SUMO and the DNA damage response.

The preservation of genome integrity requires specialised DNA damage repair (DDR) signalling pathways to respond to each type of DNA damage. A key feature of DDR is the integration of numerous post-translational modification signals with DNA repair factors. These modifications influence DDR factor recruitment to damaged DNA, activity, protein-protein interactions, and ultimately eviction to enable access for subsequent repair factors or termination of DDR signalling. SUMO1-3 (small ubiquitin-like modifier 1-3) conjugation has gained much recent attention. The SUMO-modified proteome is enriched with DNA repair factors. Here we provide a snapshot of our current understanding of how SUMO signalling impacts the major DNA repair pathways in mammalian cells. We highlight repeating themes of SUMO signalling used throughout DNA repair pathways including the assembly of protein complexes, competition with ubiquitin to promote DDR factor stability and ubiquitin-dependent degradation or extraction of SUMOylated DDR factors. As SUMO 'addiction' in cancer cells is protective to genomic integrity, targeting components of the SUMO machinery to potentiate DNA damaging therapy or exacerbate existing DNA repair defects is a promising area of study.

How to fix DNA breaks: new insights into the mechanism of non-homologous end joining.

Non-homologous end joining (NHEJ) is the major pathway for the repair of ionizing radiation-induced DNA double-strand breaks (DSBs) in human cells and is essential for the generation of mature T and B cells in the adaptive immune system via the process of V(D)J recombination. Here, we review how recently determined structures shed light on how NHEJ complexes function at DNA DSBs, emphasizing how multiple structures containing the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) may function in NHEJ. Together, these studies provide an explanation for how NHEJ proteins assemble to detect and protect DSB ends, then proceed, through DNA-PKcs-dependent autophosphorylation, to a ligation-competent complex.

Genetic modifiers of repeat expansion disorders.

Repeat expansion disorders (REDs) are monogenic diseases caused by a sequence of repetitive DNA expanding above a pathogenic threshold. A common feature of the REDs is a strong genotype-phenotype correlation in which a major determinant of age at onset (AAO) and disease progression is the length of the inherited repeat tract. Over a disease-gene carrier's life, the length of the repeat can expand in somatic cells, through the process of somatic expansion which is hypothesised to drive disease progression. Despite being monogenic, individual REDs are phenotypically variable, and exploring what genetic modifying factors drive this phenotypic variability has illuminated key pathogenic mechanisms that are common to this group of diseases. Disease phenotypes are affected by the cognate gene in which the expansion is found, the location of the repeat sequence in coding or non-coding regions and by the presence of repeat sequence interruptions. Human genetic data, mouse models and in vitro models have implicated the disease-modifying effect of DNA repair pathways via the mechanisms of somatic mutation of the repeat tract. As such, developing an understanding of these pathways in the context of expanded repeats could lead to future disease-modifying therapies for REDs.

Incorporation of inosine into DNA by human polymerase eta (Polη): kinetics of nucleotide misincorporation and structural basis for the mutagenicity.

Inosine, a purine nucleoside containing the hypoxanthine (HX) nucleobase, can form in DNA via hydrolytic deamination of adenine. Due to its structural similarity to guanine and the geometry of Watson-Crick base pairs, inosine can mispair with cytosine upon catalysis by DNA polymerases, leading to AT → GC mutations. Additionally, inosine plays an essential role in purine nucleotide biosynthesis, and inosine triphosphate is present in living cells. In a recent publication, Averill and Jung examined the possibility of polη catalyzed incorporation of deoxyinosine triphosphate (dITP) across dC and dT in a DNA template. They found that dITP can be incorporated across C or T, with the ratio of 13.7. X ray crystallography studies revealed that the mutagenic incorporation of dITP by human polη was affected by several factors including base pair geometry in the active site of the polymerase, tautomerization of nucleobases, and the interaction of the incoming dITP nucleotide with active site residues of polη. This study demonstrates that TLS incorporation of inosine monophosphate (IMP) into growing DNA chains contributes to its mutagenic potential in cells.

The function and regulation of ADP-ribosylation in the DNA damage response.

ADP-ribosylation is a post-translational modification involved in DNA damage response (DDR). In higher organisms it is synthesised by PARP 1-3, DNA strand break sensors. Recent advances have identified serine residues as the most common targets for ADP-ribosylation during DDR. To ADP-ribosylate serine, PARPs require an accessory factor, HPF1 which completes the catalytic domain. Through ADP-ribosylation, PARPs recruit a variety of factors to the break site and control their activities. However, the timely removal of ADP-ribosylation is also key for genome stability and is mostly performed by two hydrolases: PARG and ARH3. Here, we describe the key writers, readers and erasers of ADP-ribosylation and their contribution to the mounting of the DDR. We also discuss the use of PARP inhibitors in cancer therapy and the ways to tackle PARPi treatment resistance.

cGAS-STING signalling in cancer: striking a balance with chromosomal instability.

