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It was not until the ingenious works of Anton van Leeuwenhoek in the 17th century that bacteria were first discovered and understood to be a “new” form of life on Earth (1). For the next 200 y, bacteria were largely ignored and thought to simply be “bags of proteins,” even though it was recognized that they could have enormous impacts on human health. It is now widely appreciated that bacteria are incredibly complex organisms that can function in a manner that mimics a multicellular organism (2, 3). The ability of bacterial cells to constantly produce and remodel their cell wall is no exception to this, and myriad crucial findings about this process have been made over the last century. One of the most seminal of these discoveries came in the 1940s when β-lactam−containing molecules were found to combat bacterial infections. More recently, the targets of these drugs, the penicillin-binding proteins, as well as other protein machinery have been identified as essential elements for construction of the complex mesh that keeps these cells intact, the peptidoglycan (PG). Still, many mysteries remain about exactly how bacterial cells grow and divide. In particular, while enzymes that cleave and tailor portions of the PG are known to play important roles in microbial survival, they are dramatically understudied. Taguchi et al. (4) shed light on two enzymes in Streptococcus pneumoniae , MpgA and MpgB, shown to cleave a nascent carbohydrate strand to liberate newly synthesized PG. While previous studies postulated that this occurred in a fashion similar to that utilized by a close relative, the lytic glycosylase MltG in Escherichia coli [1,6-anhydroMurNac product (5)], Taguchi et al. instead discover that MpgA and MpgB act as muramidases [saccharide product with reducing end (6)]. Amazingly, they determined that a single amino acid is largely responsible for … [↵][1]1To whom correspondence may be addressed. Email: carlsone{at}umn.edu. [1]: #xref-corresp-1-1

Significance The collection of large amounts of whole-genome sequencing data allowed for identification of mutational signatures, which are characteristic combinations of substitutions in the context of neighboring bases. The clinical significance of these mutational signatures is still largely unknown. In neuroblastoma, we showed that high levels of cytosine > adenine (C > A) substitutions are associated with poor survival. We identified that these high levels of C > A substitutions result from defects in 8-oxo-guanine repair, specifically from copy number loss of the DNA glycosylases MUTYH and OGG1. The high frequency of C > A substitutions in neuroblastoma contributes to the increased adaptive capacity of these tumors. Thereby, we link basic molecular genetic mutation patterns to clinically significant tumor evolution processes., Neuroblastomas are childhood tumors with frequent fatal relapses after induction treatment, which is related to tumor evolution with additional genomic events. Our whole-genome sequencing data analysis revealed a high frequency of somatic cytosine > adenine (C > A) substitutions in primary neuroblastoma tumors, which was associated with poor survival. We showed that increased levels of C > A substitutions correlate with copy number loss (CNL) of OGG1 or MUTYH. Both genes encode DNA glycosylases that recognize 8-oxo-guanine (8-oxoG) lesions as a first step of 8-oxoG repair. Tumor organoid models with CNL of OGG1 or MUTYH show increased 8-oxoG levels compared to wild-type cells. We used CRISPR-Cas9 genome editing to create knockout clones of MUTYH and OGG1 in neuroblastoma cells. Whole-genome sequencing of single-cell OGG1 and MUTYH knockout clones identified an increased accumulation of C > A substitutions. Mutational signature analysis of these OGG1 and MUTYH knockout clones revealed enrichment for C > A signatures 18 and 36, respectively. Clustering analysis showed that the knockout clones group together with tumors containing OGG1 or MUTYH CNL. In conclusion, we demonstrate that defects in 8-oxoG repair cause accumulation of C > A substitutions in neuroblastoma, which contributes to mutagenesis and tumor evolution.

Thymine DNA glycosylase (TDG) is a pivotal enzyme with dual roles in both genome maintenance and epigenetic regulation. TDG is involved in cytosine demethylation at CpG sites in DNA. Here we have used molecular modeling to delineate the lesion search and DNA base interrogation mechanisms of TDG. First, we examined the capacity of TDG to interrogate not only DNA substrates with 5-carboxyl cytosine modifications but also G:T mismatches and nonmismatched (A:T) base pairs using classical and accelerated molecular dynamics. To determine the kinetics, we constructed Markov state models. Base interrogation was found to be highly stochastic and proceeded through insertion of an arginine-containing loop into the DNA minor groove to transiently disrupt Watson-Crick pairing. Next, we employed chain-of-replicas path-sampling methodologies to compute minimum free energy paths for TDG base extrusion. We identified the key intermediates imparting selectivity and determined effective free energy profiles for the lesion search and base extrusion into the TDG active site. Our results show that DNA sculpting, dynamic glycosylase interactions, and stabilizing contacts collectively provide a powerful mechanism for the detection and discrimination of modified bases and epigenetic marks in DNA.

Downstream metabolic events can contribute to the lethality of drugs or agents that interact with a primary cellular target. In bacteria, the production of reactive oxygen species (ROS) has been associated with the lethal effects of a variety of stresses including bactericidal antibiotics, but the relative contribution of this oxidative component to cell death depends on a variety of factors. Experimental evidence has suggested that unresolvable DNA problems caused by incorporation of oxidized nucleotides into nascent DNA followed by incomplete base excision repair contribute to the ROS-dependent component of antibiotic lethality. Expression of the chimeric periplasmic-cytoplasmic MalE-LacZ72-47 protein is an historically important lethal stress originally identified during seminal genetic experiments that defined the SecY-dependent protein translocation system. Multiple, independent lines of evidence presented here indicate that the predominant mechanism for MalE-LacZ lethality shares attributes with the ROS-dependent component of antibiotic lethality. MalE-LacZ lethality requires molecular oxygen, and its expression induces ROS production. The increased susceptibility of mutants sensitive to oxidative stress to MalE-LacZ lethality indicates that ROS contribute causally to cell death rather than simply being produced by dying cells. Observations that support the proposed mechanism of cell death include MalE-LacZ expression being bacteriostatic rather than bactericidal in cells that overexpress MutT, a nucleotide sanitizer that hydrolyzes 8-oxo-dGTP to the monophosphate, or that lack MutM and MutY, DNA glycosylases that process base pairs involving 8-oxo-dGTP. Our studies suggest stress-induced physiological changes that favor this mode of ROS-dependent death.

Global distribution of hepatocellular carcinomas (HCCs) is dominated by its incidence in developing countries, accounting for >700,000 estimated deaths per year, with dietary exposures to aflatoxin (AFB1) and subsequent DNA adduct formation being a significant driver. Genetic variants that increase individual susceptibility to AFB1-induced HCCs are poorly understood. Herein, it is shown that the DNA base excision repair (BER) enzyme, DNA glycosylase NEIL1, efficiently recognizes and excises the highly mutagenic imidazole ring-opened AFB1-deoxyguanosine adduct (AFB1-Fapy-dG). Consistent with this in vitro result, newborn mice injected with AFB1 show significant increases in the levels of AFB1-Fapy-dG in Neil1-/- vs. wild-type liver DNA. Further, Neil1-/- mice are highly susceptible to AFB1-induced HCCs relative to WT controls, with both the frequency and average size of hepatocellular carcinomas being elevated in Neil1-/- The magnitude of this effect in Neil1-/- mice is greater than that previously measured in Xeroderma pigmentosum complementation group A (XPA) mice that are deficient in nucleotide excision repair (NER). Given that several human polymorphic variants of NEIL1 are catalytically inactive for their DNA glycosylase activity, these deficiencies may increase susceptibility to AFB1-associated HCCs.

NEIL1 (Nei-like 1) is a DNA repair glycosylase guarding the mammalian genome against oxidized DNA bases. As the first enzymes in the base-excision repair pathway, glycosylases must recognize the cognate substrates and catalyze their excision. Here we present crystal structures of human NEIL1 bound to a range of duplex DNA. Together with computational and biochemical analyses, our results suggest that NEIL1 promotes tautomerization of thymine glycol (Tg)-a preferred substrate-for optimal binding in its active site. Moreover, this tautomerization event also facilitates NEIL1-catalyzed Tg excision. To our knowledge, the present example represents the first documented case of enzyme-promoted tautomerization for efficient substrate recognition and catalysis in an enzyme-catalyzed reaction.

