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Summary DNA repair enzymes are essential for the maintenance of the neuronal genome and thereby proper brain functions. Emerging evidence links DNA repair to epigenetic gene regulation; however, its contribution to different transcriptional programs required for neuronal functions remains elusive. In this study, we identified a role of the DNA repair enzyme NEIL3 in modulating the maturation and function of hippocampal CA1 neurons by shaping the CA1 transcriptome during postnatal development and in association with spatial behavior. We observed a delayed maturation in Neil3-/- CA1 and identified differentially regulated genes required for hippocampal development. We revealed impaired spatial stability in Neil3-/- CA1 place cells and found spatial experience-induced gene expression essential for synaptic plasticity. This is the first study that links molecular underpinnings of DNA repair to the neural basis of spatial cognition beyond animals' behavioral phenotypes, thus shedding light on the molecular determinants enabling a stable neural representation of space., Graphical abstract, Highlights • NEIL3 impacts CA1 maturation by shaping transcription during development • NEIL3 depletion leads to impaired function of CA1 place cells • NEIL3 shapes transcription in hippocampal CA1 during behavior • NEIL3 impacts experience-induced expression of immediate early genes (IEGs)., Neurology; Nucleic acids; Transcriptomics

8-Oxoguanine DNA glycosylase 1 (OGG1) initiates the base excision repair pathway by removing one of the most abundant DNA lesions, 8-oxo-7,8-dihydroguanine (8-oxoG). Recent data showed that 8-oxoG not only is a pro-mutagenic genomic base lesion, but also functions as an epigenetic mark and that consequently OGG1 acquire distinct roles in modulation of gene expression. In support, lack of functional OGG1 in Ogg1-/- mice led to an altered expression of genes including those responsible for the aberrant innate and adaptive immune responses and susceptibility to metabolic disorders. Therefore, the present study examined stimulus-driven OGG1-DNA interactions at whole genome level using chromatin immunoprecipitation (ChIP)-coupled sequencing, and the roles of OGG1 enriched on the genome were validated by molecular and system-level approaches. Results showed that signaling levels of cellular ROS generated by TNFα, induced enrichment of OGG1 at specific sites of chromatinized DNA, primarily in the regulatory regions of genes. OGG1-ChIP-ed genes are associated with important cellular and biological processes and OGG1 enrichment was limited to a time scale required for immediate cellular responses. Prevention of OGG1-DNA interactions by siRNA depletion led to modulation of NF-κB's DNA occupancy and differential expression of genes. Taken together these data show TNFα-ROS-driven enrichment of OGG1 at gene regulatory regions in the chromatinized DNA, which is a prerequisite to modulation of gene expression for prompt cellular responses to oxidant stress., Graphical abstract The repair protein 8-oxoguanine DNA glycosylase 1 (OGG1) modulates gene expression upon its stimulus-driven binding to gene regulatory regions. ROS generated by receptor ligand interactions, metabolic processes, or environmental exposures produces 8-oxoguanine (8-oxoG) primarily in guanine-rich gene regulatory regions and inactivates OGG1's enzymatic activity by oxidizing it at cysteine residue(s) (OGG1-SOH). OGG1-SOH flips 8-oxoG out of the DNA double helix and induces alterations in adjacent DNA sequences, by which it facilitates binding of transcription factors. This leads to expression of genes and downstream cellular biological responses. Upon the cellular redox is reestablished, genomic 8-oxoG is repaired via the BER pathway. OGG1-SH, enzymatically active OGG1; OGG1-SOH, enzyatically inactive OGG1; BER, base excision repair; red star: 8-oxoguanine; red bar: gene regulatory region; blue bars: exons.fx1