Chromosomal instability (CIN) is a hallmark of cancer that drives tumour evolution. It is now recognised that CIN in cancer leads to the constitutive production of misplaced DNA in the form of micronuclei and chromatin bridges. These structures are detected by the nucleic acid sensor cGAS, leading to the production of the second messenger 2'3'-cGAMP and activation of the critical hub of innate immune signalling STING. Activation of this immune pathway should instigate the influx and activation of immune cells, resulting in the eradication of cancer cells. That this does not universally occur in the context of CIN remains an unanswered paradox in cancer. Instead, CIN-high cancers are notably adept at immune evasion and are highly metastatic with typically poor outcomes. In this review, we discuss the diverse facets of the cGAS-STING signalling pathway, including emerging roles in homeostatic processes and their intersection with genome stability regulation, its role as a driver of chronic pro-tumour inflammation, and crosstalk with the tumour microenvironment, which may collectively underlie its apparent maintenance in cancers. A better understanding of the mechanisms whereby this immune surveillance pathway is commandeered by chromosomally unstable cancers is critical to the identification of new vulnerabilities for therapeutic exploitation.

Evolving DNA repair synthetic lethality targets in cancer.

DNA damage signaling response and repair (DDR) is a critical defense mechanism against genomic instability. Impaired DNA repair capacity is an important risk factor for cancer development. On the other hand, up-regulation of DDR mechanisms is a feature of cancer chemotherapy and radiotherapy resistance. Advances in our understanding of DDR and its complex role in cancer has led to several translational DNA repair-targeted investigations culminating in clinically viable precision oncology strategy using poly(ADP-ribose) polymerase (PARP) inhibitors in breast, ovarian, pancreatic, and prostate cancers. While PARP directed synthetic lethality has improved outcomes for many patients, the lack of sustained clinical response and the development of resistance pose significant clinical challenges. Therefore, the search for additional DDR-directed drug targets and novel synthetic lethality approaches is highly desirable and is an area of intense preclinical and clinical investigation. Here, we provide an overview of the mammalian DNA repair pathways and then focus on current state of PARP inhibitors (PARPi) and other emerging DNA repair inhibitors for synthetic lethality in cancer.

Mitochondrial DNA maintenance in Drosophila melanogaster.

All 37 mitochondrial DNA (mtDNA)-encoded genes involved with oxidative phosphorylation and intramitochondrial protein synthesis, and several nuclear-encoded genes involved with mtDNA replication, transcription, repair and recombination are conserved between the fruit fly Drosophila melanogaster and mammals. This, in addition to its easy genetic tractability, has made Drosophila a useful model for our understanding of animal mtDNA maintenance and human mtDNA diseases. However, there are key differences between the Drosophila and mammalian systems that feature the diversity of mtDNA maintenance processes inside animal cells. Here, we review what is known about mtDNA maintenance in Drosophila, highlighting areas for which more research is warranted and providing a perspective preliminary in silico and in vivo analyses of the tissue specificity of mtDNA maintenance processes in this model organism. Our results suggest new roles (or the lack thereof) for well-known maintenance proteins, such as the helicase Twinkle and the accessory subunit of DNA polymerase γ, and for other Drosophila gene products that may even aid in shedding light on mtDNA maintenance in other animals. We hope to provide the reader some interesting paths that can be taken to help our community show how Drosophila may impact future mtDNA maintenance research.

Divergent binding mode for a protozoan BRC repeat to RAD51.

Interaction of BRCA2 through ca. 30 amino acid residue motifs, BRC repeats, with RAD51 is a conserved feature of the double-strand DNA break repair by homologous recombination in eukaryotes. In humans the binding of the eight BRC repeats is defined by two sequence motifs, FxxA and LFDE, interacting with distinct sites on RAD51. Little is known of the interaction of BRC repeats in other species, especially in protozoans, where variable number of BRC repeats are found in BRCA2 proteins. Here, we have studied in detail the interactions of the two BRC repeats in Leishmania infantum BRCA2 with RAD51. We show LiBRC1 is a high-affinity repeat and determine the crystal structure of its complex with LiRAD51. Using truncation mutagenesis of the LiBRC1 repeat, we demonstrate that high affinity binding is maintained in the absence of an LFDE-like motif and suggest compensatory structural features. These observations point towards a divergent evolution of BRC repeats, where a common FxxA-binding ancestor evolved additional contacts for affinity maturation and fine-tuning.

