Defective Repair of Uracil Causes Telomere Defects in Mouse Hematopoietic Cells

Uracil in the genome can result from misincorporation of dUTP instead of dTTP during DNA synthesis, and is primarily removed by uracil DNA glycosylase (UNG) during base excision repair. Telomeres contain long arrays of TTAGGG repeats and may be susceptible to uracil misincorporation. Using model telomeric DNA substrates, we showed that the position and number of uracil substitutions of thymine in telomeric DNA decreased recognition by the telomere single-strand binding protein, POT1. In primary mouse hematopoietic cells, uracil was detectable at telomeres, and UNG deficiency further increased uracil loads and led to abnormal telomere lengthening. In UNG-deficient cells, the frequencies of sister chromatid exchange and fragility in telomeres also significantly increased in the absence of telomerase. Thus, accumulation of uracil and/or UNG deficiency interferes with telomere maintenance, thereby underscoring the necessity of UNG-initiated base excision repair for the preservation of telomere integrity.     

Structural Basis for Avoidance of Promutagenic DNA Repair by MutY Adenine DNA Glycosylase

The highly mutagenic A:oxoG (8-oxoguanine) base pair in DNA most frequently arises by aberrant replication of the primary oxidative lesion C:oxoG. This lesion is particularly insidious because neither of its constituent nucleobases faithfully transmit genetic information from the original C:G base pair. Repair of A:oxoG is initiated by adenine DNA glycosylase, which catalyzes hydrolytic cleavage of the aberrant A nucleobase from the DNA backbone. These enzymes, MutY in bacteria and MUTYH in humans, scrupulously avoid processing of C:oxoG because cleavage of the C residue in C:oxoG would actually promote mutagenic conversion to A:oxoG. Here we analyze the structural basis for rejection of C:oxoG by MutY, using a synthetic crystallography approach to capture the enzyme in the process of inspecting the C:oxoG anti-substrate, with which it ordinarily binds only fleetingly. We find that MutY uses two distinct strategies to avoid presentation of C to the enzyme active site. Firstly, MutY possesses an exo-site that serves as a decoy for C, and secondly, repulsive forces with a key active site residue prevent stable insertion of C into the nucleobase recognition pocket within the enzyme active site.     

Coordinated Interactions of Multiple POT1-TPP1 Proteins with Telomere DNA

Telomeres are macromolecular nucleoprotein complexes that protect the ends of eukaryotic chromosomes from degradation, end-to-end fusion events, and from engaging the DNA damage response. However, the assembly of this essential DNA-protein complex is poorly understood. Telomere DNA consists of the repeated double-stranded sequence 5′-TTAGGG-3′ in vertebrates, followed by a single-stranded DNA overhang with the same sequence. Both double- and single-stranded regions are coated with high specificity by telomere end-binding proteins, including POT1 and TPP1, that bind as a heterodimer to single-stranded telomeric DNA. Multiple POT1-TPP1 proteins must fully coat the single-stranded telomere DNA to form a functional telomere. To better understand the mechanism of multiple binding, we mutated or deleted the two guanosine nucleotides residing between adjacent POT1-TPP1 recognition sites in single-stranded telomere DNA that are not required for multiple POT1-TPP1 binding events. Circular dichroism demonstrated that spectra from the native telomere sequence are characteristic of a G-quadruplex secondary structure, whereas the altered telomere sequences were devoid of these signatures. The altered telomere strands, however, facilitated more cooperative loading of multiple POT1-TPP1 proteins compared with the wild-type telomere sequence. Finally, we show that a 48-nucleotide DNA with a telomere sequence is more susceptible to nuclease digestion when coated with POT1-TPP1 proteins than when it is left uncoated. Together, these data suggest that POT1-TPP1 binds telomeric DNA in a coordinated manner to facilitate assembly of the nucleoprotein complexes into a state that is more accessible to enzymatic activity.     

Base Excision by Thymine DNA Glycosylase Mediates DNA-Directed Cytotoxicity of 5-Fluorouracil

5-Fluorouracil (5-FU), a chemotherapeutic drug commonly used in cancer treatment, imbalances nucleotide pools, thereby favoring misincorporation of uracil and 5-FU into genomic DNA. The processing of these bases by DNA repair activities was proposed to cause DNA-directed cytotoxicity, but the underlying mechanisms have not been resolved. In this study, we investigated a possible role of thymine DNA glycosylase (TDG), one of four mammalian uracil DNA glycosylases (UDGs), in the cellular response to 5-FU. Using genetic and biochemical tools, we found that inactivation of TDG significantly increases resistance of both mouse and human cancer cells towards 5-FU. We show that excision of DNA-incorporated 5-FU by TDG generates persistent DNA strand breaks, delays S-phase progression, and activates DNA damage signaling, and that the repair of 5-FU–induced DNA strand breaks is more efficient in the absence of TDG. Hence, excision of 5-FU by TDG, but not by other UDGs (UNG2 and SMUG1), prevents efficient downstream processing of the repair intermediate, thereby mediating DNA-directed cytotoxicity. The status of TDG expression in a cancer is therefore likely to determine its response to 5-FU–based chemotherapy.     