Oxidative stress and the resulting damage to genomic DNA are inevitable consequences of endogenous physiological processes, and they are amplified by cellular responses to environmental exposures. One of the most frequent reactions of reactive oxygen species with DNA is the oxidation of guanine to pre-mutagenic 8-oxo-7,8-dihydroguanine (8-oxoG). Despite the vulnerability of guanine to oxidation, vertebrate genes are primarily embedded in GC-rich genomic regions, and over 72% of the promoters of human genes belong to a class with a high GC content. In the promoter, 8-oxoG may serve as an epigenetic mark, and when complexed with the oxidatively inactivated repair enzyme 8-oxoguanine DNA glycosylase 1, provide a platform for the coordination of the initial steps of DNA repair and the assembly of the transcriptional machinery to launch the prompt and preferential expression of redox-regulated genes. Deviations/variations from this artful coordination may be the etiological links between guanine oxidation and various cellular pathologies and diseases during ageing processes., Graphical abstract Binding of OGG1 to genomic 8-oxoguanine facilitates gene expression. Proposed actions of OGG1 on open chromatin region (left), OGG1 binding to its substrate facilitates DNA occupancy of transcription factors (NF-κB is shown as example) and promote transcription. If guanine oxidation occurs at closed chromatin region (right), binding of OGG1 could block the interaction of methyl binding proteins (BMPs) with their substrates, subsequently recruit transcription machinery components to activate transcription.fx1

Nei2 (Rv3297) is a DNA Base Excision Repair (BER) glycosylase that is essential for survival of Mycobacterium tuberculosis in primates. We show that MtbNei2 is a bifunctional glycosylase that specifically acts on oxidized pyrimidine-containing single-stranded, double-stranded, 5’/3’ fork and bubble DNA substrates. MtbNei2 possesses Uracil DNA glycosylase activity unlike E. coli Nei. Mutational studies demonstrate that Pro2 and Glu3 located in the active site are essential for glycosylase activity of MtbNei2. Mutational analysis demonstrated that an unstructured C-terminal zinc finger domain that was important for activity in E. coli Nei and Fpg, was not required for the glycosylase activity of MtbNei2. Lastly, we screened the NCI natural product compound database and identified three natural product inhibitors with IC50 values ranging between 41.8 μM-92.7 μM against MtbNei2 in in vitro inhibition assays. Surface Plasmon Resonance (SPR) experiments showed that the binding affinity of the best inhibitor, NSC31867, was 74 nM. The present results set the stage for exploiting this important target in developing new therapeutic strategies that target Mycobacterial BER., Highlights • Biochemical characterization of MtbNei2 (Rv3297). • Pro2 and Glu3 residues are essential for MtbNei2 activity. • Zinc finger domain is dispensable for activity. • 3 natural product inhibitors identified against MtbNei2.

Summary Oxidative damage to telomere DNA compromises telomere integrity. We recently reported that the DNA glycosylase NEIL3 preferentially repairs oxidative lesions in telomere sequences in vitro. Here, we show that loss of NEIL3 causes anaphase DNA bridging because of telomere dysfunction. NEIL3 expression increases during S phase and reaches maximal levels in late S/G2. NEIL3 co-localizes with TRF2 and associates with telomeres during S phase, and this association increases upon oxidative stress. Mechanistic studies reveal that NEIL3 binds to single-stranded DNA via its intrinsically disordered C terminus in a telomere-sequence-independent manner. Moreover, NEIL3 is recruited to telomeres through its interaction with TRF1, and this interaction enhances the enzymatic activity of purified NEIL3. Finally, we show that NEIL3 interacts with AP Endonuclease 1 (APE1) and the long-patch base excision repair proteins PCNA and FEN1. Taken together, we propose that NEIL3 protects genome stability through targeted repair of oxidative damage in telomeres during S/G2 phase.

Uracil-DNA glycosylase (UNG) initiates the base excision repair pathway by excising uracil from DNA. We have previously shown that Trypanosoma brucei cells defective in UNG exhibit reduced infectivity thus demonstrating the relevance of this glycosylase for survival within the mammalian host. In the early steps of the immune response, nitric oxide (NO) is released by phagocytes, which in combination with oxygen radicals produce reactive nitrogen species (RNS). These species can react with DNA generating strand breaks and base modifications including deaminations. Since deaminated cytosines are the main substrate for UNG, we hypothesized that the glycosylase might confer protection towards nitrosative stress. Our work establishes the occurrence of genotoxic damage in Trypanosoma brucei upon exposure to NO in vitro and shows that deficient base excision repair results in increased levels of damage in DNA and a hypermutator phenotype. We also evaluate the incidence of DNA damage during infection in vivo and show that parasites recovered from mice exhibit higher levels of DNA strand breaks, base deamination and repair foci compared to cells cultured in vitro. Notably, the absence of UNG leads to reduced infectivity and enhanced DNA damage also in animal infections. By analysing mRNA and protein levels, we found that surviving UNG-KO trypanosomes highly express tryparedoxin peroxidase involved in trypanothione/tryparedoxin metabolism. These observations suggest that the immune response developed by the host enhances the activation of genes required to counteract oxidative stress and emphasize the importance of DNA repair pathways in the protection to genotoxic and oxidative stress in trypanosomes., This work was funded by the Junta de Andalucía (BIO-199); the Plan Nacionalde Investigación Científica, Instituto de Salud Carlos III-Subdirección General de Redes y Centros de Investigación Cooperativa-Red de Investigación Cooperativa en Enfermedades Tropicales (RICET: RD16/0027/0014) and FEDER; and the Plan Nacional (SAF2016-79957-R). We thank Dr Carlos Robello for kindly providing the anti-TbTRYP1 antibody and Dr David Horn for the anti-TbγH2A antibody.

