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DNA is the source of genetic information, and preserving its integrity is essential in order to sustain life. The genome is continuously threatened by different types of DNA lesions, such as abasic sites, mismatches, interstrand crosslinks, or single-stranded and double-stranded breaks. As a consequence, cells have evolved specialized DNA damage response (DDR) mechanisms to sustain genome integrity. By orchestrating multilayer signaling cascades specific for the type of lesion that occurred, the DDR ensures that genetic information is preserved overtime. In the last decades, DNA repair mechanisms have been thoroughly investigated to untangle these complex networks of pathways and processes. As a result, key factors have been identified that control and coordinate DDR circuits in time and space. In the first part of this review, we describe the critical processes encompassing DNA damage sensing and resolution. In the second part, we illustrate the consequences of partial or complete failure of the DNA repair machinery. Lastly, we will report examples in which this knowledge has been instrumental to develop novel therapies based on genome editing technologies, such as CRISPR-Cas.

Ageing of the immune system, or immunosenescence, contributes to the morbidity and mortality of the elderly 1 , 2 . To define the contribution of immune system ageing to organism ageing, here we selectively deleted Ercc1 , which encodes a crucial DNA repair protein 3 , 4 , in mouse haematopoietic cells to increase the burden of endogenous DNA damage and thereby senescence 5 – 7 in the immune system only. We show that Vav-iCre +/− ;Ercc1 −/fl mice were healthy into adulthood, then displayed premature onset of immunosenescence characterized by attrition and senescence of specific immune cell populations and impaired immune function, similar to changes that occur during ageing in wild-type mice 8 – 10 . Notably, non-lymphoid organs also showed increased senescence and damage, which suggests that senescent, aged immune cells can promote systemic ageing. The transplantation of splenocytes from Vav-iCre +/− ;Ercc1 −/fl or aged wild-type mice into young mice induced senescence in trans , whereas the transplantation of young immune cells attenuated senescence. The treatment of Vav-iCre +/− ;Ercc1 −/fl mice with rapamycin reduced markers of senescence in immune cells and improved immune function 11 , 12 . These data demonstrate that an aged, senescent immune system has a causal role in driving systemic ageing and therefore represents a key therapeutic target to extend healthy ageing. An aged, senescent immune system has a causal role in driving systemic ageing, and the targeting of senescent immune cells with senolytic drugs has the potential to suppress morbidities associated with old age.

Colibactins are hybrid polyketide-nonribosomal peptides produced by Escherichia coli , Klebsiella pneumoniae , and other Enterobacteriaceae harboring the pks genomic island. These genotoxic metabolites are produced by pks -encoded peptide-polyketide synthases as inactive prodrugs called precolibactins, which are then converted to colibactins by deacylation for DNA-damaging effects. Colibactins are bona fide virulence factors and are suspected of promoting colorectal carcinogenesis when produced by intestinal E. coli . Natural active colibactins have not been isolated, and how they induce DNA damage in the eukaryotic host cell is poorly characterized. Here, we show that DNA strands are cross-linked covalently when exposed to enterobacteria producing colibactins. DNA cross-linking is abrogated in a clbP mutant unable to deacetylate precolibactins or by adding the colibactin self-resistance protein ClbS, confirming the involvement of the mature forms of colibactins. A similar DNA-damaging mechanism is observed in cellulo , where interstrand cross-links are detected in the genomic DNA of cultured human cells exposed to colibactin-producing bacteria. The intoxicated cells exhibit replication stress, activation of ataxia-telangiectasia and Rad3-related kinase (ATR), and recruitment of the DNA cross-link repair Fanconi anemia protein D2 (FANCD2) protein. In contrast, inhibition of ATR or knockdown of FANCD2 reduces the survival of cells exposed to colibactin-producing bacteria. These findings demonstrate that DNA interstrand cross-linking is the critical mechanism of colibactin-induced DNA damage in infected cells. IMPORTANCE Colorectal cancer is the third-most-common cause of cancer death. In addition to known risk factors such as high-fat diets and alcohol consumption, genotoxic intestinal Escherichia coli bacteria producing colibactin are proposed to play a role in colon cancer development. Here, by using transient infections with genotoxic E. coli , we showed that colibactins directly generate DNA cross-links in cellulo . Such lesions are converted into double-strand breaks during the repair response. DNA cross-links, akin to those induced by metabolites of alcohol and high-fat diets and by widely used anticancer drugs, are both severely mutagenic and profoundly cytotoxic lesions. This finding of a direct induction of DNA cross-links by a bacterium should facilitate delineating the role of E. coli in colon cancer and engineering new anticancer agents. Colorectal cancer is the third-most-common cause of cancer death. In addition to known risk factors such as high-fat diets and alcohol consumption, genotoxic intestinal Escherichia coli bacteria producing colibactin are proposed to play a role in colon cancer development. Here, by using transient infections with genotoxic E. coli , we showed that colibactins directly generate DNA cross-links in cellulo . Such lesions are converted into double-strand breaks during the repair response. DNA cross-links, akin to those induced by metabolites of alcohol and high-fat diets and by widely used anticancer drugs, are both severely mutagenic and profoundly cytotoxic lesions. This finding of a direct induction of DNA cross-links by a bacterium should facilitate delineating the role of E. coli in colon cancer and engineering new anticancer agents.