DNA Repair Pathway Selection Caused by Defects in TEL1, SAE2, and De Novo Telomere Addition Generates Specific Chromosomal Rearrangement Signatures

Whole genome sequencing of cancer genomes has revealed a diversity of recurrent gross chromosomal rearrangements (GCRs) that are likely signatures of specific defects in DNA damage response pathways. However, inferring the underlying defects has been difficult due to insufficient information relating defects in DNA metabolism to GCR signatures. By analyzing over 95 mutant strains of Saccharomyces cerevisiae, we found that the frequency of GCRs that deleted an internal CAN1/URA3 cassette on chrV L while retaining a chrV L telomeric hph marker was significantly higher in tel1Δ, sae2Δ, rad53Δ sml1Δ, and mrc1Δ tof1Δ mutants. The hph-retaining GCRs isolated from tel1Δ mutants contained either an interstitial deletion dependent on non-homologous end-joining or an inverted duplication that appeared to be initiated from a double strand break (DSB) on chrV L followed by hairpin formation, copying of chrV L from the DSB toward the centromere, and homologous recombination to capture the hph-containing end of chrV L. In contrast, hph-containing GCRs from other mutants were primarily interstitial deletions (mrc1Δ tof1Δ) or inverted duplications (sae2Δ and rad53Δ sml1Δ). Mutants with impaired de novo telomere addition had increased frequencies of hph-containing GCRs, whereas mutants with increased de novo telomere addition had decreased frequencies of hph-containing GCRs. Both types of hph-retaining GCRs occurred in wild-type strains, suggesting that the increased frequencies of hph retention were due to the relative efficiencies of competing DNA repair pathways. Interestingly, the inverted duplications observed here resemble common GCRs in metastatic pancreatic cancer.     

Dynamic Processing of a Common Oxidative DNA Lesion by the First Two Enzymes of the Base Excision Repair Pathway

Base excision repair (BER) is the primary pathway by which eukaryotic cells resolve single base damage. One common example of single base damage is 8-oxo-7,8-dihydro-2ʹ-deoxoguanine (8-oxoG). High incidence and mutagenic potential of 8-oxoG necessitate rapid and efficient DNA repair. How BER enzymes coordinate their activities to resolve 8-oxoG damage while limiting cytotoxic BER intermediates from propagating genomic instability remains unclear. Here we use single-molecule Förster resonance energy transfer (smFRET) and ensemble-level techniques to characterize the activities and interactions of consecutive BER enzymes important for repair of 8-oxoG. In addition to characterizing the damage searching and processing mechanisms of human 8-oxoguanine glycosylase 1 (hOGG1), our data support the existence of a ternary complex between hOGG1, the damaged DNA substrate, and human AP endonuclease 1 (APE1). Our results indicate that hOGG1 is actively displaced from its abasic site containing product by protein–protein interactions with APE1 to ensure timely repair of damaged DNA.     

The Recombination Protein RAD52 Cooperates with the Excision Repair Protein OGG1 for the Repair of Oxidative Lesions in Mammalian Cells