DNA polymerase β plays a central role in the base excision DNA repair pathway that cleanses the genome of apurinic/apyrimidinic (AP) sites. AP sites arise in DNA from spontaneous base loss and DNA damage-specific glycosylases that hydrolyze the N-glycosidic bond between the deoxyribose and damaged base. AP sites are deleterious lesions because they can be mutagenic and/or cytotoxic. DNA polymerase β contributes two enzymatic activities, DNA synthesis and lyase, during the repair of AP sites; these activities reside on carboxyl- and amino-terminal domains, respectively. Accordingly, its cellular, structural, and kinetic attributes have been extensively characterized and it serves as model enzyme for the nucleotidyl transferase reaction utilized by other replicative, repair, and trans-lesion DNA polymerases.

Ogg1 and Mutyh DNA glycosylases cooperate to prevent mutations caused by 8-oxoG, a major premutagenic DNA lesion associated with cognitive decline. We have examined behavior and cognitive function in mice deficient of these glycosylases. Ogg1−/−Mutyh−/− mice were more active and less anxious, with impaired learning ability. In contrast, Mutyh−/− mice showed moderately improved memory. We observed no apparent change in genomic 8-oxoG levels, suggesting that Ogg1 and Mutyh play minor roles in global repair in adult brain. Notably, transcriptome analysis of hippocampus revealed that differentially expressed genes in the mutants belong to pathways known to be involved in anxiety and cognition. Esr1 targets were upregulated, suggesting a role of Ogg1 and Mutyh in repression of Esr1 signaling. Thus, beyond their involvement in DNA repair, Ogg1 and Mutyh regulate hippocampal gene expression related to cognition and behavior, suggesting a role for the glycosylases in regulating adaptive behavior. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Mitochondrial DNA (mtDNA) resides in close proximity to metabolic reactions, and is maintained by the 8-oxoguanine DNA glycosylase (Ogg1) and other members of the base excision repair pathway. Here, we tested the hypothesis that changes in liver metabolism as under fasting/feeding conditions would be sensed by liver mtDNA, and that Ogg1 deficient mice might unravel a metabolic phenotype. Wild type (WT) and ogg1−/− mice were either fed ad libitum or subjected to fasting for 24 h, and the corresponding effects on liver gene expression, DNA damage, as well as serum values were analyzed. Ogg1 deficient mice fed ad libitum exhibited hyperglycemia, elevated insulin levels and higher liver glycogen content as well as increased accumulation of 8oxoG in mtDNA compared to age- and gender matched WT mice. Interestingly, these phenotypes were absent in ogg1−/− mice during fasting. Gene expression and functional analyses suggest that the diabetogenic phenotype in the ogg1−/− mice is due to a failure to suppress gluconeogensis in the fed state. The ogg1−/− mice exhibited reduced mitochondrial electron transport chain (ETC) capacity and a combined low activity of the pyruvate dehydrogenase (PDH), alluding to inefficient channeling of glycolytic products into the citric acid cycle. Our data demonstrate a physiological role of base excision repair that goes beyond DNA maintenance, and implies that DNA repair is involved in regulating metabolism. © 2017. This is the authors’ accepted and refereed manuscript to the article. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/