On fertilization in Arabidopsis thaliana, one maternal gamete, the central cell, forms a placenta-like tissue, the endosperm. The DNA glycosylase DEMETER (DME) excises 5-methylcytosine via the base excision repair pathway in the central cell before fertilization, creating patterns of asymmetric DNA methylation and maternal gene expression across DNA replications in the endosperm lineage (EDL). Active DNA demethylation in the central cell is essential for transcriptional activity in the EDL of a set of genes, including FLOWERING WAGENINGEN (FWA). A DME-binding motif for iron-sulfur (Fe-S) cluster cofactors is indispensable for its catalytic activity. We used an FWA-GFP reporter to find mutants defective in maternal activation of FWA-GFP in the EDL, and isolated an allele of the yeast Dre2/human antiapoptotic factor CIAPIN1 homolog, encoding an enzyme previously implicated in the cytosolic Fe-S biogenesis pathway (CIA), which we named atdre2-2. We found that AtDRE2 acts in the central cell to regulate genes maternally activated in the EDL by DME. Furthermore, the FWA-GFP expression defect in atdre2-2 was partially suppressed genetically by a mutation in the maintenance DNA methyltransferase MET1; the DNA methylation levels at four DME targets increased in atdre2-2 seeds relative to WT. Although atdre2-2 shares zygotic seed defects with CIA mutants, it also uniquely manifests dme phenotypic hallmarks. These results demonstrate a previously unidentified epigenetic function of AtDRE2 that may be separate from the CIA pathway.

Somatic hypermutation (SHM) of antibody variable region genes is initiated in germinal center B cells during an immune response by activation-induced cytidine deaminase (AID), which converts cytosines to uracils. During accurate repair in nonmutating cells, uracil is excised by uracil DNA glycosylase (UNG), leaving abasic sites that are incised by AP endonuclease (APE) to create single-strand breaks, and the correct nucleotide is reinserted by DNA polymerase β. During SHM, for unknown reasons, repair is error prone. There are two APE homologs in mammals and, surprisingly, APE1, in contrast to its high expression in both resting and in vitro-activated splenic B cells, is expressed at very low levels in mouse germinal center B cells where SHM occurs, and APE1 haploinsufficiency has very little effect on SHM. In contrast, the less efficient homolog, APE2, is highly expressed and contributes not only to the frequency of mutations, but also to the generation of mutations at A:T base pair (bp), insertions, and deletions. In the absence of both UNG and APE2, mutations at A:T bp are dramatically reduced. Single-strand breaks generated by APE2 could provide entry points for exonuclease recruited by the mismatch repair proteins Msh2-Msh6, and the known association of APE2 with proliferating cell nuclear antigen could recruit translesion polymerases to create mutations at AID-induced lesions and also at A:T bp. Our data provide new insight into error-prone repair of AID-induced lesions, which we propose is facilitated by down-regulation of APE1 and up-regulation of APE2 expression in germinal center B cells.

DNA methylation is a conserved epigenetic mark in eukaryotes involved in many important biological processes, such as genome integrity, gene imprinting, and gene regulation (1). In genomes, DNA methylation marks can be added through the DNA methylation pathway and can be removed through the DNA demethylation pathway (1). The REPRESSOR OF SILENCING 1 (ROS1)/DEMETER (DME; DEMETER-like, DML) family of DNA demethylases were first reported in plants (2, 3) and now are known to be involved in DNA demethylation in all eukaryotes (1). The bifunctional glycosylase/lyase activities of the ROS1 family of enzymes could initiate DNA demethylation through removal of methylcytosine from the DNA backbone, leaving a single-nucleotide gap that is filled with an unmethylated cytosine through a base excision repair (BER) mechanism (1). In both plants and mammals, the active DNA demethylation pathway depends on the BER mechanism, although different enzymes initiate the pathway in the two systems (1). In recent years, several additional enzymes and regulators involved in the active DNA demethylation pathway have been identified and studied in plants (1). In contrast to the extensive mechanistic understanding of DNA demethylation pathways, however, the developmental function of DNA demethylation factors is largely unknown … [↵][1]1To whom correspondence may be addressed. Email: lang.zhaobo{at}foxmail.com or gongzz{at}cau.edu.cn. [1]: #xref-corresp-1-1

Base excision repair (BER) removes at least 20,000 DNA lesions per human cell per day and is critical for the maintenance of genomic stability. We hypothesize that aberrant BER, resulting from mutations in BER genes, can lead to genomic instability and cancer. The first step in BER is catalyzed by DNA N -glycosylases. One of these, n th endonuclease III-like (NTH1), removes oxidized pyrimidines from DNA, including thymine glycol. The rs3087468 single nucleotide polymorphism of the NTH1 gene is a G-to-T base substitution that results in the NTH1 D239Y variant protein that occurs in ∼6.2% of the global population and is found in Europeans, Asians, and sub-Saharan Africans. In this study, we functionally characterize the effect of the D239Y variant expressed in immortal but nontransformed human and mouse mammary epithelial cells. We demonstrate that expression of the D239Y variant in cells also expressing wild-type NTH1 leads to genomic instability and cellular transformation as assessed by anchorage-independent growth, focus formation, invasion, and chromosomal aberrations. We also show that cells expressing the D239Y variant are sensitive to ionizing radiation and hydrogen peroxide and accumulate double strand breaks after treatment with these agents. The DNA damage response is also activated in D239Y-expressing cells. In combination, our data suggest that individuals possessing the D239Y variant are at risk for genomic instability and cancer.

Recent findings suggest that neurons can efficiently repair oxidatively damaged DNA, and that both DNA damage and repair are enhanced by activation of excitatory glutamate receptors. However, in pathological conditions such as ischemic stroke, excessive DNA damage can trigger the death of neurons. Oxidative DNA damage is mainly repaired by base excision repair (BER), a process initiated by DNA glycosylases that recognize and remove damaged DNA bases. Endonuclease VIII-like 1 (NEIL1) is a DNA glycosylase that recognizes a broad range of oxidative lesions. Here, we show that mice lacking NEIL1 exhibit impaired memory retention in a water maze test, but no abnormalities in tests of motor performance, anxiety, or fear conditioning. NEIL1 deficiency results in increased brain damage and a defective functional outcome in a focal ischemia/reperfusion model of stroke. The incision capacity on a 5-hydroxyuracil–containing bubble substrate was lower in the ipsilateral side of ischemic brains and in the mitochondrial lysates of unstressed old NEIL1-deficient mice. These results indicate that NEIL1 plays an important role in learning and memory and in protection of neurons against ischemic injury.

DNA base excision repair is essential for maintaining genomic integrity and for active DNA demethylation, a central element of epigenetic regulation. A key player is thymine DNA glycosylase (TDG), which excises thymine from mutagenic G·T mispairs that arise by deamination of 5-methylcytosine (mC). TDG also removes 5-formylcytosine and 5-carboxylcytosine, oxidized forms of mC produced by Tet enzymes. Recent studies show that the glycosylase activity of TDG is essential for active DNA demethylation and for embryonic development. Our understanding of how repair enzymes excise modified bases without acting on undamaged DNA remains incomplete, particularly for mismatch glycosylases such as TDG. We solved a crystal structure of TDG (catalytic domain) bound to a substrate analog and characterized active-site residues by mutagenesis, kinetics, and molecular dynamics simulations. The studies reveal how TDG binds and positions the nucleophile (water) and uncover a previously unrecognized catalytic residue (Thr197). Remarkably, mutation of two active-site residues (Ala145 and His151) causes a dramatic enhancement in G·T glycosylase activity but confers even greater increases in the aberrant removal of thymine from normal A·T base pairs. The strict conservation of these residues may reflect a mechanism used to strike a tolerable balance between the requirement for efficient repair of G·T lesions and the need to minimize aberrant action on undamaged DNA, which can be mutagenic and cytotoxic. Such a compromise in G·T activity can account in part for the relatively weak G·T activity of TDG, a trait that could potentially contribute to the hypermutability of CpG sites in cancer and genetic disease.