Oxidized bases are common types of DNA modifications. Their accumulation in the genome is linked to aging and degenerative diseases. These modifications are commonly repaired by the base excision repair (BER) pathway. Oxoguanine DNA glycosylase (OGG1) initiates BER of oxidized purine bases. A small number of protein interactions have been identified for OGG1, while very few appear to have functional consequences. We report here that OGG1 interacts with the recombination protein RAD52 in vitro and in vivo. This interaction has reciprocal functional consequences as OGG1 inhibits RAD52 catalytic activities and RAD52 stimulates OGG1 incision activity, likely increasing its turnover rate. RAD52 colocalizes with OGG1 after oxidative stress to cultured cells, but not after the direct induction of double-strand breaks by ionizing radiation. Human cells depleted of RAD52 via small interfering RNA knockdown, and mouse cells lacking the protein via gene knockout showed increased sensitivity to oxidative stress. Moreover, cells depleted of RAD52 show higher accumulation of oxidized bases in their genome than cells with normal levels of RAD52. Our results indicate that RAD52 cooperates with OGG1 to repair oxidative DNA damage and enhances the cellular resistance to oxidative stress. Our observations suggest a coordinated action between these proteins that may be relevant when oxidative lesions positioned close to strand breaks impose a hindrance to RAD52 catalytic activities.Oxidative DNA damage is generated at high levels in mammalian cells, even in cells not exposed to exogenous sources of reactive oxygen species. Several kinds of DNA modifications are formed upon oxidative stress (8). The most prevalent modifications, quantitatively, are single-strand breaks and oxidized bases. Clustered DNA damage, when two or more modifications are closely positioned in opposite strands, is detectable after gamma irradiation and has recently been shown to be generated by normal oxidative metabolism (3, 35). One unique aspect of such clustered lesions is that they can be converted into double-strand breaks (DSB) if a DNA glycosylase removes the two opposite bases and an apurinic/apyrimidinic (AP)-endonuclease cleaves the resulting abasic sites. Thus, although quantitatively minor, DSB are possible outcomes of oxidative DNA damage.Oxidized DNA bases are repaired primarily by the base excision repair pathway (BER) (22, 39). BER is initiated by a lesion-specific DNA N-glycosylase that recognizes and excises the damaged base. Eight-hydroxyguanine (8-oxoG) is one of the most abundant oxidized bases detected in cellular DNA. This adduct is easily bypassed by replicative polymerases; however, it can direct the misincorporation of adenine opposite 8-oxoG, thus leading to G·C-to-T·A transversion mutations (31). 8-oxoG accumulation has been causally associated with carcinogenesis and aging in several experimental models (1, 12). In eukaryotes, oxoguanine DNA glycosylase (OGG1) is the major 8-oxoG DNA glycosylase. OGG1 possesses an associated AP-lyase activity, such that it removes 8-oxoG and cleaves the DNA backbone. Human cells express two distinct OGG1 isoforms, α and β, which share the first 316 amino acids but differ significantly in their C termini (25). While OGG1-α is a bone fide DNA glycosylase (5) and localizes both to nuclei and mitochondria, OGG1-β localizes exclusively to mitochondria. We recently showed that the recombinant OGG1-β protein has no DNA glycosylase activity (13). The high degree of conservation of repair pathways for 8-oxoG, from bacteria to humans, along with epidemiological data correlating OGG1 polymorphisms and activity with predisposition to some cancers (11, 27, 33) attest to the biological importance of the repair of 8-oxoGs and other oxidative DNA lesions.Until recently, distinct classes of DNA lesions were believed to be metabolized by different and independent repair pathways. However, experimental evidence indicates that these pathways can interact and that there is a considerable degree of overlap in their substrate specificity and in the proteins that participate in each pathway. Experiments using yeast strains lacking one or more distinct DNA repair genes suggest that DSB repair pathways may play a role in repair of oxidative DNA damage. Swanson et al. showed that while yeast cells lacking ntg1 and ntg2 (homologues of Escherichia coli endonuclease III, a DNA glycosylase specific for pyrimidine lesions formed by oxidation) and apn1 (the major yeast abasic site endonuclease) are not overtly sensitive to oxidative stress, the additional disruption of the rad52 gene significantly increases sensitivity to H₂O₂ and menadione (36). Similarly, yeast cells expressing decreased levels of frataxin, which leads to elevated oxidative stress, show accumulation of oxidative damage in nuclear DNA only in a rad52 mutant background (18). RAD52 is a member of the RAD51 epistatic group. These proteins are believed to be involved in the early steps of homologous recombination, contributing to homology search and strand invasion; disruption of the corresponding genes renders cells deficient in DSB repair and hyper-recombinogenic (19).These results suggested a possible role for RAD52 in the repair of oxidative DNA damage. Moreover, an in vitro screening of protein partners that interact physically with OGG1-β performed in our lab (unpublished data) showed that human RAD52 strongly interacted with this glycosylase, again suggesting a possible function for RAD52 in the oxidative DNA damage response. Thus, we investigated whether RAD52 plays a role in the repair of oxidative DNA damage in human cells. We show here that human RAD52 physically interacts with both OGG1-α and -β, in vitro and in cell extracts. We also show that OGG1-α and -β inhibit RAD52 enzymatic activities. Conversely, RAD52 stimulates OGG1-α 8-oxoG incision activity. RAD52 colocalizes with OGG1-α in cells, and this colocalization increases after oxidative stress. Moreover, lower RAD52 expression, via gene knockdown (KD) or disruption of the RAD52 gene, render cells sensitive to oxidative stress. Based on our results, we discuss a model in which OGG1 and RAD52 cooperate to repair 8-oxoG lesions.     