Human 8-oxoguanine DNA glycosylase 1 (hOGG1) is an important DNA repair enzyme that can excise the 8-oxo-7,8-dihydroguanine (8-oxoG) from damaged DNA and is implicated in the etiology of many kinds of cancers. Here, we report an electrochemical method to assess the activity of hOGG1 by using a carefully designed DNA probe, which contains an internal 8-oxoG site and an electrochemical active tag. In this method, hOGG1 can selectively recognize the 8-oxoG site and cleave the probe DNA, releasing a methylene blue (MB)-labeled signal fragment. The signal fragment can then hybridize with the DNA molecules that have been previously immobilized on a gold electrode surface. Consequently, a sensitive electrochemical technique, square wave voltammetry, can be employed for the signal readout, thus characterizing the activity of hOGG1. This method is simple, sensitive and specific, and it has been used for human serum samples, so it may imply a potential new method for the assay of hOGG1. Keywords: Human 8-oxoguanine DNA glycosylase 1, DNA repair, Cancer development, Electrochemical assay, Square wave voltammetry

Base excision repair (BER) is a key genome maintenance pathway that removes endogenously damaged DNA bases that arise in cells at very high levels on a daily basis. Failure to remove these damaged DNA bases leads to increased levels of mutagenesis and chromosomal instability, which have the potential to drive carcinogenesis. Next Generation sequencing efforts of the germline and tumors genomes of thousands of individuals has uncovered many rare mutations in BER genes. Given that BER is critical for genome maintenance, it is important to determine whether BER genomic variants have functional phenotypes. In this chapter we present our in silico methods for the identification and prioritization of BER variants for further study. We also provide detailed instructions and commentary on the initial cellular assays we employ to dissect potentially important phenotypes of human BER variants and highlight the strengths and weaknesses of our approaches. BER variants possessing interesting functional phenotypes can then be studied in more detail to provide important mechanistic insights regarding the role of aberrant BER in carcinogenesis.

Endogenous and exogenous reactive species react with DNA in living organisms by numerous mechanisms and cause oxidatively induced DNA damage with multiple lesions. If not repaired, DNA lesions can cause genetic instability, leading to mutagenesis and cell death, and consequently to disease processes including carcinogenesis. Living organisms have evolved to repair DNA damage by numerous repair mechanisms, the activities of which depend on the nature of DNA lesions. The basic steps of DNA repair consist of lesion recognition and excision, followed by DNA synthesis and ligation. DNA repair pathways are drug targets in cancer therapy for enzyme inhibition to prevent drug resistance and to achieve optimal treatment. An overview is presented on the mechanisms of oxidatively induced DNA damage with resulting DNA lesions and on the repair pathways that process this type of damage to restore DNA structure.

Base excision repair (BER) is a conserved and ubiquitous pathway that is initiated by DNA glycosylases, which recognize and remove damaged or mismatched nucleobases, setting the stage for restoration of the correct DNA sequence by follow-on BER enzymes. DNA glycosylases employ a nucleotide-flipping step prior to cleavage of the N-glycosyl bond, and most exhibit slow release of the abasic DNA product and/or strong product inhibition. As such, studying the catalytic mechanism of these enzymes requires care in the design, execution, and interpretation of single- and multiple-turnover kinetics experiments, which is the topic of this chapter.

Enzymes that modify nucleobases in double-stranded genomic DNA, either as part of a DNA repair pathway or as an epigenetic modifying pathway, adopt a multistep pathway to locate target sites and reconfigure the DNA to gain access. Work on several different enzymes has shown that in almost all cases base flipping, also known as nucleotide flipping, is a key feature of specific site recognition. In this chapter, we discuss some of the strategies that can be used to perform a kinetic characterization for DNA binding and nucleotide flipping. The resulting kinetic and thermodynamic framework provides a platform for understanding substrate specificity, mechanisms of inhibition, and the roles of important amino acids. We use a human DNA repair glycosylase called alkyladenine DNA glycosylase as a case study, because this is one of the best-characterized nucleotide-flipping enzymes. However, the approaches that are described can be readily adapted to study other enzymes, and future studies are needed to understand the mechanism of substrate recognition in each individual case. As more enzymes are characterized, we can hope to uncover which features of DNA searching and nucleotide flipping are fundamental features shared by many different families of DNA modifying enzymes, and which features are specific to a particular enzyme. Such an understanding provides reasonable models for less characterized enzymes that are important for epigenetic DNA modification and DNA repair pathways.