Fuchs endothelial corneal dystrophy (FECD) is a leading cause of corneal endothelial (CE) degeneration resulting in impaired visual acuity. It is a genetically complex and age-related disorder, with higher incidence in females. In this study, we established a nongenetic FECD animal model based on the physiologic outcome of CE susceptibility to oxidative stress by demonstrating that corneal exposure to ultraviolet A (UVA) recapitulates the morphological and molecular changes of FECD. Targeted irradiation of mouse corneas with UVA induced reactive oxygen species (ROS) production in the aqueous humor, and caused greater CE cell loss, including loss of ZO-1 junctional contacts and corneal edema, in female than male mice, characteristic of late-onset FECD. UVA irradiation caused greater mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) damage in female mice, indicative of the sex-driven differential response of the CE to UVA, thus accounting for more severe phenotype in females. The sex-dependent effect of UVA was driven by the activation of estrogen-metabolizing enzyme CYP1B1 and formation of reactive estrogen metabolites and estrogen-DNA adducts in female but not male mice. Supplementation of N-acetylcysteine (NAC), a scavenger of reactive oxygen species (ROS), diminished the morphological and molecular changes induced by UVA in vivo. This study investigates the molecular mechanisms of environmental factors in FECD pathogenesis and demonstrates a strong link between UVA-induced estrogen metabolism and increased susceptibility of females for FECD development.

R-loops are three-stranded structures that harbour an RNA–DNA hybrid and frequently form during transcription. R-loop misregulation is associated with DNA damage, transcription elongation defects, hyper-recombination and genome instability. In contrast to such ‘unscheduled’ R-loops, evidence is mounting that cells harness the presence of RNA–DNA hybrids in scheduled, ‘regulatory’ R-loops to promote DNA transactions, including transcription termination and other steps of gene regulation, telomere stability and DNA repair. R-loops formed by cellular RNAs can regulate histone post-translational modification and may be recognized by dedicated reader proteins. The two-faced nature of R-loops implies that their formation, location and timely removal must be tightly regulated. In this Perspective, we discuss the cellular processes that regulatory R-loops modulate, the regulation of R-loops and the potential differences that may exist between regulatory R-loops and unscheduled R-loops.R-loops (three-stranded RNA–DNA structures) are often associated with transcription defects, DNA damage and genome instability, but ‘regulatory’ R-loops can promote gene regulation, telomere stability and DNA repair. This dual functionality of R-loops requires tight control of their formation, location and timely removal.

Reactive oxygen species (ROS) are continuously produced in living cells due to metabolic and biochemical reactions and due to exposure to physical, chemical and biological agents. Excessive ROS cause oxidative stress and lead to oxidative DNA damage. Within ROS-mediated DNA lesions, 8-oxoguanine (8-oxoG) and its nucleotide 8-oxo-2′-deoxyguanosine (8-oxodG)—the guanine and deoxyguanosine oxidation products, respectively, are regarded as the most significant biomarkers for oxidative DNA damage. The quantification of 8-oxoG and 8-oxodG in urine, blood, tissue and saliva is essential, being employed to determine the overall effects of oxidative stress and to assess the risk, diagnose, and evaluate the treatment of autoimmune, inflammatory, neurodegenerative and cardiovascular diseases, diabetes, cancer and other age-related diseases. High-performance liquid chromatography with electrochemical detection (HPLC–ECD) is largely employed for 8-oxoG and 8-oxodG determination in biological samples due to its high selectivity and sensitivity, down to the femtomolar range. This review seeks to provide an exhaustive analysis of the most recent reports on the HPLC–ECD determination of 8-oxoG and 8-oxodG in cellular DNA and body fluids, which is relevant for health research.