The capacity of human poly(ADP-ribose) polymerase-1 (PARP-1) to interact with intact apurinic/apyrimidinic (AP) sites in DNA has been demonstrated. In cell extracts, sodium borohydride reduction of the PARP-1/AP site DNA complex resulted in covalent cross-linking of PARP-1 to DNA; the identity of cross-linked PARP-1 was confirmed by mass spectrometry. Using purified human PARP-1, the specificity of PARP-1 binding to AP site-containing DNA was confirmed in competition binding experiments. PARP-1 was only weakly activated to conduct poly(ADP-ribose) synthesis upon binding to AP site-containing DNA, but was strongly activated for poly(ADP-ribose) synthesis upon strand incision by AP endonuclease 1 (APE1). By virtue of its binding to AP sites, PARP-1 could be poised for its role in base excision repair, pending DNA strand incision by APE1 or the 5′-dRP/AP lyase activity in PARP-1.

Editing of the pre-mRNA for the DNA repair enzyme NEIL1 causes a lysine to arginine change in the lesion recognition loop of the protein. The two forms of NEIL1 are shown here to have distinct enzymatic properties. The edited form removes thymine glycol from duplex DNA 30 times more slowly than the form encoded in the genome, whereas editing enhances repair of the guanidinohydantoin lesion by NEIL1. In addition, we show that the NEIL1 recoding site is a preferred editing site for the RNA editing adenosine deaminase ADAR1. The edited adenosine resides in an A-C mismatch in a hairpin stem formed by pairing of exon 6 to the immediate upstream intron 5 sequence. As expected for an ADAR1 site, editing at this position is increased in human cells treated with interferon α. These results suggest a unique regulatory mechanism for DNA repair and extend our understanding of the impact of RNA editing.

Human tyrosyl-DNA phosphodiesterase (TDP1) hydrolyzes the phosphodiester bond at a DNA 3′-end linked to a tyrosyl moiety and has been implicated in the repair of topoisomerase I (Top1)-DNA covalent complexes. TDP1 can also hydrolyze other 3′-end DNA alterations including 3′-phosphoglycolate and 3′-abasic sites, and exhibits 3′-nucleosidase activity indicating it may function as a general 3′-end-processing DNA repair enzyme. Here, using laser confocal microscopy, subcellular fractionation and biochemical analyses we demonstrate that a fraction of the TDP1 encoded by the nuclear TDP1 gene localizes to mitochondria. We also show that mitochondrial base excision repair depends on TDP1 activity and provide evidence that TDP1 is required for efficient repair of oxidative damage in mitochondrial DNA. Together, our findings provide evidence for TDP1 as a novel mitochondrial enzyme.

Vision loss affects >3 million Americans and many more people worldwide. Although predisposing genes have been identified their link to known environmental factors is unclear. In wild-type animals DNA alkylating agents induce photoreceptor apoptosis and severe retinal degeneration. Alkylation-induced retinal degeneration is totally suppressed in the absence of the DNA repair protein alkyladenine DNA glycosylase (Aag) in both differentiating and postmitotic retinas. Moreover, transgenic expression of Aag activity restores the alkylation sensitivity of photoreceptors in Aag null animals. Aag heterozygotes display an intermediate level of retinal degeneration, demonstrating haploinsufficiency and underscoring that Aag expression confers a dominant retinal degeneration phenotype.

8-Oxoguanine (8OG) is efficiently bypassed by RNA polymerases in vitro and in bacterial cells in vivo , leading to mutant transcripts by directing incorporation of an incorrect nucleotide during transcription. Such transcriptional mutagenesis (TM) may produce a pool of mutant proteins. In contrast, transcription-coupled repair safeguards against DNA damage, contingent upon the ability of lesions to arrest elongating RNA polymerase. In mammalian cells, the Cockayne syndrome B protein (Csb) mediates transcription-coupled repair, and its involvement in the repair of 8OG is controversial. The DNA glycosylase Ogg1 initiates base excision repair of 8OG, but its influence on TM is unknown. We have developed a mammalian system for TM in congenic mouse embryonic fibroblasts (MEFs), either WT or deficient in Ogg1 (ogg −/− ), Csb (csb −/− ), or both. This system uses expression of the Ras oncogene in which an 8OG replaces guanine in codon 61. Repair of 8OG restores the WT sequence; however, bypass and misinsertion opposite this lesion during transcription leads to a constitutively active mutant Ras protein and activation of downstream signaling events, including increased phosphorylation of ERK kinase. Upon transfection of MEFs with replication-incompetent 8OG constructs, we observed a marked increase in phospho-ERK in ogg −/− and csb −/− ogg −/− cells at 6 h, indicating persistence of the lesion and the occurrence of TM. This effect is absent in WT and csb −/− cells, suggesting rapid repair. These studies provide evidence that 8OG causes TM in mammalian cells, leading to a phenotypic change with important implications for the role of TM in tumorigenesis.

Polycyclic aromatic hydrocarbons (PAHs) are tobacco carcinogens implicated in the causation of human lung cancer. Metabolic activation is a key prerequisite for PAHs to cause their deleterious effects. Using human lung adenocarcinoma (A549) cells, we provide evidence for the metabolic activation of (±)- trans -7,8dihydroxy-7,8-dihydrobenzo[ a ]pyrene (B[ a ]P-7,8- trans -dihydrodiol) by aldo-keto reductases (AKRs) to yield benzo[ a ]pyrene-7,8-dione (B[ a ]P-7,8-dione), a redox-active o -quinone. We show that B[ a ]P-7,8- trans -dihydrodiol (AKR substrate) and B[ a ]P-7,8-dione (AKR product) lead to the production of intracellular reactive oxygen species (ROS) (measured as an increase in dichlorofluorescin diacetate fluores-cence) and that similar changes were not observed with the regioisomer (±)- trans -4,5-dihydroxy-4,5-dihydrobenzo[ a ]pyrene or the diol-epoxide, (±)- anti -7,8-dihydroxy-9α,10β-epoxy-7,8,9,10-tetrahydro-B[ a ]P. B[ a ]P-7,8- trans -dihydrodiol and B[ a ]P-7,8-dione also caused a decrease in glutathione levels and an increase in NADP + /NADPH ratios, with a concomitant increase in single-strand breaks (as measured by the comet assay) and 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxo-dGuo). The specificity of the comet assay was validated by coupling it to human 8-oxo-guanine glycosylase (hOGG1), which excises 8-oxo-Gua to yield single-strand breaks. The levels of 8-oxo-dGuo observed were confirmed by an immunoaffinity purification stable isotope dilution ([ 15 N 5 ]-8-oxo-dGuo) liquid chromatography-electrospray ionization/multiple reaction monitoring/mass spectrometry (LC-ESI/MRM/MS) assay. B[ a ]P-7,8- trans -dihydrodiol produced DNA strand breaks in the hOGG1-coupled comet assay as well as 8-oxo-dGuo (as measured by LC-ESI/MRM/MS) and was enhanced by a catechol O -methyl transferase (COMT) inhibitor, suggesting that COMT protects against o -quinone-mediated redox cycling. We conclude that activation of PAH- trans -dihydrodiols by AKRs in lung cells leads to ROS-mediated genotoxicity and contributes to lung carcinogenesis.