DNA methylation is now accepted as an important epigenetic mechanism in the regulation of gene activity during nervous system development, as well as in brain functions such as learning and memory, and the etiology of brain disorders. Ten-eleven translocation (TET) enzymes can oxidize 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC), and the oxidation derivatives 5fC and 5caC can be further catalyzed by thymine DNA glycosylase in conjunction with base excision repair. Recent data suggest that 5fC and 5caC could represent active DNA demethylation at enhancers and promoters, giving us a new perspective on cytosine modification–dependent regulatory mechanisms. Here we describe general aspects of the epigenetic regulation of gene activity by active DNA demethylation and then discuss the latest research into 5fC and 5caC. Finally, we discuss the potential roles of 5fC and 5caC in brain development and the need for further scientific investigation.

DNA is under constant attack from a variety of endogenous and exogenous sources, creating DNA damage that must be repaired in order to maintain the genetic code. One critical DNA repair pathway by which this is accomplished is base excision repair. This pathway corrects small DNA base lesions by recognizing and excising the damage, processing the phosphodiester backbone so that the correct nucleotide can be inserted, and finally closing the nick in the DNA backbone to restore genome integrity. A deficiency in base excision repair increases genomic instability, and has been associated with the development of human diseases.

General comments about the survival factors and signaling pathways involved in cancer resistance to drugs or ionizing radiation are followed by the relevant multidrug resistance (MDR) phenotype associated with the overexpression of transport proteins, their mechanisms, and the development of blockers. The effects on tumor cells sensitivity of inhibition of metabolic enzymes such as γ-glutamylcysteine synthetase and glutathione S -transferases, as well as the effects of intracellular glutathione depletion, are also discussed. The changes in the DNA repair pathways produced in various chemoresistant and radioresistant phenotypes and the therapeutic efficacy of the disruption of these processes with targeted chemosensitizers are analyzed with emphasis on inhibitors of O 6 -alkylguanine DNA alkyltransferase, antitumor adjuvants targeting the BER process, inhibitors of DNA glycosylases, poly(ADP-ribose) polymerase inhibitors, and inhibitors of enzymes involved in double-strand DNA break repair pathways. The role of antitumor drug resistance related to cellular adhesion molecules, carbonic anhydrase and SPARC protein is studied, followed by comments on tumor radiosensitization, and chemoresistance to anti-VEGF, anti-EGFR and ALK mutations.

Among the DNA viruses, the so-called nucleo-cytoplasmic large DNA viruses (NCLDV) constitute a monophyletic group that currently consists of seven families of viruses infecting a very broad variety of eukaryotes, from unicellular marine protists to humans. Many recent papers have analyzed the sequence and structure of NCLDV genomes and their phylogeny, providing detailed analysis about their genomic structure and evolutionary history and proposing their inclusion in a new viral order named Megavirales that, according to some authors, should be considered as a fourth domain of life, aside from Bacteria, Archaea and Eukarya. The maintenance of genetic information protected from environmental attacks and mutations is essential not only for the survival of cellular organisms but also viruses. In cellular organisms, damaged DNA bases are removed in two major repair pathways: base excision repair (BER) and nucleotide incision repair (NIR) that constitute the major pathways responsible for repairing most endogenous base lesions and abnormal bases in the genome by precise repair procedures. Like cells, many NCLDV encode proteins that might constitute viral DNA repair pathways that would remove damages through BER/NIR pathways. However, the molecular mechanisms and, specially, the biological roles of those viral repair pathways have not been deeply addressed in the literature so far. In this paper, we review viral-encoded BER proteins and the genetic and biochemical data available about them. We propose and discuss probable viral-encoded DNA repair mechanisms and pathways, as compared with the functional and molecular features of known homologs proteins. This work was funded by a grant from the Spanish Ministerio de Economía y Competitividad (AGL2010-22229-C03-02) and by an institutional grant from Fundación Ramón Areces to Centro de Biología Molecular Severo Ochoa.

tThe most common mutations in cancer are C to T transitions, but their origin has remained elusive.Recently, mutational signatures of APOBEC-family cytosine deaminases were identified in many com-mon cancers, suggesting off-target deamination of cytosine to uracil as a common mutagenic mechanism.Here we present evidence from mass spectrometric quantitation of deoxyuridine in DNA that shows sig-nificantly higher genomic uracil content in B-cell lymphoma cell lines compared to non-lymphoma cancercell lines and normal circulating lymphocytes. The genomic uracil levels were highly correlated with AIDmRNA and protein expression, but not with expression of other APOBECs. Accordingly, AID knockdownsignificantly reduced genomic uracil content. B-cells stimulated to express endogenous AID and undergoclass switch recombination displayed a several-fold increase in total genomic uracil, indicating that Bcells may undergo widespread cytosine deamination after stimulation. In line with this, we found thatclustered mutations (kataegis) in lymphoma and chronic lymphocytic leukemia predominantly carryAID-hotspot mutational signatures. Moreover, we observed an inverse correlation of genomic uracil withuracil excision activity and expression of the uracil-DNA glycosylases UNG and SMUG1. In conclusion,AID-induced mutagenic U:G mismatches in DNA may be a fundamental and common cause of mutationsin B-cell malignancies. © 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-NDlicense (http://creativecommons.org/licenses/by-nc-nd/3.0/).