Evidence for Base Excision Repair of Oxidative DNA Damage in Chloroplasts of Arabidopsis thaliana

Chloroplasts are the sites of photosynthesis in plants, and they contain their own multicopy, requisite genome. Chloroplasts are also major sites for production of reactive oxygen species, which can damage essential components of the chloroplast, including the chloroplast genome. Compared with mitochondria in animals, relatively little is known about the potential to repair oxidative DNA damage in chloroplasts. Here we provide evidence of DNA glycosylase-lyase/endonuclease activity involved in base excision repair of oxidized pyrimidines in chloroplast protein extracts of Arabidopsis thaliana. Three base excision repair components (two endonuclease III homologs and an apurinic/apyrimidinic endonuclease) that might account for this activity were identified by bioinformatics. Transient expression of protein-green fluorescent protein fusions showed that all three are targeted to the chloroplast and co-localized with chloroplast DNA in nucleoids. The glycosylase-lyase/endonuclease activity of one of the endonuclease III homologs, AtNTH2, which had not previously been characterized, was confirmed in vitro. T-DNA insertions in each of these genes were identified, and the physiological and biochemical phenotypes of the single, double, and triple mutants were analyzed. This mutant analysis revealed the presence of a third glycosylase activity and potentially another pathway for repair of oxidative DNA damage in chloroplasts.Reactive oxygen species (ROS)² are inevitable by-products of metabolism in all aerobic organisms (1). Plants and algae are especially prone to photo-oxidative stress because of ROS generated during oxygenic photosynthesis. Several types of ROS are generated at various sites in the photosynthetic electron transport chain in chloroplasts, and their production is enhanced by such factors as excess or varying light intensities and extremes of temperature, drought, nutrient deficiencies, and herbicides (2). These ROS can damage many chloroplast constituents, including lipids, proteins, pigments, and the multicopy genome.Plants have evolved numerous mechanisms to deal with photo-oxidative stress, including dissipation of excess light energy, synthesis of antioxidant molecules and scavenging enzymes, and targeted repair (2). DNA repair of oxidized bases, such as thymine glycol (TG) or 8-oxoguanine, can be hypothesized as an important element of chloroplast photoprotection. Although there is considerable overlap in both the types of DNA lesions caused by different insults and the targeting of different DNA repair mechanisms, base excision repair (BER) is considered to be the main repair pathway for oxidative DNA damage, at least in the nucleus and mitochondrion (3, 4).BER repairs single damaged bases (because of oxidation, deamination, alkylation, etc.) in DNA by removing them, breaking the phosphodiester backbone, excising the sugar residue at the abasic site, and filling the gap (reviewed in Refs. 5, 6). BER begins with a DNA glycosylase or glycosylase-lyase. There are many types of glycosylases in any given organism and across taxa, and they are distinguishable by their substrate specificity, whether they are monofunctional (glycosylase activity only) or bifunctional (glycosylase plus apurinic/apyrimidinic (AP) lyase activities; see below), by the phylogenetic family in which they reside, and/or by conserved structural characteristics (reviewed in Refs. 6–8). The glycosylases involved in BER of oxidative DNA damage can be roughly divided into those that target either oxidized purines or oxidized pyrimidines (4, 9). For example, TG is a common type of oxidized pyrimidine, which is removed primarily by endonuclease III (Nth), endonuclease VIII (Nei), or their homologs (10). TG is only poorly mutagenic, but it strongly blocks polymerases, inducing cell cycle arrest and potentially cell death if it is not removed.After an appropriate glycosylase cleaves the N-glycosyl bond attaching a damaged base to deoxyribose, leaving an abasic site, the sugar-phosphate backbone is nicked. Bifunctional glycosylases also have an AP lyase activity that cleaves on the 3′ side of the AP site. However, the site still requires the function of a separate AP endonuclease that cuts on the 5′ side of the AP site to remove the 3′-deoxyribose residue at the nick site (11) before repair can continue. In the case of a monofunctional glycosylase, an AP endonuclease nicks the strand on the 5′ side of the AP site. Escherichia coli has two unrelated AP endonucleases, exonuclease III (Xth) and endonuclease IV (Nfo). In humans Ape1/Ref-1 is an Xth homolog, and in yeast Apn1p is an Nfo homolog (5, 12). Following generation of the AP site and nicking of the backbone, the gap is filled by a polymerase in either a short or long patch and then sealed by a ligase.BER of oxidative DNA lesions such TG has been studied intensively in E. coli, yeast, and mammals, whereas comparatively little is known about BER in plants. For example, only two genes involved in BER of oxidized pyrimidines have been characterized previously in the model plant Arabidopsis thaliana (13, 14), and their localization within the plant cell is unknown. An Nth homolog in Arabidopsis, AtNTH1 (At2g31450), has the expected bifunctional glycosylase-lyase activity in vitro (14). The ARP gene (At2g41460) in Arabidopsis encodes an enzyme with AP endonuclease activity (13).Here we present the results of experiments conducted to address whether there is BER of oxidized pyrimidines in the Arabidopsis chloroplast. Chloroplast protein extracts were assayed for glycosylase-lyase/endonuclease activity. The chloroplast localization of ARP, AtNTH1, and AtNTH2, a second Arabidopsis homolog of Nth, was tested experimentally, and the predicted activity of AtNTH2 was confirmed in vitro. In addition, an analysis of T-DNA insertion mutants affecting each of these three BER genes was performed.     

