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Caloric restriction without malnutrition extends life span in a range of organisms including insects and mammals and lowers free radical production by the mitochondria. However, the mechanism responsible for this adaptation are poorly understood. The current study was undertaken to examine muscle mitochondrial bioenergetics in response to caloric restriction alone or in combination with exercise in 36 young (36.8 +/- 1.0 y), overweight (body mass index, 27.8 +/- 0.7 kg/m(2)) individuals randomized into one of three groups for a 6-mo intervention: Control, 100% of energy requirements; CR, 25% caloric restriction; and CREX, caloric restriction with exercise (CREX), 12.5% CR + 12.5% increased energy expenditure (EE). In the controls, 24-h EE was unchanged, but in CR and CREX it was significantly reduced from baseline even after adjustment for the loss of metabolic mass (CR, -135 +/- 42 kcal/d, p = 0.002 and CREX, -117 +/- 52 kcal/d, p = 0.008). Participants in the CR and CREX groups had increased expression of genes encoding proteins involved in mitochondrial function such as PPARGC1A, TFAM, eNOS, SIRT1, and PARL (all, p < 0.05). In parallel, mitochondrial DNA content increased by 35% +/- 5% in the CR group (p = 0.005) and 21% +/- 4% in the CREX group (p < 0.004), with no change in the control group (2% +/- 2%). However, the activity of key mitochondrial enzymes of the TCA (tricarboxylic acid) cycle (citrate synthase), beta-oxidation (beta-hydroxyacyl-CoA dehydrogenase), and electron transport chain (cytochrome C oxidase II) was unchanged. DNA damage was reduced from baseline in the CR (-0.56 +/- 0.11 arbitrary units, p = 0.003) and CREX (-0.45 +/- 0.12 arbitrary units, p = 0.011), but not in the controls. In primary cultures of human myotubes, a nitric oxide donor (mimicking eNOS signaling) induced mitochondrial biogenesis but failed to induce SIRT1 protein expression, suggesting that additional factors may regulate SIRT1 content during CR. The observed increase in muscle mitochondrial DNA in association with a decrease in whole body oxygen consumption and DNA damage suggests that caloric restriction improves mitochondrial function in young non-obese adults.

Boron nitride nanotubes (BNNTs) represent an extremely interesting class of nanomaterials, and recent findings have suggested a number of applications in the biomedical field. Anyhow, extensive biocompatibility investigations are mandatory before any further advancement toward preclinical testing. Here, we report on the effects of multiwalled BNNTs in freshwater planarians, one of the best-characterized in vivo models for developmental biology and regeneration research. Obtained results indicate that BNNTs are biocompatible in the investigated model, since they do not induce oxidative DNA damage and apoptosis, and do not show adverse effects on planarian stem cell biology and on de novo tissue regeneration. In summary, collected findings represent another important step toward BNNT realistic applications in nanomedicine.

Regular physical exercise has been shown to be one of the most important lifestyle influences on improving functional performance, decreasing morbidity and all causes of mortality among older people. However, it is known that acute physical exercise may induce an increase in oxidative stress and oxidative damage in several structures, including DNA. Considering this, the purpose of this study was to identify the effects of 16 weeks of combined physical exercise in DNA damage and repair capacity in lymphocytes. In addition, we aimed to investigate the role of oxidative stress involved in those changes. Fifty-seven healthy men (40 to 74 years) were enrolled in this study. The sample was divided into two groups: the experimental group (EG), composed of 31 individuals, submitted to 16 weeks of combined physical exercise training; and the control group (CG), composed of 26 individuals, who did not undergo any specifically orientated physical activity. We observed an improvement of overall physical performance in the EG, after the physical exercise training. A significant decrease in DNA strand breaks and FPG-sensitive sites was found after the physical exercise training, with no significant changes in 8-oxoguanine DNA glycosylase enzyme activity. An increase was observed in antioxidant activity, and a decrease was found in lipid peroxidation levels after physical exercise training. These results suggest that physical exercise training induces protective effects against DNA damage in lymphocytes possibly related to the increase in antioxidant capacity.

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.

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.

