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Ageing is a complex, multifaceted process leading to widespread functional decline that affects every organ and tissue, but it remains unknown whether ageing has a unifying causal mechanism or is grounded in multiple sources. Phenotypically, the ageing process is associated with a wide variety of features at the molecular, cellular and physiological level—for example, genomic and epigenomic alterations, loss of proteostasis, declining overall cellular and subcellular function and deregulation of signalling systems. However, the relative importance, mechanistic interrelationships and hierarchical order of these features of ageing have not been clarified. Here we synthesize accumulating evidence that DNA damage affects most, if not all, aspects of the ageing phenotype, making it a potentially unifying cause of ageing. Targeting DNA damage and its mechanistic links with the ageing phenotype will provide a logical rationale for developing unified interventions to counteract age-related dysfunction and disease. This Review examines the evidence showing that DNA damage is associated with ageing phenotypes, suggesting that it may have a central role as the cause of ageing.

Expanded CGG-repeats have been linked to neurodevelopmental and neurodegenerative disorders, including the fragile X syndrome and fragile X-associated tremor/ataxia syndrome (FXTAS). We hypothesized that as of yet uncharacterised CGG-repeat expansions within the genome contribute to human disease. To catalogue the CGG-repeats, 544 human whole genomes were analyzed. In total, 6101 unique CGG-repeats were detected of which more than 93% were highly variable in repeat length. Repeats with a median size of 12 repeat units or more were always polymorphic but shorter repeats were often polymorphic, suggesting a potential intergenerational instability of the CGG region even for repeats units with a median length of four or less. 410 of the CGG repeats were associated with known neurodevelopmental disease genes or with strong candidate genes. Based on their frequency and genomic location, CGG repeats may thus be a currently overlooked cause of human disease.

Key Points Exploration for biomarkers for drugs that block immune checkpoints should be rationally conducted based on knowledge of the mechanism of action of the targeted pathway. The programmed cell death protein 1 (PD1) and cytotoxic T lymphocyte associated antigen 4 (CTLA4) pathways are unique, and there are special considerations based on mechanisms of action for developing biomarkers for drugs blocking each of these pathways. Biomarkers for immune checkpoint-blocking drugs currently fall into three major categories: immunological, genetic and virological. Future work may reveal additional markers related to metabolism and the microbiome. Immunological biomarkers offer the advantage of applicability across multiple tumour types amenable to immune checkpoint blockade. In the case of anti-PD1 drugs, tumour PD1 ligand 1 (PDL1) expression is a pretreatment biomarker that predicts a greater likelihood of response to therapy. Despite technical pitfalls that make clinical application challenging, two PDL1 immunohistochemistry tests are currently approved by the US Food and Drug Administration for guiding treatment decisions in patients with non-small-cell lung cancer and melanoma. Although no specific oncogene or driver mutation has yet been correlated with clinical response to immune checkpoint blockade, overall tumour mutational burden reflecting neoantigenic diversity may have predictive value. This is exemplified by the high anti-PD1 response rate in DNA mismatch repair deficient colorectal cancers (which have a large mutational burden and which account for ∼15% of all colon cancers), whereas mismatch repair proficient colon cancers are unlikely to respond. Virus-associated cancers, which account for more than 20% of cancers worldwide, express viral neoantigens that are strongly immunogenic. Early evidence demonstrates expression of PD1–PDL1 in these cancers, and suggests responsiveness to anti-PD1 therapies. Combination treatment regimens based on immune checkpoint-blocking drugs are emerging as the next step in clinical development to improve efficacy and response durability. Biomarker considerations for these regimens are complex and are likely to involve multifactorial assessments. This Review assesses the mechanism-based biomarkers in use and in development for immune checkpoint inhibition, identifying cancer types and cancer phenotypes that are most likely to respond to immune checkpoint blockade, and considerations for future biomarkers of immune checkpoint response. With recent approvals for multiple therapeutic antibodies that block cytotoxic T lymphocyte associated antigen 4 (CTLA4) and programmed cell death protein 1 (PD1) in melanoma, non-small-cell lung cancer and kidney cancer, and additional immune checkpoints being targeted clinically, many questions still remain regarding the optimal use of drugs that block these checkpoint pathways. Defining biomarkers that predict therapeutic effects and adverse events is a crucial mandate, highlighted by recent approvals for two PDL1 diagnostic tests. Here, we discuss biomarkers for anti-PD1 therapy based on immunological, genetic and virological criteria. The unique biology of the CTLA4 immune checkpoint, compared with PD1, requires a different approach to biomarker development. Mechanism-based insights from such studies may guide the design of synergistic treatment combinations based on immune checkpoint blockade.