Conformational Dynamics of Damage Processing by Human DNA Glycosylase NEIL1

Endonuclease VIII-like protein 1 (NEIL1) is a DNA repair enzyme found in higher eukaryotes, including humans. It belongs to the helix–two turn–helix (H2TH) structural superfamily together with Escherichia coli formamidopyrimidine–DNA glycosylase (Fpg) and endonuclease VIII (Nei), and removes a variety of oxidized purine and pyrimidine bases from DNA. Structural, modeling and kinetic studies have established that the bacterial H2TH superfamily enzymes proceed through several conformational intermediates while recognizing and removing their cognate lesions. Here we apply stopped-flow kinetics with detection of intrinsic Trp fluorescence and Förster resonance energy transfer fluorescence to follow the conformational dynamics of human NEIL1 and DNA when the enzyme interacts with undamaged DNA, or DNA containing cleavable or non-cleavable abasic sites, or dihydrouracil lesions. NEIL1 processed a natural abasic site and a damaged base in DNA equally well but showed an additional fluorescently discernible step when DHU was present, likely reflecting additional rearrangements during base eversion into the enzyme's active site. With undamaged DNA and DNA containing a non-cleavable abasic site analog, (3-hydroxytetrahydrofuran-2-yl)methyl phosphate, NEIL1 was diverted to a non-productive DNA conformation early in the reaction. Our results support the view of NEIL1 as an enzyme that actively destabilizes damaged DNA and uses multiple checkpoints along the reaction coordinate to drive substrate lesions into the active site while rejecting normal bases and non-substrate lesions.     

Repair of Oxidative DNA Base Damage in the Host Genome Influences the HIV Integration Site Sequence Preference

Host base excision repair (BER) proteins that repair oxidative damage enhance HIV infection. These proteins include the oxidative DNA damage glycosylases 8-oxo-guanine DNA glycosylase (OGG1) and mutY homolog (MYH) as well as DNA polymerase beta (Polβ). While deletion of oxidative BER genes leads to decreased HIV infection and integration efficiency, the mechanism remains unknown. One hypothesis is that BER proteins repair the DNA gapped integration intermediate. An alternative hypothesis considers that the most common oxidative DNA base damages occur on guanines. The subtle consensus sequence preference at HIV integration sites includes multiple G:C base pairs surrounding the points of joining. These observations suggest a role for oxidative BER during integration targeting at the nucleotide level. We examined the hypothesis that BER repairs a gapped integration intermediate by measuring HIV infection efficiency in Polβ null cell lines complemented with active site point mutants of Polβ. A DNA synthesis defective mutant, but not a 5′dRP lyase mutant, rescued HIV infection efficiency to wild type levels; this suggeted Polβ DNA synthesis activity is not necessary while 5′dRP lyase activity is required for efficient HIV infection. An alternate hypothesis that BER events in the host genome influence HIV integration site selection was examined by sequencing integration sites in OGG1 and MYH null cells. In the absence of these 8-oxo-guanine specific glycosylases the chromatin elements of HIV integration site selection remain the same as in wild type cells. However, the HIV integration site sequence preference at G:C base pairs is altered at several positions in OGG1 and MYH null cells. Inefficient HIV infection in the absence of oxidative BER proteins does not appear related to repair of the gapped integration intermediate; instead oxidative damage repair may participate in HIV integration site preference at the sequence level.     