Drugs that block the activity of the methyltransferase EZH2 are in clinical development for the treatment of non-Hodgkin lymphomas harboring EZH2 gain-of-function mutations that enhance its polycomb repressive function. We have previously reported that EZH2 can act as a transcriptional activator in castration-resistant prostate cancer (CRPC). Now we show that EZH2 inhibitors can also block the transactivation activity of EZH2 and inhibit the growth of CRPC cells. Gene expression and epigenomics profiling of cells treated with EZH2 inhibitors demonstrated that in addition to derepressing gene expression, these compounds also robustly down-regulate a set of DNA damage repair (DDR) genes, especially those involved in the base excision repair (BER) pathway. Methylation of the pioneer factor FOXA1 by EZH2 contributes to the activation of these genes, and interaction with the transcriptional coactivator P300 via the transactivation domain on EZH2 directly turns on the transcription. In addition, CRISPR-Cas9–mediated knockout screens in the presence of EZH2 inhibitors identified these BER genes as the determinants that underlie the growth-inhibitory effect of EZH2 inhibitors. Interrogation of public data from diverse types of solid tumors expressing wild-type EZH2 demonstrated that expression of DDR genes is significantly correlated with EZH2 dependency and cellular sensitivity to EZH2 inhibitors. Consistent with these findings, treatment of CRPC cells with EZH2 inhibitors dramatically enhances their sensitivity to genotoxic stress. These studies reveal a previously unappreciated mechanism of action of EZH2 inhibitors and provide a mechanistic basis for potential combination cancer therapies.

Plants from the Brassicaceae family are characterized by their high content of glucosinolates (GSLs), whose hydrolysis products, isothiocyanates (ITC) or indole compounds, have been found to have anti-inflammatory, antioxidant and metabolic regulatory functions. In this work, we used a model of transformation using the MCF10A cell line, a non-tumorigenic breast fibrocystic disease cell line, treated with benzo(a)pyrene (B(a)P), a potent carcinogen known to induce the production of reactive oxygen species (ROS) and DNA damage. Broccoli sprout (BSE) or red cabbage aqueous (RCA) extracts were rich in ITC and indole compounds. Their use decreased B(a)P induced cellular proliferation and ROS production. in addition, RCA extract induced autophagy in MCF10A cells. Our results indicate a potential use of BSE or RCA for the prevention of carcinogen-induced transformation and of RCA as a method for autophagy, a tumor suppressor pathway, induction.

Resistance to chemo- and radiotherapy is a common event among cancer patients and a reason why new cancer therapies and therapeutic strategies need to be in continuous investigation and development. DNA damage response (DDR) comprises several pathways that eliminate DNA damage to maintain genomic stability and integrity, but different types of cancers are associated with DDR machinery defects. Many improvements have been made in recent years, providing several drugs and therapeutic strategies for cancer patients, including those targeting the DDR pathways. Currently, poly (ADP-ribose) polymerase inhibitors (PARP inhibitors) are the DDR inhibitors (DDRi) approved for several cancers, including breast, ovarian, pancreatic, and prostate cancer. However, PARPi resistance is a growing issue in clinical settings that increases disease relapse and aggravate patients’ prognosis. Additionally, resistance to other DDRi is also being found and investigated. The resistance mechanisms to DDRi include reversion mutations, epigenetic modification, stabilization of the replication fork, and increased drug efflux. This review highlights the DDR pathways in cancer therapy, its role in the resistance to conventional treatments, and its exploitation for anticancer treatment. Biomarkers of treatment response, combination strategies with other anticancer agents, resistance mechanisms, and liabilities of treatment with DDR inhibitors are also discussed.

Cells possess an armamentarium of DNA repair pathways to counter DNA damage and prevent mutation. Here we use C. elegans whole genome sequencing to systematically quantify the contributions of these factors to mutational signatures. We analyse 2,717 genomes from wild-type and 53 DNA repair defective backgrounds, exposed to 11 genotoxins, including UV-B and ionizing radiation, alkylating compounds, aristolochic acid, aflatoxin B1, and cisplatin. Combined genotoxic exposure and DNA repair deficiency alters mutation rates or signatures in 41% of experiments, revealing how different DNA alterations induced by the same genotoxin are mended by separate repair pathways. Error-prone translesion synthesis causes the majority of genotoxin-induced base substitutions, but averts larger deletions. Nucleotide excision repair prevents up to 99% of point mutations, almost uniformly across the mutation spectrum. Our data show that mutational signatures are joint products of DNA damage and repair and suggest that multiple factors underlie signatures observed in cancer genomes. Recent research has shown that mutational signatures reflective of the history of a cancer can be detected in a cancer genome. Here, using whole genome sequencing of DNA repair deficient and proficient nematodes exposed to genotoxins, the authors show that these mutational signatures reflect both the initial DNA damage that was inflicted and the repair processes that ensue.