The Vpr accessory protein of HIV-1 induces a response similar to that of DNA damage. In cells expressing Vpr, the DNA damage sensing kinase, ATR, is activated, resulting in G 2 arrest and apoptosis. In addition, Vpr causes rapid degradation of the uracil-DNA glycosylases UNG2 and SMUG1. Although several cellular proteins have been reported to bind to Vpr, the mechanism by which Vpr mediates its biological effects is unknown. Using tandem affinity purification and mass spectrometry, we identified a predominant cellular protein that binds to Vpr as the damage-specific DNA-binding protein 1 (DDB1). In addition to its role in the repair of damaged DNA, DDB1 is a component of an E3 ubiquitin ligase that degrades numerous cellular substrates. Interestingly, DDB1 is targeted by specific regulatory proteins of other viruses, including simian virus 5 and hepatitis B. We show that the interaction with DDB1 mediates Vpr-induced apoptosis and UNG2/SMUG1 degradation and impairs the repair of UV-damaged DNA, which could account for G 2 arrest and apoptosis. The interaction with DDB1 may explain several of the diverse biological functions of Vpr and suggests potential roles for Vpr in HIV-1 replication.

1, N 6 -ethanoadenine (EA) forms through the reaction of adenine in DNA with the antitumor agent 1,3-bis(2-chloroethyl)-1-nitrosourea, a chemotherapeutic used to combat various brain, head, and neck tumors. Previous studies of the toxic and mutagenic properties of the DNA adduct EA have been limited to in vitro experiments using mammalian polymerases and have revealed the lesion to be both miscoding and genotoxic. This work explores lesion bypass and mutagenicity of EA replicated in vivo and demonstrates that EA is neither toxic nor mutagenic in wild-type Escherichia coli . Although the base excision repair glycosylase enzymes of both humans and E. coli possess a weak ability to act on the lesion in vitro , an in vivo repair pathway has not yet been demonstrated. Here we show that an enzyme mechanistically unrelated to DNA glycosylases, the adaptive response protein AlkB, is capable of acting on EA via its canonical mechanism of oxidative dealkylation. The reaction alleviates the unrepaired adduct's potent toxicity through metabolism at the C8 position (attached to N1 of adenine), producing a nontoxic and weakly mutagenic N 6 adduct. AlkB is shown here to be a geno-protective agent that reduces the toxicity of DNA damage by converting the primary adduct to a less toxic secondary product.

We have determined the crystal structure of the human base excision repair enzyme DNA polymerase β (Pol β) in complex with a 1-nt gapped DNA substrate containing a template N 2 -guanine adduct of the tumorigenic (−)-benzo[ c ]phenanthrene 4 R ,3 S -diol 2 S ,1 R -epoxide in the gap. Nucleotide insertion opposite this adduct favors incorrect purine nucleotides over the correct dCMP and hence can be mutagenic. The structure reveals that the phenanthrene ring system is stacked with the base pair immediately 3′ to the modified guanine, thereby occluding the normal binding site for the correct incoming nucleoside triphosphate. The modified guanine base is displaced downstream and prevents the polymerase from achieving the catalytically competent closed conformation. The incoming nucleotide binding pocket is distorted, and the adducted deoxyguanosine is in a syn conformation, exposing its Hoogsteen edge, which can hydrogen-bond with dATP or dGTP. In a reconstituted base excision repair system, repair of a deaminated cytosine (i.e., uracil) opposite the adducted guanine was dramatically decreased at the Pol β insertion step, but not blocked. The efficiency of gap-filling dCMP insertion opposite the adduct was diminished by >6 orders of magnitude compared with an unadducted templating guanine. In contrast, significant misinsertion of purine nucleotides (but not dTMP) opposite the adducted guanine was observed. Pol β also misinserts a purine nucleotide opposite the adduct with ungapped DNA and exhibits limited bypass DNA synthesis. These results indicate that Pol β-dependent base excision repair of uracil opposite, or replication through, this bulky DNA adduct can be mutagenic.

DNA methylation is a stable epigenetic mark for transcriptional gene silencing in diverse organisms including plants and many animals. In contrast to the well characterized mechanism of DNA methylation by methyltransferases, the mechanisms and function of active DNA demethylation have been controversial. Genetic evidence suggested that the DNA glycosylase domain-containing protein ROS1 of Arabidopsis is a putative DNA demethylase, because loss-of-function ros1 mutations cause DNA hypermethylation and enhance transcriptional gene silencing. We report here the biochemical characterization of ROS1 and the effect of its overexpression on the DNA methylation of target genes. Our data suggest that the DNA glycosylase activity of ROS1 removes 5-methylcytosine from the DNA backbone and then its lyase activity cleaves the DNA backbone at the site of 5-methylcytosine removal by successive β- and δ-elimination reactions. Overexpression of ROS1 in transgenic plants led to a reduced level of cytosine methylation and increased expression of a target gene. These results demonstrate that ROS1 is a 5-methylcytosine DNA glycosylase/lyase important for active DNA demethylation in Arabidopsis .

Cytosine methylation is an epigenetic mark that promotes gene silencing and plays important roles in development and genome defense against transposons. Methylation patterns are established and maintained by DNA methyltransferases that catalyze transfer of a methyl group from S -adenosyl- l -methionine to cytosine bases in DNA. Erasure of cytosine methylation occurs during development, but the enzymatic basis of active demethylation remains controversial. In Arabidopsis thaliana , DEMETER ( DME ) activates the maternal expression of two imprinted genes silenced by methylation, and REPRESSOR OF SILENCING 1 ( ROS1 ) is required for release of transcriptional silencing of a hypermethylated transgene. DME and ROS1 encode two closely related DNA glycosylase domain proteins, but it is unknown whether they participate directly in a DNA demethylation process or counteract silencing through an indirect effect on chromatin structure. Here we show that DME and ROS1 catalyze the release of 5-methylcytosine (5-meC) from DNA by a glycosylase/lyase mechanism. Both enzymes also remove thymine, but not uracil, mismatched to guanine. DME and ROS1 show a preference for 5-meC over thymine in the symmetric dinucleotide CpG context, where most plant DNA methylation occurs. Nevertheless, they also have significant activity on both substrates at CpApG and asymmetric sequences, which are additional methylation targets in plant genomes. These findings suggest that a function of ROS1 and DME is to initiate erasure of 5-meC through a base excision repair process and provide strong biochemical evidence for the existence of an active DNA demethylation pathway in plants.

Endogenously formed reactive oxygen species continuously damage cellular constituents including DNA. These challenges, coupled with exogenous exposure to agents that generate reactive oxygen species, are both associated with normal aging processes and linked to cardiovascular disease, cancer, cataract formation, and fatty liver disease. Although not all of these diseases have been definitively shown to originate from mutations in nuclear DNA or mitochondrial DNA, repair of oxidized, saturated, and ring-fragmented bases via the base excision repair pathway is known to be critical for maintaining genomic stability. One enzyme that initiates base excision repair of ring-fragmented purines and some saturated pyrimidines is NEIL1, a mammalian homolog to Escherichia coli endonuclease VIII. To investigate the organismal consequences of a deficiency in NEIL1, a knockout mouse model was created. In the absence of exogenous oxidative stress, neil1 knockout ( neil1 −/− ) and heterozygotic ( neil1 +/− ) mice develop severe obesity, dyslipidemia, and fatty liver disease and also have a tendency to develop hyperinsulinemia. In humans, this combination of clinical manifestations, including hypertension, is known as the metabolic syndrome and is estimated to affect >40 million people in the United States. Additionally, mitochondrial DNA from neil1 −/− mice show increased levels of steady-state DNA damage and deletions relative to wild-type controls. These data suggest an important role for NEIL1 in the prevention of the diseases associated with the metabolic syndrome.