RAS Transformation Requires CUX1-Dependent Repair of Oxidative DNA Damage

The Cut homeobox 1 (CUX1) gene is a target of loss-of-heterozygosity in many cancers, yet elevated CUX1 expression is frequently observed and is associated with shorter disease-free survival. The dual role of CUX1 in cancer is illustrated by the fact that most cell lines with CUX1 LOH display amplification of the remaining allele, suggesting that decreased CUX1 expression facilitates tumor development while increased CUX1 expression is needed in tumorigenic cells. Indeed, CUX1 was found in a genome-wide RNAi screen to identify synthetic lethal interactions with oncogenic RAS. Here we show that CUX1 functions in base excision repair as an ancillary factor for the 8-oxoG-DNA glycosylase, OGG1. Single cell gel electrophoresis (comet assay) reveals that Cux1^+/− MEFs are haploinsufficient for the repair of oxidative DNA damage, whereas elevated CUX1 levels accelerate DNA repair. In vitro base excision repair assays with purified components demonstrate that CUX1 directly stimulates OGG1''s enzymatic activity. Elevated reactive oxygen species (ROS) levels in cells with sustained RAS pathway activation can cause cellular senescence. We show that elevated expression of either CUX1 or OGG1 prevents RAS-induced senescence in primary cells, and that CUX1 knockdown is synthetic lethal with oncogenic RAS in human cancer cells. Elevated CUX1 expression in a transgenic mouse model enables the emergence of mammary tumors with spontaneous activating Kras mutations. We confirmed cooperation between Kras^G12V and CUX1 in a lung tumor model. Cancer cells can overcome the antiproliferative effects of excessive DNA damage by inactivating a DNA damage response pathway such as ATM or p53 signaling. Our findings reveal an alternate mechanism to allow sustained proliferation in RAS-transformed cells through increased DNA base excision repair capability. The heightened dependency of RAS-transformed cells on base excision repair may provide a therapeutic window that could be exploited with drugs that specifically target this pathway.     

Nucleosomes Suppress the Formation of Double-strand DNA Breaks during Attempted Base Excision Repair of Clustered Oxidative Damages

Exposure to ionizing radiation can produce multiple, clustered oxidative lesions in DNA. The near simultaneous excision of nearby lesions in opposing DNA strands by the base excision repair (BER) enzymes can produce double-strand DNA breaks (DSBs). This attempted BER accounts for many of the potentially lethal or mutagenic DSBs that occur in vivo. To assess the impact of nucleosomes on the frequency and pattern of BER-dependent DSB formation, we incubated nucleosomes containing oxidative damages in opposing DNA strands with selected DNA glycosylases and human apurinic/apyrimidinic endonuclease 1. Overall, nucleosomes substantially suppressed DSB formation. However, the degree of suppression varied as a function of (i) the lesion type and DNA glycosylase tested, (ii) local sequence context and the stagger between opposing strand lesions, (iii) the helical orientation of oxidative lesions relative to the underlying histone octamer, and (iv) the distance between the lesion cluster and the nucleosome edge. In some instances the binding of a BER factor to one nucleosomal lesion appeared to facilitate binding to the opposing strand lesion. DSB formation did not invariably lead to nucleosome dissolution, and in some cases, free DNA ends resulting from DSB formation remained associated with the histone octamer. These observations explain how specific structural and dynamic properties of nucleosomes contribute to the suppression of BER-generated DSBs. These studies also suggest that most BER-generated DSBs will occur in linker DNA and in genomic regions associated with elevated rates of nucleosome turnover or remodeling.     

Germ Line Variants of Human N-Methylpurine DNA Glycosylase Show Impaired DNA Repair Activity and Facilitate 1,N6-Ethenoadenine-induced Mutations

Human N-methylpurine DNA glycosylase (hMPG) initiates base excision repair of a number of structurally diverse purine bases including 1,N⁶-ethenoadenine, hypoxanthine, and alkylation adducts in DNA. Genetic studies discovered at least eight validated non-synonymous single nucleotide polymorphisms (nsSNPs) of the hMPG gene in human populations that result in specific single amino acid substitutions. In this study, we tested the functional consequences of these nsSNPs of hMPG. Our results showed that two specific arginine residues, Arg-141 and Arg-120, are important for the activity of hMPG as the germ line variants R120C and R141Q had reduced enzymatic activity in vitro as well as in mammalian cells. Expression of these two variants in mammalian cells lacking endogenous MPG also showed an increase in mutations and sensitivity to an alkylating agent compared with the WT hMPG. Real time binding experiments by surface plasmon resonance spectroscopy suggested that these variants have substantial reduction in the equilibrium dissociation constant of binding (K_D) of hMPG toward 1,N⁶-ethenoadenine-containing oligonucleotide (ϵA-DNA). Pre-steady-state kinetic studies showed that the substitutions at arginine residues affected the turnover of the enzyme significantly under multiple turnover condition. Surface plasmon resonance spectroscopy further showed that both variants had significantly decreased nonspecific (undamaged) DNA binding. Molecular modeling suggested that R141Q substitution may have resulted in a direct loss of the salt bridge between ϵA-DNA and hMPG, whereas R120C substitution redistributed, at a distance, the interactions among residues in the catalytic pocket. Together our results suggest that individuals carrying R120C and R141Q MPG variants may be at risk for genomic instability and associated diseases as a consequence.     