Identification of the target and mode of action for the prokaryotic nucleotide excision repair inhibitor ATBC.

In bacteria, nucleotide excision repair (NER) plays a major role in repairing DNA damage from a wide variety of sources. Therefore, its inhibition offers potential to develop a new antibacterial in combination with adjuvants, such as UV light. To date, only one known chemical inhibitor of NER is 2-(5-amino-1,3,4-thiadiazol-2-yl)benzo(f)chromen-3-one (ATBC) exists and targets Mycobacterium tuberculosis NER. To enable the design of future drugs, we need to understand its mechanism of action. To determine the mechanism of action, we used in silico structure-based prediction, which identified the ATP-binding pocket of Escherichia coli UvrA as a probable target. Growth studies in E. coli showed it was nontoxic alone, but able to impair growth when combined with DNA-damaging agents, and as we predicted, it reduced by an approximately 70% UvrA's ATPase rate. Since UvrA's ATPase activity is necessary for effective DNA binding, we used single-molecule microscopy to directly observe DNA association. We measured an approximately sevenfold reduction in UvrA molecules binding to a single molecule of dsDNA suspended between optically trapped beads. These data provide a clear mechanism of action for ATBC, and show that targeting UvrA's ATPase pocket is effective and ATBC provides an excellent framework for the derivation of more soluble inhibitors that can be tested for activity.

Spectrum of DNA mismatch repair failures viewed through the lens of cancer genomics and implications for therapy.

Genome sequencing can be used to detect DNA repair failures in tumors and learn about underlying mechanisms. Here, we synthesize findings from genomic studies that examined deficiencies of the DNA mismatch repair (MMR) pathway. The impairment of MMR results in genome-wide hypermutation and in the 'microsatellite instability' (MSI) phenotype-occurrence of indel mutations at short tandem repeat (microsatellite) loci. The MSI status of tumors was traditionally assessed by molecular testing of a selected set of MS loci or by measuring MMR protein expression levels. Today, genomic data can provide a more complete picture of the consequences on genomic instability. Multiple computational studies examined somatic mutation distributions that result from failed DNA repair pathways in tumors. These include analyzing the commonly studied trinucleotide mutational spectra of single-nucleotide variants (SNVs), as well as of other features such as indels, structural variants, mutation clusters and regional mutation rate redistribution. The identified mutation patterns can be used to rigorously measure prevalence of MMR failures across cancer types, and potentially to subcategorize the MMR deficiencies. Diverse data sources, genomic and pre-genomic, from human and from experimental models, suggest there are different ways in which MMR can fail, and/or that the cell-type or genetic background may result in different types of MMR mutational patterns. The spectrum of MMR failures may direct cancer evolution, generating particular sets of driver mutations. Moreover, MMR affects outcomes of therapy by DNA damaging drugs, antimetabolites, nonsense-mediated mRNA decay (NMD) inhibitors, and immunotherapy by promoting either resistance or sensitivity, depending on the type of therapy.

Mechanism of action of nucleotide excision repair machinery.

Nucleotide excision repair (NER) is a versatile DNA repair pathway essential for the removal of a broad spectrum of structurally diverse DNA lesions arising from a variety of sources, including UV irradiation and environmental toxins. Although the core factors and basic stages involved in NER have been identified, the mechanisms of the NER machinery are not well understood. This review summarizes our current understanding of the mechanisms and order of assembly in the core global genome (GG-NER) pathway.

Beyond base excision repair: an evolving picture of mitochondrial DNA repair.