UVA (320-400 nm) radiation constitutes >90% of the environmentally relevant solar UV radiation, and it has been proposed to have a role in skin cancer and aging. Because of the popularity of UVA tanning beds and prolonged periods of sunbathing, the potential deleterious effect of UVA has emerged as a source of concern for public health. Although generally accepted, the impact of DNA damage on the cytotoxic, mutagenic, and carcinogenic effect of UVA radiation remains unclear. In the present study, we investigated the sensitivity of a panel of yeast mutants affected in the processing of DNA damage to the lethal and mutagenic effect of UVA radiation. The data show that none of the major DNA repair pathways, such as base excision repair, nucleotide excision repair, homologous recombination, and postreplication repair, efficiently protect yeast from the lethal action of UVA radiation. In contrast, the results show that the Ogg1 DNA glycosylase efficiently prevents UVA-induced mutagenesis, suggesting the formation of oxidized guanine residues. Furthermore, sequence analysis of UVA-induced canavanine-resistant mutations reveals a bias in favor of GC→TA events when compared with spontaneous or H 2 O 2 -, UVC-, and γ-ray- induced canavanine-resistant mutations in the WT strain. Taken together, our data point out a major role of oxidative DNA damage, mostly 7,8-dihydro-8-oxoguanine, in the genotoxicity of UVA radiation in the yeast Saccharomyces cerevisiae . Therefore, the capacity of skin cells to repair 7,8-dihydro-8-oxoguanine may be a key parameter in the mutagenic and carcinogenic effect of UVA radiation in humans.

Activation-induced cytidine deaminase (AID) is required for the DNA cleavage step of Ig somatic hypermutation (SHM). However, its molecular mechanism is controversial. The RNA editing hypothesis postulates that AID deaminates cytosine in an unknown mRNA to generate a new mRNA encoding SHM endonuclease. On the other hand, the DNA deamination hypothesis explains DNA cleavage by cytosine deamination in DNA, followed by uracil removal by uracil DNA glycosylase (UNG). By using the protein synthesis inhibitor cycloheximide, we showed that SHM requiresde novoprotein synthesis in accord with predictions by the RNA editing hypothesis. In addition, we found that cycloheximide but not Ugi (the specific inhibitor of UNG) inhibited AID-dependent DNA cleavage in the Ig gene during SHM, by using histone H2AX focus formation as a marker of DNA cleavage. The results indicate the following order of events: AID expression, protein synthesis, DNA cleavage, and SHM. The requirement of protein synthesis but not of UNG for the DNA cleavage step of SHM forces us to reconsider the DNA deamination hypothesis and strengthens the RNA editing hypothesis.

Mutagenesis of protein-encoding sequences occurs ubiquitously; it enables evolution, accumulates during aging, and is associated with disease. Many biotechnological methods exploit random mutations to evolve novel proteins. To quantitate protein tolerance to random change, it is vital to understand the probability that a random amino acid replacement will lead to a protein's functional inactivation. We define this probability as the “ x factor.” Here, we develop a broadly applicable approach to calculate x factors and demonstrate this method using the human DNA repair enzyme 3-methyladenine DNA glycosylase (AAG). Three gene-wide mutagenesis libraries were created, each with 10 5 diversity and averaging 2.2, 4.6, and 6.2 random amino acid changes per mutant. After determining the percentage of functional mutants in each library using high-stringency selection (>19,000-fold), the x factor was found to be 34% ± 6%. Remarkably, reanalysis of data from studies of diverse proteins reveals similar inactivation probabilities. To delineate the nature of tolerated amino acid substitutions, we sequenced 244 surviving AAG mutants. The 920 tolerated substitutions were characterized by substitutability index and mapped onto the AAG primary, secondary, and known tertiary structures. Evolutionarily conserved residues show low substitutability indices. In AAG, β strands are on average less substitutable than α helices; and surface loops that are not involved in DNA binding are the most substitutable. Our results are relevant to such diverse topics as applied molecular evolution, the rate of introduction of deleterious alleles into genomes in evolutionary history, and organisms' tolerance of mutational burden.

Escherichia coli nucleoside diphosphate kinase (Ndk) catalyzes ATP-dependent synthesis of ribo- and deoxyribonucleoside triphosphates from the cognate diphosphate precursor. Recently, the Ndk polypeptide was reported to be a multifunctional base excision repair nuclease that processed uracil residues in DNA by acting sequentially as a uracil-DNA glycosylase inhibitor protein (Ugi)-sensitive uracil-DNA glycosylase, an apurinic/apyrimidiniclyase, and a 3′-phosphodiesterase [Postel, E. H. & Abramczyk, B. M. (2003) Proc. Natl. Acad. Sci. USA 100, 13247–13252]. Here we demonstrate that the E. coli Ndk polypeptide lacked detectable uracil-DNA glycosylase activity and, hence, was incapable of acting as a uracil-processing DNA repair nuclease. This finding was based on the following observations: ( i ) uracil-DNA glycosylase activity did not copurify with Ndk activity; ( ii ) Ndk purified from E. coli ung - cells showed no detectable uracil-DNA glycosylase activity; and ( iii ) Ndk failed to bind to a Ugi-Sepharose affinity column that tightly bound E. coli uracil-DNA glycosylase (Ung). Collectively, these observations demonstrate that the E. coli Ndk polypeptide does not possess inherent uracil-DNA glycosylase activity.

Escherichia coli nucleoside diphosphate kinase (eNDK) is an XTP:XDP phosphotransferase that plays an important role in the regulation of cellular nucleoside triphosphate concentrations. It is also one of several recently discovered DNases belonging to the NM23/NDK family. E. coli cells disrupted in the ndk gene display a spontaneous mutator phenotype, which has been attributed to the mutagenic effects of imbalanced nucleotide pools and errors made by replicative DNA polymerases. Another explanation for the increased mutation rates is that e ndk - cells lack the nuclease activity of the NDK protein that is essential for a DNA repair pathway. Here, we show that purified, cloned endk is a DNA repair nuclease whose substrate is uracil misincorporated into DNA. We have identified three new catalytic activities in eNDK that act sequentially to repair the uracil lesion: ( i ) uracil-DNA glycosylase that excises uracil from single-stranded and from U/A and U/G mispairs in double-stranded DNA; ( ii ) apyrimidinic endonuclease that cleaves double-stranded DNA as a lyase by forming a covalent enzyme-DNA intermediate complex with the apyrimidinic site created by the glycosylase; and ( iii ) DNA repair phosphodiesterase that removes 3′-blocking residues from the ends of duplex DNA. All three of these activities, as well as the nucleoside-diphosphate kinase, reside in the same protein. Based on these findings, we propose an editing function for eNDK as a mechanism by which the enzyme prevents mutations in DNA.

The methyl–CpG dinucleotide containing a symmetrical 5-methylcytosine (mC) is involved in gene regulation and genome stability. We report here that methylation-mediated transcriptional repressor methylated DNA-binding domain 1 (MBD1) interacts with methylpurine–DNA glycosylase (MPG), which excises damaged bases from substrate DNA. MPG itself actively represses transcription and has a synergistic effect on gene silencing together with MBD1. Chromatin immunoprecipitation analysis reveals the molecular movement of MBD1 and MPG in vivo : ( i ) The MBD1–MPG complex normally exists on the methylated gene promoter; ( ii ) treatment of cells with alkylating agent methylmethanesulfonate (MMS) induces the dissociation of MBD1 from the methylated promoter, and MPG is located on both methylated and unmethylated promoters; and ( iii ) after completion of the repair, the MBD1–MPG complex is restored on the methylated promoter. Mobility-shift and structural analyses show that the MBD of MBD1 binds a methyl–CpG pair (mCpG × mCpG) but not the methyl–CpG pair containing a single 7-methylguanine (N) (mCpG × mCpN) that is known as one of the major lesions caused by MMS. We further demonstrate that knockdown of MBD1 by specific small interfering RNAs significantly increases cell sensitivity to MMS. These data suggest that MBD1 cooperates with MPG for transcriptional repression and DNA repair. We hypothesize that MBD1 functions as a reservoir for MPG and senses the base damage in chromatin.