Arabidopsis Uracil DNA Glycosylase (UNG) Is Required for Base Excision Repair of Uracil and Increases Plant Sensitivity to 5-Fluorouracil

Uracil in DNA arises by misincorporation of dUMP during replication and by hydrolytic deamination of cytosine. This common lesion is actively removed through a base excision repair (BER) pathway initiated by a uracil DNA glycosylase (UDG) activity that excises the damage as a free base. UDGs are classified into different families differentially distributed across eubacteria, archaea, yeast, and animals, but remain to be unambiguously identified in plants. We report here the molecular characterization of AtUNG (Arabidopsis thaliana uracil DNA glycosylase), a plant member of the Family-1 of UDGs typified by Escherichia coli Ung. AtUNG exhibits the narrow substrate specificity and single-stranded DNA preference that are characteristic of Ung homologues. Cell extracts from atung^−/− mutants are devoid of UDG activity, and lack the capacity to initiate BER on uracil residues. AtUNG-deficient plants do not display any apparent phenotype, but show increased resistance to 5-fluorouracil (5-FU), a cytostatic drug that favors dUMP misincorporation into DNA. The resistance of atung^−/− mutants to 5-FU is accompanied by the accumulation of uracil residues in DNA. These results suggest that AtUNG excises uracil in vivo but generates toxic AP sites when processing abundant U:A pairs in dTTP-depleted cells. Altogether, our findings point to AtUNG as the major UDG activity in Arabidopsis.     

Accelerated Repair and Reduced Mutagenicity of DNA Damage Induced by Cigarette Smoke in Human Bronchial Cells Transfected with E.coli Formamidopyrimidine DNA Glycosylase

Cigarette smoke (CS) is associated to a number of pathologies including lung cancer. Its mutagenic and carcinogenic effects are partially linked to the presence of reactive oxygen species and polycyclic aromatic hydrocarbons (PAH) inducing DNA damage. The bacterial DNA repair enzyme formamidopyrimidine DNA glycosylase (FPG) repairs both oxidized bases and different types of bulky DNA adducts. We investigated in vitro whether FPG expression may enhance DNA repair of CS-damaged DNA and counteract the mutagenic effects of CS in human lung cells. NCI-H727 non small cell lung carcinoma cells were transfected with a plasmid vector expressing FPG fused to the Enhanced Green Fluorescent Protein (EGFP). Cells expressing the fusion protein EGFP-FPG displayed accelerated repair of adducts and DNA breaks induced by CS condensate. The mutant frequencies induced by low concentrations of CS condensate to the Na⁺K⁺-ATPase locus (oua^r) were significantly reduced in cells expressing EGFP-FPG. Hence, expression of the bacterial DNA repair protein FPG stably protects human lung cells from the mutagenic effects of CS by improving cells’ capacity to repair damaged DNA.     

人线粒体tRNALeu(UUR)基因氧化损伤与修复研究

人mtDNA比核DNA更易受到自由基的氧化损伤,这些损伤可以被线粒体内的DNA修复机制所修复,损伤与修复是决定突变是否产生的两个重要因素.为了确定氧化损伤与损伤后修复对mtDNA突变的具体影响,采用四氧嘧啶处理LO2细胞,这种试剂进入细胞后,经氧化还原反应生成的自由基与线粒体自身代谢产生的自由基类似,然后观察自由基对细胞mtDNA的氧化损伤与损伤后DNA修复的动力学变化.由于线粒体的正常功能为修复机制所必需,采用MTT细胞活力实验检测不同浓度四氧嘧啶处理下线粒体酶活力,发现9 mmol/L四氧嘧啶培养细胞1h后,线粒体琥珀酸脱氢酶功能在撤去药物后0,2,8和24 h时间点均无明显变化.提取各组细胞的mtDNA,用EndoⅢ和Fgp两种酶切除受氧化损伤的核苷酸,然后用碱性琼脂糖凝胶电泳分离大小不等的mtDNA,进行DNA印迹实验,地高辛-抗体-碱性磷酸酶系统显色,检测完整与断裂的mtDNA量,利用Poisson公式(s=-lnP0/P,P0为未断裂链光密度值,P为所有链光密度值总和)计算一个mtDNA分子的平均损伤频率,结果显示,9 mmol/L四氧嘧啶处理细胞1 h,链平均损伤频率由对照的0.11个/分子增加至5.60个/分子,明显增加了mtDNA上核苷酸的氧化损伤,除去药物后8 h,绝大部分损伤可被修复,损伤频率减至0.40个/分子,除去药物后24h核苷酸的氧化损伤恢复至正常水平.采用接头介导PCR(LM-PCR)检测MTTL1基因区域内单个核苷酸的损伤与修复动力学.这种方法可以检测各组mtDNA上MTTL1基因75 bp区域内单个核苷酸损伤的部位及频率.结果显示,人MTTL1基因存在20个易受氧化损伤的核苷酸热点,经与相应区域内文献报道的16个突变热点比较,有12个热点部位重合,而修复未显示热点部位或区域.结果提示,自由基对核苷酸的选择性氧化损伤是决定mtDNA点突变发生及发生部位的主要原因.     