DNA G-quadruplexes (G4s) are guanine-rich sequences that fold into four-stranded structures. Recent progress in the detection and mapping of genomic G4 structures has provided new insights into their functions in regulating transcription and genome stability, and has revealed their potential relevance for cancer therapy. Single-stranded guanine-rich DNA sequences can fold into four-stranded DNA structures called G-quadruplexes (G4s) that arise from the self-stacking of two or more guanine quartets. There has been considerable recent progress in the detection and mapping of G4 structures in the human genome and in biologically relevant contexts. These advancements, many of which align with predictions made previously in computational studies, provide important new insights into the functions of G4 structures in, for example, the regulation of transcription and genome stability, and uncover their potential relevance for cancer therapy.

The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic/apyrimidinic sites), alkylated and oxidized nucleobases, DNA-DNA crosslinks, UV-induced cyclobutane pyrimidine dimers and some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Various applications of the comet assay have been validated by research groups in academia, industry and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility.

A key mutational process in cancer is structural variation, in which rearrangements delete, amplify or reorder genomic segments that range in size from kilobases to whole chromosomes 1 – 7 . Here we develop methods to group, classify and describe somatic structural variants, using data from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA), which aggregated whole-genome sequencing data from 2,658 cancers across 38 tumour types 8 . Sixteen signatures of structural variation emerged. Deletions have a multimodal size distribution, assort unevenly across tumour types and patients, are enriched in late-replicating regions and correlate with inversions. Tandem duplications also have a multimodal size distribution, but are enriched in early-replicating regions—as are unbalanced translocations. Replication-based mechanisms of rearrangement generate varied chromosomal structures with low-level copy-number gains and frequent inverted rearrangements. One prominent structure consists of 2–7 templates copied from distinct regions of the genome strung together within one locus. Such cycles of templated insertions correlate with tandem duplications, and—in liver cancer—frequently activate the telomerase gene TERT . A wide variety of rearrangement processes are active in cancer, which generate complex configurations of the genome upon which selection can act. Whole-genome sequencing data from more than 2,500 cancers of 38 tumour types reveal 16 signatures that can be used to classify somatic structural variants, highlighting the diversity of genomic rearrangements in cancer.

In animals, PIWI-interacting RNAs (piRNAs) of 21–35 nucleotides in length silence transposable elements, regulate gene expression and fight viral infection. piRNAs guide PIWI proteins to cleave target RNA, promote heterochromatin assembly and methylate DNA. The architecture of the piRNA pathway allows it both to provide adaptive, sequence-based immunity to rapidly evolving viruses and transposons and to regulate conserved host genes. piRNAs silence transposons in the germ line of most animals, whereas somatic piRNA functions have been lost, gained and lost again across evolution. Moreover, most piRNA pathway proteins are deeply conserved, but different animals employ remarkably divergent strategies to produce piRNA precursor transcripts. Here, we discuss how a common piRNA pathway allows animals to recognize diverse targets, ranging from selfish genetic elements to genes essential for gametogenesis.

Topoisomerases introduce transient DNA breaks to relax supercoiled DNA, thereby mediating chromatin dynamics and stability, transcription, replication and DNA damage repair. Topoisomerases are targets of various anticancer drugs, and their deregulation can cause, in addition to cancer, neurodegenerative diseases and immune disorders. Topoisomerases introduce transient DNA breaks to relax supercoiled DNA, remove catenanes and enable chromosome segregation. Human cells encode six topoisomerases (TOP1, TOP1mt, TOP2α, TOP2β, TOP3α and TOP3β), which act on a broad range of DNA and RNA substrates at the nuclear and mitochondrial genomes. Their catalytic intermediates, the topoisomerase cleavage complexes (TOPcc), are therapeutic targets of various anticancer drugs. TOPcc can also form on damaged DNA during replication and transcription, and engage specific repair pathways, such as those mediated by tyrosyl-DNA phosphodiesterase 1 (TDP1) and TDP2 and by endonucleases (MRE11, XPF–ERCC1 and MUS81). Here, we review the roles of topoisomerases in mediating chromatin dynamics, transcription, replication, DNA damage repair and genomic stability, and discuss how deregulation of topoisomerases can cause neurodegenerative diseases, immune disorders and cancer.