The long-term effect of exposure to DNA alkylating agents is entwined with the cell's genetic capacity for DNA repair and appropriate DNA damage responses. A unique combination of environmental exposure and deficiency in these responses can lead to genomic instability; this “gene–environment interaction” paradigm is a theme for research on chronic disease etiology. In the present study, we used mouse embryonic fibroblasts with a gene deletion in the base excision repair (BER) enzymes DNA β-polymerase (β-pol) and alkyladenine DNA glycosylase (AAG), along with exposure to methyl methanesulfonate (MMS) to study mutagenesis as a function of a particular gene–environment interaction. The β-pol null cells, defective in BER, exhibit a modest increase in spontaneous mutagenesis compared with wild-type cells. MMS exposure increases mutant frequency in β-pol null cells, but not in isogenic wild-type cells; UV light exposure or N -methyl- N ′-nitro- N -nitrosoguanidine exposure increases mutant frequency similarly in both cell lines. The MMS-induced increase in mutant frequency in β-pol null cells appears to be caused by DNA lesions that are AAG substrates, because overexpression of AAG in β-pol null cells eliminates the effect. In contrast, β-pol/AAG double null cells are slightly more mutable than the β-pol null cells after MMS exposure. These results illustrate that BER plays a role in protecting mouse embryonic fibroblast cells against methylation-induced mutations and characterize the effect of a particular combination of BER gene defect and environmental exposure.

DNA–protein crosslinks (DPCs) arise in biological systems as a result of exposure to a variety of chemical and physical agents, many of which are known or suspected carcinogens. The biochemical pathways for the recognition and repair of these lesions are not well understood in part because of methodological difficulties in creating site-specific DPCs. Here, a strategy for obtaining site-specific DPCs is presented, and in vitro interactions of the Escherichia coli nucleotide excision repair (NER) UvrABC nuclease at sites of DPCs are investigated. To create site-specific DPCs, the catalytic chemistry of the T4 pyrimidine dimer glycosylase/apurinic/apyrimidinic site lyase (T4-pdg) has been exploited, namely, its ability to be covalently trapped to apurinic/apyrimidinic sites within duplex DNA under reducing conditions. Incubation of the DPCs with UvrABC proteins resulted in DNA incision at the 8th phosphate 5′ and the 5th and 6th phosphates 3′ to the protein-adducted site, generating as a major product of the reaction a 12-mer DNA fragment crosslinked with the protein. The incision occurred only in the presence of all three protein subunits, and no incisions were observed in the nondamaged complementary strand. The UvrABC nuclease incises DPCs with a moderate efficiency. The proper assembly and catalytic function of the NER complex on DNA containing a covalently attached 16-kDa protein suggest that the NER pathway may be involved in DPC repair and that at least some subset of DPCs can be removed by this mechanism without prior proteolytic degradation.

We have shown that the DNA demethylation complex isolated from chicken embryos has a G⋅T mismatch DNA glycosylase that also possesses 5-methylcytosine DNA glycosylase (5-MCDG) activity. Herein we show that human embryonic kidney cells stably transfected with 5-MCDG cDNA linked to a cytomegalovirus promoter overexpress 5-MCDG. A 15- to 20-fold overexpression of 5-MCDG results in the specific demethylation of a stably integrated ecdysone-retinoic acid responsive enhancer-promoter linked to a β-galactosidase reporter gene. Demethylation occurs in the absence of the ligand ponasterone A (an analogue of ecdysone). The state of methylation of the transgene was investigated by Southern blot analysis and by the bisulfite genomic sequencing reaction. Demethylation occurs downstream of the hormone response elements. No genome-wide demethylation was observed. The expression of an inactive mutant of 5-MCDG or the empty vector does not elicit any demethylation of the promoter-enhancer of the reporter gene. An increase in 5-MCDG activity does not influence the activity of DNA methyltransferase(s) when tested in vitro with a hemimethylated substrate. There is no change in the transgene copy number during selection of the clones with antibiotics. Immunoprecipitation combined with Western blot analysis showed that an antibody directed against 5-MCDG precipitates a complex containing the retinoid X receptor α. The association between retinoid receptor and 5-MCDG is not ligand dependent. These results suggest that a complex of the hormone receptor with 5-MCDG may target demethylation of the transgene in this system.

The oxidation of thymine in DNA can generate a base pair between 5-hydroxymethyluracil (HmU) and adenine, whereas the oxidation and deamination of 5-methylcytosine (5mC) in DNA can generate a base pair between HmU and guanine. Using synthetic oligonucleotides containing HmU at a defined site, HmU-DNA glycosylase activities in HeLa cell and human fibroblast cell extracts have been observed. An HmU-DNA glycosylase activity that removes HmU mispaired with guanine has been measured. Surprisingly, the HmU:G excision activity is 60 times greater than the corresponding HmU:A activity, even though the expected rate of formation of the HmU:A base pair exceeds that of the HmU:G base pair by a factor of 10 7 . The HmU:G mispair would arise from the 5mC:G base pair, and, if unrepaired, would give rise to a transition mutation. The observation of an unexpectedly high HmU:G glycosylase activity suggests that human cells may encounter the HmU:G mispair much more frequently than expected. The conversion of 5mC to HmU must be considered as a potential pathway for the generation of 5mC to T transition mutations, which are often found in human tumors.

The human 3-methyladenine DNA glycosylase [alkyladenine DNA glycosylase (AAG)] catalyzes the first step of base excision repair by cleaving damaged bases from DNA. Unlike other DNA glycosylases that are specific for a particular type of damaged base, AAG excises a chemically diverse selection of substrate bases damaged by alkylation or deamination. The 2.1-Å crystal structure of AAG complexed to DNA containing 1, N 6 -ethenoadenine suggests how modified bases can be distinguished from normal DNA bases in the enzyme active site. Mutational analyses of residues contacting the alkylated base in the crystal structures suggest that the shape of the damaged base, its hydrogen-bonding characteristics, and its aromaticity all contribute to the selective recognition of damage by AAG.

To assess the role of the Ogg1 DNA glycosylase in the transcription-coupled repair (TCR) of the mutagenic lesion, 7,8-dihydro-8oxoguanine (8-OxoG), we have investigated the removal of this lesion in wild-type and ogg1 −/− null mouse embryo fibroblast (MEF) cell lines. We used nonreplicating plasmids containing a single 8-OxoG·C base pair in a different assay that allowed us to study the removal of 8-OxoG located in a transcribed sequence (TS) or in a nontranscribed sequence (NTS). The results show that the removal of 8-OxoG in a wild-type MEF cell line is faster in the TS than in the NTS, indicating TCR of 8-OxoG in murine cells. In the homozygous ogg1 −/− MEF cell line, 8-OxoG was not removed from the NTS whereas there was still efficient 8-OxoG repair in the TS. Expression of the mouse Ogg1 protein in the homozygous ogg1 −/− cell line restored the ability to remove 8-OxoG in the NTS. Therefore, we have demonstrated that Ogg1 is essential for the repair of 8-OxoG in the NTS but is not required in the TS. These results indicate the existence of an Ogg1-independent pathway for the TCR of 8-OxoG in vivo .

In vitro evolution was used to develop a DNA enzyme that catalyzes the site-specific depurination of DNA with a catalytic rate enhancement of about 10 6 -fold. The reaction involves hydrolysis of the N -glycosidic bond of a particular deoxyguanosine residue, leading to DNA strand scission at the apurinic site. The DNA enzyme contains 93 nucleotides and is structurally complex. It has an absolute requirement for a divalent metal cation and exhibits optimal activity at about pH 5. The mechanism of the reaction was confirmed by analysis of the cleavage products by using HPLC and mass spectrometry. The isolation and characterization of an N -glycosylase DNA enzyme demonstrates that single-stranded DNA, like RNA and proteins, can form a complex tertiary structure and catalyze a difficult biochemical transformation. This DNA enzyme provides a new approach for the site-specific cleavage of DNA molecules.

