of DNA resection over DNA structures with high complexity. Here, we found evidence that the human helicase PIF1 has a role in DNA resection, specifically for defined DNA regions, such as those prone to form G-quadruplexes. Indeed, PIF1 is recruited to the site of DNA damage and physically interacts with proteins involved in DNA resection, and its depletion causes DNA damage sensitivity and a reduction of HR efficiency. Moreover, G4 stabilization by itself hampers DNA resection, a phenomenon suppressed by PIF1 overexpression.

Phosphatidylserine-Liposomes Promote Tolerogenic Features on Dendritic Cells in Human Type 1 Diabetes by Apoptotic Mimicry.

Type 1 diabetes (T1D) is a metabolic disease caused by the autoimmune destruction of insulin-producing ß-cells. With its incidence increasing worldwide, to find a safe approach to permanently cease autoimmunity and allow ß-cell recovery has become vital. Relying on the inherent ability of apoptotic cells to induce immunological tolerance, we demonstrated that liposomes mimicking apoptotic ß-cells arrested autoimmunity to ß-cells and prevented experimental T1D through tolerogenic dendritic cell (DC) generation. These liposomes contained phosphatidylserine (PS)-the main signal of the apoptotic cell membrane-and ß-cell autoantigens. To move toward a clinical application, PS-liposomes with optimum size and composition for phagocytosis were loaded with human insulin peptides and tested on DCs from patients with T1D and control age-related subjects. PS accelerated phagocytosis of liposomes with a dynamic typical of apoptotic cell clearance, preserving DCs viability. After PS-liposomes phagocytosis, the expression pattern of molecules involved in efferocytosis, antigen presentation, immunoregulation, and activation in DCs concurred with a tolerogenic functionality, both in patients and control subjects. Furthermore, DCs exposed to PS-liposomes displayed decreased ability to stimulate autologous T cell proliferation. Moreover, transcriptional changes in DCs from patients with T1D after PS-liposomes phagocytosis pointed to an immunoregulatory prolife. Bioinformatics analysis showed 233 differentially expressed genes. Genes involved in antigen presentation were downregulated, whereas genes pertaining to tolerogenic/anti-inflammatory pathways were mostly upregulated. In conclusion, PS-liposomes phagocytosis mimics efferocytosis and leads to phenotypic and functional changes in human DCs, which are accountable for tolerance induction. The herein reported results reinforce the potential of this novel immunotherapy to re-establish immunological tolerance, opening the door to new therapeutic approaches in the field of autoimmunity.

Histone H2A phosphorylation and H3 methylation are required for a novel Rad9 DSB repair function following checkpoint activation.

In budding yeast, the Rad9 protein is an important player in the maintenance of genomic integrity and has a well-characterised role in DNA damage checkpoint activation. Recently, roles for different post-translational histone modifications in the DNA damage response, including H2A serine 129 phosphorylation and H3 lysine 79 methylation, have also been demonstrated. Here, we show that Rad9 recruitment to foci and bulk chromatin occurs specifically after ionising radiation treatment in G2 cells. This stable recruitment correlates with late stages of double strand break (DSB) repair and, surprisingly, it is the hypophosphorylated form of Rad9 that is retained on chromatin rather than the hyperphosphorylated, checkpoint-associated, form. Stable Rad9 accumulation in foci requires the Mec1 kinase and two independently regulated histone modifications, H2A phosphorylation and Dot1-dependent H3 methylation. In addition, Rad9 is selectively recruited to a subset of Rad52 repair foci. These results, together with the observation that rad9Delta cells are defective in repair of IR breaks in G2, strongly indicate a novel post checkpoint activation role for Rad9 in promoting efficient repair of DNA DSBs by homologous recombination.