DNA base excision repair (BER) prevents mutagenesis or cell death by correcting most of the hydrolytic, oxidative, and alkylation DNA lesions due to both cellular reactions and environmental agents. Two branches of BER carry out the repair process by replacing one or multiple nucleotides, respectively. DNA N-glycosylases (11 distinct proteins in human cells) remove many different damaged or inappropriately inserted bases. The DNA glycosylases generate abasic (AP) sites then enter the central BER pathway, along with abasic lesions formed by hydrolysis or oxidative attack. These are cleaved by a hydrolytic AP endonuclease. In some cases, the DNA glycosylase may itself also cleave the AP site in a β-elimination reaction; the resulting 3′-abasic product is also excised by an AP endonuclease. DNA synthesis then replaces at least the missing nucleotide. In mammalian cells, repair synthesis carried out by DNA polymerase β, which then also excises the 5′-abasic residue, and DNA ligase III completes the repair. In some cases, as with the oxidative lesion 2-deoxyribonolactone, excision by the polymerase is not possible, and additional DNA synthesis takes place involving the replication polymerases δ or e. The resulting displaced flap must then be excised by another replication protein, FEN1, before ligase I can complete the repair. The single-nucleotide and ‘long-patch’ BER pathways function both in the nucleus and in mitochondria.

Maintaining the integrity of the genome is essential for life. However, nucleic acids, and particularly DNA, are prone to damage accumulation from chemical and physical agents. Among those damages, oxidative lesions are some of the more abundant types of modifications, owing, in part, to the generation of oxidants by normal metabolism in aerobic cells. Oxidation to the nucleotide bases and to the sugar can lead to mutagenesis and cell death. Most living organisms have several enzymatic pathways to repair DNA lesions. Oxidative damage is repaired primarily by the base excision repair (BER) pathway. This pathway is accomplished by sequential enzymatic reactions as follows: the damaged base is recognized and excised, generating an abasic site; the abasic site is further cleaved at the sugar–phosphate backbone, generating a single-strand break; a new nucleotide is inserted, and if needed the 5′ and 3′ termini are processed to generate ligatable ends; and finally, the nick is sealed. Some minor types of oxidized lesions, such as cyclic base modifications (8,5′-cyclopurine-2′-deoxynucleosides) are not processed by BER and need to be repaired by other pathways, such as nucleotide excision repair.

DNA glycosylases recognize and remove damaged bases from DNA by cleaving the base–sugar ( N -glycosylic) bond, and downstream base excision repair enzymes restore the correct nucleotide. DNA bases are chemically reactive and are continuously subject to damage by mechanisms including alkylation, deamination, oxidation, and ultraviolet radiation, giving a variety of lesions that can be mutagenic and/or cytotoxic. DNA glycosylases use a remarkable nucleotide-flipping mechanism to extrude damaged nucleotides from the DNA helix, thereby coupling lesion recognition and catalysis. Some glycosylases are specific for a single lesion, while others handle multiple forms of damage. DNA glycosylases are also important for transcriptional regulation and immunity.

5-Methylcytosine (5meC) is found in the DNA of many organisms including mammals. Water-mediated deamination of 5meC produces thymine which – if unrepaired – causes C:G to T:A mutations. Such mutations are reduced in Escherichia coli by the action of very short patch repair system which employs an endonuclease to initiate the replacement of the mismatched thymine with a cytosine. In mammals, a different repair pathway is utilized which may be initiated by an enzyme that excises the T in a T:G mispair. Inactivation of these repair pathways results in an increase in 5meC to T mutations in the relevant organisms and in mouse models makes them more susceptible to tumor formation.