We previously have shown that DNA demethylation by chicken embryo 5-methylcytosine DNA glycosylase (5-MCDG) needs both RNA and proteins. One of these proteins is a RNA helicase. Further peptides were sequenced, and three of them are identical to the mammalian G/T mismatch DNA glycosylase. A 3,233-bp cDNA coding for the chicken homologue of human G/T mismatch DNA glycosylase was isolated and sequenced. The derived amino acid sequence (408 aa) shows 80% identity with the human G/T mismatch DNA glycosylase, and both the C and N-terminal parts have about 50% identity. As for the highly purified chicken embryo DNA demethylation complex the recombinant protein expressed in Escherichia coli has both G/T mismatch and 5-MCDG activities. The recombinant protein has the same substrate specificity as the chicken embryo 5-MCDG where hemimethylated DNA is a better substrate than symmetrically methylated CpGs. The activity ratio of G/T mismatch and 5-MCDG is about 30:1 for the recombinant protein expressed in E. coli and 3:1 for the purified enzyme from chicken embryos. The incubation of a recombinant CpG-rich RNA isolated from the purified DNA demethylation complex with the recombinant enzyme strongly inhibits G/T mismatch glycosylase while slightly stimulating the activity of 5-MCDG. Deletion mutations indicate that G/T mismatch and 5-MCDG activities share the same areas of the N- and C-terminal parts of the protein. In reconstitution experiments RNA helicase in the presence of recombinant RNA and ATP potentiates the activity of 5-MCDG.

Deamination of cytosine to uracil is the most common promutagenic change in DNA, and it is greatly increased at the elevated growth temperatures of hyperthermophilic archaea. If not repaired to cytosine prior to replication, uracil in a template strand directs incorporation of adenine, generating a G⋅C → A⋅U transition mutation in half the progeny. Surprisingly, genomic analysis of archaea has so far failed to reveal any homologues of either of the known families of uracil-DNA glycosylases responsible for initiating the base-excision repair of uracil in DNA, which is otherwise universal. Here we show that DNA polymerases from several hyperthermophilic archaea (including Vent and Pfu ) specifically recognize the presence of uracil in a template strand and stall DNA synthesis before mutagenic misincorporation of adenine. A specific template-checking function in a DNA polymerase has not been observed previously, and it may represent the first step in a pathway for the repair of cytosine deamination in archaea.

Increased spontaneous mutation is associated with increased cancer risk. Here, by using a model system, we show that spontaneous mutation can be increased several hundred-fold by a simple imbalance between the first two enzymes involved in DNA base excision repair. The Saccharomyces cerevisiae MAG1 3-methyladenine (3MeA) DNA glycosylase, when expressed at high levels relative to the apurinic/apyrimidinic endonuclease, increases spontaneous mutation by up to ≈600-fold in S. cerevisiae and ≈200-fold in Escherichia coli . Genetic evidence suggests that, in yeast, the increased spontaneous mutation requires the generation of abasic sites and the processing of these sites by the REV1 / REV3 / REV7 lesion bypass pathway. Comparison of the mutator activity produced by Mag1, which has a broad substrate range, with that produced by the E. coli Tag 3MeA DNA glycosylase, which has a narrow substrate range, indicates that the removal of endogenously produced 3MeA is unlikely to be responsible for the mutator effect of Mag1. Finally, the human AAG 3-MeA DNA glycosylase also can produce a small (≈2-fold) but statistically significant increase in spontaneous mutation, a result which could have important implications for carcinogenesis.

Exocyclic DNA adducts are generated in cellular DNA by various industrial pollutants such as the carcinogen vinyl chloride and by endogenous products of lipid peroxidation. The etheno derivatives of purine and pyrimidine bases 3, N 4 -ethenocytosine (ɛC), 1, N 6 -ethenoadenine (ɛA), N 2 ,3-ethenoguanine, and 1, N 2 -ethenoguanine cause mutations. The ɛA residues are excised by the human and the Escherichia coli 3-methyladenine-DNA glycosylases (ANPG and AlkA proteins, respectively), but the enzymes repairing ɛC residues have not yet been described. We have identified two homologous proteins present in human cells and E. coli that remove ɛC residues by a DNA glycosylase activity. The human enzyme is an activity of the mismatch-specific thymine-DNA glycosylase (hTDG). The bacterial enzyme is the double-stranded uracil-DNA glycosylase (dsUDG) that is the homologue of the hTDG. In addition to uracil and ɛC-DNA glycosylase activity, the dsUDG protein repairs thymine in a G/T mismatch. The fact that ɛC is recognized and efficiently excised by the E. coli dsUDG and hTDG proteins in vitro suggests that these enzymes may be responsible for the repair of this mutagenic lesion in vivo and be important contributors to genetic stability.

Apurinic/apyrimidinic (AP) endonuclease (APE; EC 4.2.99.18 ) plays a central role in repair of DNA damage due to reactive oxygen species (ROS) because its DNA 3′-phosphoesterase activity removes 3′ blocking groups in DNA that are generated by DNA glycosylase/AP-lyases during removal of oxidized bases and by direct ROS reaction with DNA. The major human APE (APE-1) gene is activated selectively by sublethal levels of a variety of ROS and ROS generators, including ionizing radiation, but not by other genotoxicants—e.g., UV light and alkylating agents. Increased expression of APE mRNA and protein was observed both in the HeLa S3 tumor line and in WI 38 primary fibroblasts, and it was accompanied by translocation of the endonuclease to the nucleus. ROS-treated cells showed a significant increase in resistance to the cytotoxicity of such ROS generators as H 2 O 2 and bleomycin, but not to UV light. This “adaptive response” appears to result from enhanced repair of cytotoxic DNA lesions due to an increased activity of APE-1, which may be limiting in the base excision repair process for ROS-induced toxic lesions.

It has previously been reported that 1, N 6 -ethenoadenine (ɛA), deaminated adenine (hypoxanthine, Hx), and 7,8-dihydro-8-oxoguanine (8-oxoG), but not 3, N 4 -ethenocytosine (ɛC), are released from DNA in vitro by the DNA repair enzyme alkylpurine-DNA-N-glycosylase (APNG). To assess the potential contribution of APNG to the repair of each of these mutagenic lesions in vivo , we have used cell-free extracts of tissues from APNG-null mutant mice and wild-type controls. The ability of these extracts to cleave defined oligomers containing a single modified base was determined. The results showed that both testes and liver cells of these knockout mice completely lacked activity toward oligonucleotides containing ɛA and Hx, but retained wild-type levels of activity for ɛC and 8-oxoG. These findings indicate that ( i ) the previously identified ɛA-DNA glycosylase and Hx-DNA glycosylase activities are functions of APNG; ( ii ) the two structurally closely related mutagenic adducts ɛA and ɛC are repaired by separate gene products; and ( iii ) APNG does not contribute detectably to the repair of 8-oxoG.

The major mutagenic base lesion in DNA caused by exposure to reactive oxygen species is 8-hydroxyguanine (8-oxo-7,8-dihydroguanine). In bacteria and Saccharomyces cerevisiae , this damaged base is excised by a DNA glycosylase with an associated lyase activity for chain cleavage. We have cloned, sequenced, and expressed a human cDNA with partial sequence homology to the relevant yeast gene. The encoded 47-kDa human enzyme releases free 8-hydroxyguanine from oxidized DNA and introduces a chain break in a double-stranded oligonucleotide specifically at an 8-hydroxyguanine residue base paired with cytosine. Expression of the human protein in a DNA repair-deficient E. coli mutM mutY strain partly suppresses its spontaneous mutator phenotype. The gene encoding the human enzyme maps to chromosome 3p25. These results show that human cells have an enzyme that can initiate base excision repair at mutagenic DNA lesions caused by active oxygen.