Key Points Mammalian non-homologous DNA end joining (NHEJ) is the primary pathway for the repair of DNA double-strand breaks (DSBs) throughout the cell cycle, including during S and G2 phases. NHEJ relies on the Ku protein to thread onto each broken DNA end. Ku recruits the enzymes and complexes that are needed to trim (nucleases) or to fill in (polymerases) the ends to make them optimally ligatable by the DNA ligase IV complex. The configuration of the DNA ends determines which of several subpathways of NHEJ is able to join the ends. Because NHEJ is flexible and iterative, any of these subpathways can be used but some pathways are more efficient than others for certain DNA ends. When NHEJ is absent owing to a lack of Ku or the DNA ligase complex, alternative end joining (a-EJ) can join the ends using microhomology (usually >4 bp) and there is often some evidence of templated insertions of substantial length (>10 nucleotides). DNA polymerase θ (Pol θ) is of key importance for a-EJ. The single-strand annealing (SSA) pathway requires further end resection by exonuclease 1 (EXO1), Bloom syndrome RecQ-like helicase (BLM) or DNA replication helicase/nuclease 2 (DNA2) to generate the long 3′ single-strand DNA (ssDNA) tails (>20 nucleotides) that are bound by replication protein A (RPA) to prevent the formation of DNA secondary structures. The 3′ ssDNA tails are annealed by RAD52. In mammalian cells, DNA double-strand breaks (DSBs) are repaired predominantly by the non-homologous end joining (NHEJ) pathway, which includes subpathways that can repair different DNA-end configurations. Furthermore, the repair of some DNA-end configurations can be shunted to the auxiliary pathways of alternative end joining (a-EJ) or single-strand annealing (SSA). DNA double-strand breaks (DSBs) are the most dangerous type of DNA damage because they can result in the loss of large chromosomal regions. In all mammalian cells, DSBs that occur throughout the cell cycle are repaired predominantly by the non-homologous DNA end joining (NHEJ) pathway. Defects in NHEJ result in sensitivity to ionizing radiation and the ablation of lymphocytes. The NHEJ pathway utilizes proteins that recognize, resect, polymerize and ligate the DNA ends in a flexible manner. This flexibility permits NHEJ to function on a wide range of DNA-end configurations, with the resulting repaired DNA junctions often containing mutations. In this Review, we discuss the most recent findings regarding the relative involvement of the different NHEJ proteins in the repair of various DNA-end configurations. We also discuss the shunting of DNA-end repair to the auxiliary pathways of alternative end joining (a-EJ) or single-strand annealing (SSA) and the relevance of these different pathways to human disease.

Repetitive elements in the human genome, once considered ‘junk DNA’, are now known to adopt more than a dozen alternative (that is, non-B) DNA structures, such as self-annealed hairpins, left-handed Z-DNA, three-stranded triplexes (H-DNA) or four-stranded guanine quadruplex structures (G4 DNA). These dynamic conformations can act as functional genomic elements involved in DNA replication and transcription, chromatin organization and genome stability. In addition, recent studies have revealed a role for these alternative structures in triggering error-generating DNA repair processes, thereby actively enabling genome plasticity. As a driving force for genetic variation, non-B DNA structures thus contribute to both disease aetiology and evolution.Non-B DNA secondary structures, such as G quadruplexes, H-DNA or Z-DNA, have key roles in genetic instability and disease aetiology. The authors review the impact of non-B DNA on transcription, replication, recombination and DNA damage and repair, the mechanisms of non-B DNA-induced mutagenesis and the role of non-B DNA sequences in human disease.