Mitochondria are highly specialised organelles required for key cellular processes including ATP production through cellular respiration and controlling cell death via apoptosis. Unlike other organelles, mitochondria contain their own DNA genome which encodes both protein and RNA required for cellular respiration. Each cell may contain hundreds to thousands of copies of the mitochondrial genome, which is essential for normal cellular function - deviation of mitochondrial DNA (mtDNA) copy number is associated with cellular ageing and disease. Furthermore, mtDNA lesions can arise from both endogenous or exogenous sources and must either be tolerated or corrected to preserve mitochondrial function. Importantly, replication of damaged mtDNA can lead to stalling and introduction of mutations or genetic loss, mitochondria have adapted mechanisms to repair damaged DNA. These mechanisms rely on nuclear-encoded DNA repair proteins that are translocated into the mitochondria. Despite the presence of many known nuclear DNA repair proteins being found in the mitochondrial proteome, it remains to be established which DNA repair mechanisms are functional in mammalian mitochondria. Here, we summarise the existing and emerging research, alongside examining proteomic evidence, demonstrating that mtDNA damage can be repaired using Base Excision Repair (BER), Homologous Recombination (HR) and Microhomology-mediated End Joining (MMEJ). Critically, these repair mechanisms do not operate in isolation and evidence for interplay between pathways and repair associated with replication is discussed. Importantly, characterising non-canonical functions of key proteins and understanding the bespoke pathways used to tolerate, repair or bypass DNA damage will be fundamental in fully understanding the causes of mitochondrial genome mutations and mitochondrial dysfunction.

Reconstitution of human CMG helicase ubiquitylation by CUL2LRR1 and multiple E2 enzymes.

Cullin ubiquitin ligases drive replisome disassembly during DNA replication termination. In worm, frog and mouse cells, CUL2LRR1 is required to ubiquitylate the MCM7 subunit of the CMG helicase. Here, we show that cullin ligases also drive CMG-MCM7 ubiquitylation in human cells, thereby making the helicase into a substrate for the p97 unfoldase. Using purified human proteins, including a panel of E2 ubiquitin-conjugating enzymes, we have reconstituted CMG helicase ubiquitylation, dependent upon neddylated CUL2LRR1. The reaction is highly specific to CMG-MCM7 and requires the LRR1 substrate targeting subunit, since replacement of LRR1 with the alternative CUL2 adaptor VHL switches ubiquitylation from CMG-MCM7 to HIF1. CUL2LRR1 firstly drives monoubiquitylation of CMG-MCM7 by the UBE2D class of E2 enzymes. Subsequently, CUL2LRR1 activates UBE2R1/R2 or UBE2G1/G2 to extend a single K48-linked ubiquitin chain on CMG-MCM7. Thereby, CUL2LRR1 converts CMG into a substrate for p97, which disassembles the ubiquitylated helicase during DNA replication termination.

Primary cilia and the DNA damage response: linking a cellular antenna and nuclear signals.

The maintenance of genome stability involves integrated biochemical activities that detect DNA damage or incomplete replication, delay the cell cycle, and direct DNA repair activities on the affected chromatin. These processes, collectively termed the DNA damage response (DDR), are crucial for cell survival and to avoid disease, particularly cancer. Recent work has highlighted links between the DDR and the primary cilium, an antenna-like, microtubule-based signalling structure that extends from a centriole docked at the cell surface. Ciliary dysfunction gives rise to a range of complex human developmental disorders termed the ciliopathies. Mutations in ciliopathy genes have been shown to impact on several functions that relate to centrosome integrity, DNA damage signalling, responses to problems in DNA replication and the control of gene expression. This review covers recent findings that link cilia and the DDR and explores the various roles played by key genes in these two contexts. It outlines how proteins encoded by ciliary genes impact checkpoint signalling, DNA replication and repair, gene expression and chromatin remodelling. It discusses how these diverse activities may integrate nuclear responses with those that affect a structure of the cell periphery. Additional directions for exploration of the interplay between these pathways are highlighted, with a focus on new ciliary gene candidates that alter genome stability.

Nutrient regulation of the flow of genetic information by O-GlcNAcylation.

O-linked-ß-N-acetylglucosamine (O-GlcNAc) is a post-translational modification (PTM) that is actively added to and removed from thousands of intracellular proteins. As a PTM, O-GlcNAcylation tunes the functions of a protein in various ways, such as enzymatic activity, transcriptional activity, subcellular localization, intermolecular interactions, and degradation. Its regulatory roles often interplay with the phosphorylation of the same protein. Governed by 'the Central Dogma', the flow of genetic information is central to all cellular activities. Many proteins regulating this flow are O-GlcNAc modified, and their functions are tuned by the cycling sugar. Herein, we review the regulatory roles of O-GlcNAcylation on the epigenome, in DNA replication and repair, in transcription and in RNA processing, in protein translation and in protein turnover.

New developments in Huntington's disease and other triplet repeat diseases: DNA repair turns to the dark side.