Key Points The constant deleterious modification of DNA by reactive molecules, endogenously or exogenously generated, is offset by protective processes that are initiated by the DNA damage response. The interplay of the diverse signalling cascades (DNA damage response) that originate from the interference of DNA lesions with replication and the transcriptome leads to the activation of DNA repair, autophagy, senescence, apoptosis and necroptosis. Aspects of how post-translational modifications of the tumour suppressor p53 determine the switch between these end points are discussed. The crosstalk between autophagy, senescence, apoptosis and regulated necrosis is also discussed, focusing on the importance of thresholds for deciding cell fate. Throughout this Review, emphasis is placed on how DNA damage and DNA repair fit within the complex cellular context. Understanding how DNA damage determines cell fate — DNA repair and cell survival or death — is important for gaining insight into carcinogenesis and in promoting successful cancer therapy. This Review describes key decision-making nodes in the complex interplay between DNA damage responses and cell fate signalling. DNA is vulnerable to damage resulting from endogenous metabolites, environmental and dietary carcinogens, some anti-inflammatory drugs, and genotoxic cancer therapeutics. Cells respond to DNA damage by activating complex signalling networks that decide cell fate, promoting not only DNA repair and survival but also cell death. The decision between cell survival and death following DNA damage rests on factors that are involved in DNA damage recognition, and DNA repair and damage tolerance, as well as on factors involved in the activation of apoptosis, necrosis, autophagy and senescence. The pathways that dictate cell fate are entwined and have key roles in cancer initiation and progression. Furthermore, they determine the outcome of cancer therapy with genotoxic drugs. Understanding the molecular basis of these pathways is important not only for gaining insight into carcinogenesis, but also in promoting successful cancer therapy. In this Review, we describe key decision-making nodes in the complex interplay between cell survival and death following DNA damage.

The DNA damage response (DDR) preserves genomic integrity. Small non-coding RNAs termed DDRNAs are generated at DNA double-strand breaks (DSBs) and are critical for DDR activation. Here we show that active DDRNAs specifically localize to their damaged homologous genomic sites in a transcription-dependent manner. Following DNA damage, RNA polymerase II (RNAPII) binds to the MRE11–RAD50–NBS1 complex, is recruited to DSBs and synthesizes damage-induced long non-coding RNAs (dilncRNAs) from and towards DNA ends. DilncRNAs act both as DDRNA precursors and by recruiting DDRNAs through RNA–RNA pairing. Together, dilncRNAs and DDRNAs fuel DDR focus formation and associate with 53BP1. Accordingly, inhibition of RNAPII prevents DDRNA recruitment, DDR activation and DNA repair. Antisense oligonucleotides matching dilncRNAs and DDRNAs impair site-specific DDR focus formation and DNA repair. We propose that DDR signalling sites, in addition to sharing a common pool of proteins, individually host a unique set of site-specific RNAs necessary for DDR activation. Michelini et al.  show that RNA polymerase II is recruited to double-strand breaks to induce long non-coding RNAs and the generation of small DNA damage response RNAs that promote recruitment of DNA repair factors and repair.

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.

Upon genotoxic stress, PCNA ubiquitination allows for replication of damaged DNA by recruiting lesion-bypass DNA polymerases. However, PCNA is also ubiquitinated during normal S-phase progression. By employing 293T and RPE1 cells deficient in PCNA ubiquitination, generated through CRISPR/Cas9 gene editing, here, we show that this modification promotes cellular proliferation and suppression of genomic instability under normal growth conditions. Loss of PCNA-ubiquitination results in DNA2-dependent but MRE11-independent nucleolytic degradation of nascent DNA at stalled replication forks. This degradation is linked to defective gap-filling in the wake of the replication fork and incomplete Okazaki fragment maturation, which interferes with efficient PCNA unloading by ATAD5 and subsequent nucleosome deposition by CAF-1. Moreover, concomitant loss of PCNA-ubiquitination and the BRCA pathway results in increased nascent DNA degradation and PARP inhibitor sensitivity. In conclusion, we show that by ensuring efficient Okazaki fragment maturation, PCNA-ubiquitination protects fork integrity and promotes the resistance of BRCA-deficient cells to PARP-inhibitors. PCNA is essential for DNA replication and cellular proliferation. Here, the authors reveal that PCNA ubiquitination protects stalled replication forks from DNA2-mediated degradation via regulation of Okazaki fragment maturation and chromatin assembly.

DNA damage and disease Cellular DNA is a sitting target for many toxic agents — from ionizing radiation to any number of chemicals in the environment. To that can be added errors that arise from physiological processes. Unchecked, damaged DNA can cause disease and threaten the gene pool. The human body has evolved several systems to detect DNA damage and mediate its repair. Stephen Jackson and Jiri Bartek review recent work on how DNA lesions are dealt with at the molecular level, and show how an understanding of DNA-damage responses is providing new avenues for disease management. The prime objective for every life form is to deliver its genetic material, intact and unchanged, to the next generation. This must be achieved despite constant assaults by endogenous and environmental agents on the DNA. To counter this threat, life has evolved several systems to detect DNA damage, signal its presence and mediate its repair. Such responses, which have an impact on a wide range of cellular events, are biologically significant because they prevent diverse human diseases. Our improving understanding of DNA-damage responses is providing new avenues for disease management.