Oxidative DNA damage is generated by reactive oxygen species. The mutagenic base, 8-oxoguanine, formed by this process, is removed from oxidatively damaged DNA by base excision repair. Genes coding for DNA repair enzymes that recognize 8-oxoguanine have been reported in bacteria and yeast. We have identified and characterized mouse and human cDNAs encoding homologs of the 8-oxoguanine DNA glycosylase ( ogg1 ) gene of Saccharomyces cerevisiae. Escherichia coli doubly mutant for mutM and mutY have a mutator phenotype and are deficient in 8-oxoguanine repair. The recombinant mouse gene (mOgg1) suppresses the mutator phenotype of mutY/mutM E. coli . Extracts prepared from mutY/mutM E. coli expressing mOgg1 contain an activity that excises 8-oxoguanine from DNA and a β-lyase activity that nicks DNA 3′ to the lesion. The mouse ogg1 gene product acts efficiently on DNA duplexes in which 7,8-dihydroxy-8-oxo-2′-deoxyguanosine (8-oxodG) is paired with dC, acts weakly on duplexes in which 8-oxodG is paired with dT or dG, and is inactive against duplexes in which 8-oxodG is paired with dA. Mouse and human ogg1 genes contain a helix–hairpin–helix structural motif with conserved residues characteristic of a recently defined family of DNA glycosylases. Ogg1 mRNA is expressed in several mouse tissues; highest levels were detected in testes. Isolation of the mouse ogg1 gene makes it possible to modulate its expression in mice and to explore the involvement of oxidative DNA damage and associated repair processes in aging and cancer.

Mammalian nucleotide excision repair (NER) eliminates carcinogen–DNA adducts by double endonucleolytic cleavage and subsequent release of 24–32 nucleotide-long single-stranded fragments. Here we manipulated the deoxyribose–phosphate backbone of DNA to analyze the mechanism by which damaged strands are discriminated as substrates for dual incision. We found that human NER is completely inactive on DNA duplexes containing single C4′-modified backbone residues. However, the same C4′ backbone variants, which by themselves do not perturb complementary hydrogen bonds, induced strong NER reactions when incorporated into short segments of mispaired bases. No oligonucleotide excision was detected when DNA contained abnormal base pairs without concomitant changes in deoxyribose–phosphate composition. Thus, neither C4′ backbone lesions nor improper base pairing stimulated human NER, but the combination of these two substrate alterations constituted an extremely potent signal for double DNA incision. In summary, we used C4′-modified backbone residues as molecular tools to dissect DNA damage recognition by human NER into separate components and identified a bipartite discrimination mechanism that requires changes in DNA chemistry with concurrent disruption of Watson–Crick base pairing.

The gene of Micrococcus luteus UV endonuclease (cyclobutane pyrimidine dimer–DNA glycosylase/abasic lyase) was cloned and characterized. The cloned gene, whose product had a predicted molecular mass of 17,120 Da, was found to be capable of complementing the Escherichia coli uvrA6 mutation in vivo with respect to resistance to acetone-mediated molecular photosensitization, a treatment producing exclusively cyclobutane pyrimidine dimers in DNA. It also generated a nicking activity specific for photosensitization-treated DNA by in vitro transcription/translation. When expressed in E. coli cells, the gene produced a protein structurally identical with UV endonuclease and possessing an activity consistent with cyclobutane pyrimidine dimer–DNA glycosylase/abasic lyase with respect to the effect of inhibitors and the site of the DNA backbone scission. Furthermore, the UV endonuclease-deficient mutant DB7 was shown to regain the enzyme through transformation with the cloned gene. The deduced amino acid sequence of the gene product was at best 27% identical with that of endonuclease V of phage T4, an enzyme strikingly similar to UV endonuclease in molecular and catalytic properties. Despite this marginal overall similarity in amino acid sequence, four of the seven amino acid residues reported to be functionally important in the T4 enzyme were found to be conserved in the M. luteus enzyme. We propose that the gene be called uveA .

Spontaneous hydrolytic deamination of cytosine to uracil (U) in DNA is a constant source of genome instability in cells. This mutagenic process is greatly enhanced at high temperatures and in single-stranded DNA. If not repaired, these uracil residues give rise to C → T transitions, which are the most common spontaneous mutations occurring in living organisms and are frequently found in human tumors. In the majority of species, uracil residues are removed from DNA by specific uracil-DNA glycosylases in the base excision repair pathway. Alternatively, in certain archaeal organisms, uracil residues are eliminated by apurinic/apyrimidinic (AP) endonucleases in the nucleotide incision repair pathway. Here, we characterized the substrate specificity of the major human AP endonuclease 1, APE1, toward U in duplex DNA. APE1 cleaves oligonucleotide duplexes containing a single U⋅G base pair; this activity depends strongly on the sequence context and the base opposite to U. The apparent kinetic parameters of the reactions show that APE1 has high affinity for DNA containing U but cleaves the DNA duplex at an extremely low rate. MALDI-TOF MS analysis of the reaction products demonstrated that APE1-catalyzed cleavage of a U⋅G duplex generates the expected DNA fragments containing a 5'-terminal deoxyuridine monophosphate. The fact that U in duplex DNA is recognized and cleaved by APE1 in vitro suggests that this property of the exonuclease III family of AP endonucleases is remarkably conserved from Archaea to humans. We propose that nucleotide incision repair may act as a backup pathway to base excision repair to remove uracils arising from cytosine deamination.

The major human apurinic/apyrimidinic (AP) endonuclease (class II) is known to cleave DNA 5′ adjacent to an AP site, which is probably the most common DNA damage produced hydrolytically or by glycosylase-mediated removal of modified bases. p -Benzoquinone (pBQ), one of the major benzene metabolites, reacts with DNA to form bulky exocyclic adducts. Herein we report that the human AP endonuclease directly catalyzes incision in a defined oligonucleotide containing 3, N 4 -benzetheno-2′-deoxycytidine (pBQ-dC) without prior generation of an AP site. The enzyme incises the oligonucleotide 5′ to the adduct and generates 3′-hydroxyl and 5′-phosphoryl termini but leaves the pBQ-dC on the 5′ terminus of the cleavage fragment. The AP function of the enzyme is not involved in this action, as no preexisting AP site is present nor is a DNA glycosylase activity involved. Nicking of the pBQ-dC adduct also leads to the same “dangling base” cleavage when two Escherichia coli enzymes, exonuclease III and endonuclease IV, are used. Our finding of this unusual mode of action used by both human and bacterial AP endonucleases raises important questions regarding the requirements for substrate recognition and catalytic active site(s) for this essential cellular repair enzyme. We believe this to be the first instance of the presence of a bulky carcinogen adduct leading to this unusual mode of action.

A spontaneous mutator strain of Escherichia coli (fpg mutY) was used to clone the OGG1 gene of Saccharomyces cerevisiae, which encodes a DNA glycosylase activity that excises 7,8-dihydro-8-oxoguanine (8-OxoG). E. coli (fpg mutY) was transformed by a yeast DNA library, and clones that showed a reduced spontaneous mutagenesis were selected. The antimutator activity was associated with pYSB10, an 11-kbp recombinant plasmid. Cell-free extracts of E. coli (fpg mutY) harboring pYSB10 possess an enzymatic activity that cleaves a 34-mer oligonucleotide containing a single 8-oxoG opposite a cytosine (8-OxoG/C). The yeast DNA fragment of 1.7 kbp that suppresses spontaneous mutagenesis and overproduces the 8-OxoG/C cleavage activity was sequenced and mapped to chromosome XIII. DNA sequencing identified an open reading frame, designated OGG1, which encodes a protein of 376 amino acids with a molecular mass of 43 kDa. The OGG1 gene was inserted in plasmid pUC19, yielding pYSB110. E. coli (fpg) harboring pYSB110 was used to purify the Ogg1 protein of S. cerevisiae to apparent homogeneity. The Ogg1 protein possesses a DNA glycosylase activity that releases 8-OxoG and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine. The Ogg1 protein preferentially incises DNA that contains 8-OxoG opposite cytosine (8-OxoG/C) or thymine (8-OxoG/T). In contrast, Ogg1 protein does not incise the duplex where an adenine is placed opposite 8-OxoG (8-OxoG/A). The mechanism of strand cleavage by Ogg1 protein is probably due to the excision of 8-OxoG followed by a beta-elimination at the resulting apurinic/apyrimidinic site.