Homology Modeling and Molecular Dynamics Simulations of PBCV-1 Glycosylase Complexed with UV-damaged DNA

The UV-light damage specific DNA glycosylase from Chlorella virus strain PBCV-1 (pyrimidine dimer glycosylase; PDG) incises DNA at sites containing UV-induced thymidine dimers by catalyzing the breakage of the N-C-1 glycosyl bond. As the amino acid sequence of PDG is 41% identical to that of T4 endonuclease V (Endo V), and potential key active site residues are conserved, we used coordinates from a crystal structure of an Endo V complexed with DNA containing a cis-syn cyclobutane thymidine-dimer as a template to model a similar complex of PDG. Quantum mechanical calculations of the damaged base pair and the distance geometry based program DIAMOD were used to generate a PDG/DNA model whose backbone root mean square deviation (RMSD) to the Endo V/DNA structure was 0.5 Å, 0.5 Å, and 0.8 Å for DNA, protein, and the whole complex, respectively. To better understand structural details that could account for differences in activity of the two enzymes, molecular dynamics simulations were used to follow protein-DNA interactions in an aqueous environment. The simulations of the Endo V/DNA complex indicate new roles for Arg22 and Arg26 in the active site in recognizing irregular pairing and maintaining the strand separation needed for incision of the damaged bases. The model for the PDG/DNA complex and simulations thereof indicate a similar mechanism for DNA binding by this enzyme despite significant differences in residues maintaining the flipped-out adenine and strand separation in the area of damage. According to our model, PDGs increased affinity for substrate is probably due to a higher surface charge. Further, reduced packing density in the active site could account for PDGs activity on trans-syn II cyclobutane dimers.Electronic Supplementary Material available.     

Neurons Efficiently Repair Glutamate-induced Oxidative DNA Damage by a Process Involving CREB-mediated Up-regulation of Apurinic Endonuclease 1

Glutamate, the major excitatory neurotransmitter in the brain, activates receptors coupled to membrane depolarization and Ca²⁺ influx that mediates functional responses of neurons including processes such as learning and memory. Here we show that reversible nuclear oxidative DNA damage occurs in cerebral cortical neurons in response to transient glutamate receptor activation using non-toxic physiological levels of glutamate. This DNA damage was prevented by intracellular Ca²⁺ chelation, the mitochondrial superoxide dismutase mimetic MnTMPyP (Mn-5,10,15,20-tetra(4-pyridyl)-21H,23H-porphine chloride tetrakis(methochloride)), and blockade of the permeability transition pore. The repair of glutamate-induced DNA damage was associated with increased DNA repair activity and increased mRNA and protein levels of apurinic endonuclease 1 (APE1). APE1 knockdown induced accumulation of oxidative DNA damage after glutamate treatment, suggesting that APE1 is a key repair protein for glutamate-induced DNA damage. A cAMP-response element-binding protein (CREB) binding sequence is present in the Ape1 gene (encodes APE1 protein) promoter and treatment of neurons with a Ca²⁺/calmodulin-dependent kinase inhibitor (KN-93) blocked the ability of glutamate to induce CREB phosphorylation and APE1 expression. Selective depletion of CREB using RNA interference prevented glutamate-induced up-regulation of APE1. Thus, glutamate receptor stimulation triggers Ca²⁺- and mitochondrial reactive oxygen species-mediated DNA damage that is then rapidly repaired by a mechanism involving Ca²⁺-induced, CREB-mediated APE1 expression. Our findings reveal a previously unknown ability of neurons to efficiently repair oxidative DNA lesions after transient activation of glutamate receptors.