Huntington's disease (HD) is a fatal, inherited neurodegenerative disease that causes neuronal death, particularly in medium spiny neurons. HD leads to serious and progressive motor, cognitive and psychiatric symptoms. Its genetic basis is an expansion of the CAG triplet repeat in the HTT gene, leading to extra glutamines in the huntingtin protein. HD is one of nine genetic diseases in this polyglutamine (polyQ) category, that also includes a number of inherited spinocerebellar ataxias (SCAs). Traditionally it has been assumed that HD age of onset and disease progression were solely the outcome of age-dependent exposure of neurons to toxic effects of the inherited mutant huntingtin protein. However, recent genome-wide association studies (GWAS) have revealed significant effects of genetic variants outside of HTT. Surprisingly, these variants turn out to be mostly in genes encoding DNA repair factors, suggesting that at least some disease modulation occurs at the level of the HTT DNA itself. These DNA repair proteins are known from model systems to promote ongoing somatic CAG repeat expansions in tissues affected by HD. Thus, for triplet repeats, some DNA repair proteins seem to abandon their normal genoprotective roles and, instead, drive expansions and accelerate disease. One attractive hypothesis-still to be proven rigorously-is that somatic HTT expansions augment the disease burden of the inherited allele. If so, therapeutic approaches that lower levels of huntingtin protein may need blending with additional therapies that reduce levels of somatic CAG repeat expansions to achieve maximal effect.

RecQ helicases in DNA repair and cancer targets.

Helicases are enzymes that use the energy derived from ATP hydrolysis to catalyze the unwinding of DNA or RNA. The RecQ family of helicases is conserved through evolution from prokaryotes to higher eukaryotes and plays important roles in various DNA repair pathways, contributing to the maintenance of genome integrity. Despite their roles as general tumor suppressors, there is now considerable interest in exploiting RecQ helicases as synthetic lethal targets for the development of new cancer therapeutics. In this review, we summarize the latest developments in the structural and mechanistic study of RecQ helicases and discuss their roles in various DNA repair pathways. Finally, we consider the potential to exploit RecQ helicases as therapeutic targets and review the recent progress towards the development of small molecules targeting RecQ helicases as cancer therapeutics.

Making it or breaking it: DNA methylation and genome integrity.

Cells encounter a multitude of external and internal stress-causing agents that can ultimately lead to DNA damage, mutations and disease. A cascade of signaling events counters these challenges to DNA, which is termed as the DNA damage response (DDR). The DDR preserves genome integrity by engaging appropriate repair pathways, while also coordinating cell cycle and/or apoptotic responses. Although many of the protein components in the DDR are identified, how chemical modifications to DNA impact the DDR is poorly understood. This review focuses on our current understanding of DNA methylation in maintaining genome integrity in mammalian cells. DNA methylation is a reversible epigenetic mark, which has been implicated in DNA damage signaling, repair and replication. Sites of DNA methylation can trigger mutations, which are drivers of human diseases including cancer. Indeed, alterations in DNA methylation are associated with increased susceptibility to tumorigenesis but whether this occurs through effects on the DDR, transcriptional responses or both is not entirely clear. Here, we also highlight epigenetic drugs currently in use as therapeutics that target DNA methylation pathways and discuss their effects in the context of the DDR. Finally, we pose unanswered questions regarding the interplay between DNA methylation, transcription and the DDR, positing the potential coordinated efforts of these pathways in genome integrity. While the impact of DNA methylation on gene regulation is widely understood, how this modification contributes to genome instability and mutations, either directly or indirectly, and the potential therapeutic opportunities in targeting DNA methylation pathways in cancer remain active areas of investigation.

Structural insight into FANCI-FANCD2 monoubiquitination.

The Fanconi anemia (FA) pathway coordinates a faithful repair mechanism for DNA damage that blocks DNA replication, such as interstrand cross-links. A key step in the FA pathway is the conjugation of ubiquitin on to FANCD2 and FANCI, which is facilitated by a large E3 ubiquitin ligase complex called the FA core complex. Mutations in FANCD2, FANCI or FA core complex components cause the FA bone marrow failure syndrome. Despite the importance of these proteins to DNA repair and human disease, our molecular understanding of the FA pathway has been limited due to a deficit in structural studies. With the recent development in cryo-electron microscopy (EM), significant advances have been made in structural characterization of these proteins in the last 6 months. These structures, combined with new biochemical studies, now provide a more detailed understanding of how FANCD2 and FANCI are monoubiquitinated and how DNA repair may occur. In this review, we summarize these recent advances in the structural and molecular understanding of these key components in the FA pathway, compare the activation steps of FANCD2 and FANCI monoubiquitination and suggest molecular steps that are likely to be involved in regulating its activity.