This chapter describes some of the recent, exciting developments that have characterized and connected processes that modify DNA bases with DNA repair pathways. It begins with AID/APOBEC or TET family members that covalently modify bases within DNA. The modified bases, such as uracil or 5-formylcytosine, are then excised by DNA glycosylases including UNG or TDG to initiate base excision repair (BER). BER is known to preserve genome integrity by removing damaged bases. The newer studies underscore the necessity of BER following enzymes that deliberately damage DNA. This includes the role of BER in antibody diversification and more recently, its requirement for demethylation of 5-methylcytosine in mammalian cells. The recent advances have shed light on mechanisms of DNA demethylation, and have raised many more questions. The potential hazards of these processes have also been revealed. Dysregulation of the activity of base modifying enzymes, and resolution by unfaithful or corrupt means can be a driver of genome instability and tumorigenesis. The understanding of both DNA and histone methylation and demethylation is now revealing the true extent to which epigenetics influence normal development and cancer, an abnormal development.

Oxidative genome damage induced by reactive oxygen species includes oxidized bases, abasic (AP) sites, and single-strand breaks, all of which are repaired via the evolutionarily conserved base excision repair/single-strand break repair (BER/SSBR) pathway. BER/SSBR in mammalian cells is complex, with preferred and backup sub-pathways, and is linked to genome replication and transcription. The early BER/SSBR enzymes, namely, DNA glycosylases (DGs) and the end-processing proteins such as abasic endonuclease 1 (APE1), form complexes with downstream repair (and other noncanonical) proteins via pairwise interactions. Furthermore, a unique feature of mammalian early BER/SSBR enzymes is the presence of a disordered terminal extension that is absent in their Escherichia coli prototypes. These nonconserved segments usually contain organelle-targeting signals, common interaction interfaces, and sites of posttranslational modifications that may be involved in regulating their repair function including lesion scanning. Finally, the linkage of BER/SSBR deficiency to cancer, aging, and human neurodegenerative diseases, and therapeutic targeting of BER/SSBR are discussed.

One of the most exciting recent advances in the epigenetic field is the discovery that 5-methylcytosine (5mC) in DNA can be iteratively oxidized by a family of proteins known as Tet proteins to generate 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). These 5mC derivatives can be further processed by thymine-DNA glycosylase (TDG) followed by base excision repair or by replication-dependent dilution leading to DNA demethylation. Given the similarity between 5mC and its oxidation derivatives, many of the conventional techniques used for 5mC analysis cannot distinguish between 5mC and 5hmC/5fC/5caC. Here, we describe 2D-TLC and mass spectrometry methods that we have successfully used in differentiating 5mC from its oxidative derivatives as well as in characterizing the enzymatic activity of Tet proteins both in vitro and in vivo.

Publisher Summary This chapter focuses on the base excision repair (BER) pathway which is responsible for the repair of single-base lesions and can be reconstituted in vitro with a glycosylase, AP endonuclease, polymerase β, and a ligase. BER is the predominant pathway for the repair of oxidative and alkylation DNA damage as well as a basic or baseless sites. Without this balance of enzymatic activities, incomplete processing of the damage occurs, repair intermediates accumulate, and eventually cells will die. Inhibition of proliferation and cell death are desirable in tumor cell populations, therefore inhibitors of BER proteins are under development and currently being evaluated in the clinic. Several of these agents are being evaluated in combination with numerous existing chemotherapeutic agents and radiation therapy. Combination therapy with agents that generate DNA damage that is repaired by BER is reasonable and demonstrating efficacy both preclinically and in early clinical trials.

Publisher Summary This chapter focuses on the role of apurinic/apyrimidinic endonuclease (APE1) in redox regulation and response to oxidative stress and recent therapeutic developments targeting APE1's redox function. A key enzyme in BER, APE1,6 recognizes a basic sites produced either spontaneously or through removal of an oxidized base by a DNA glycosylase. Knock-out of APE1 in mice is embryonic lethal establishing APE1 as an essential enzyme; 7 APE1 functions in base excision repair by cleaving the phosphodiester backbone 5’ of a basic site resulting in a 3’OH and 5’ deoxyribose phosphate. Further processing by DNA polymerase beta and DNA ligase completes the DNA repair. APE1 is also a nuclear redox factor that recognizes oxidized transcription factors and reduces them, significantly stimulating their DNA-binding properties. In this regard, APE1 plays a unique role in that it repairs both oxidized DNA and a number of transcription factors, which in turn regulate expression of DNA repair genes.