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Although DNA repair is known to impact susceptibility to cancer and other diseases, relatively few population studies have been performed to evaluate DNA repair kinetics in people due to the difficulty of assessing DNA repair in a high throughput manner. Here we use the CometChip, a high throughput comet assay, to explore inter-individual variation in repair of oxidative damage to DNA, a known risk factor for aging, cancer and other diseases. DNA repair capacity after H(2)O(2)-induced DNA oxidation damage was quantified in peripheral blood mononuclear cells (PBMCs). For 10 individuals, blood was drawn at several times over the course of 4-6 weeks. In addition, blood was drawn once from each of 56 individuals. DNA damage levels were quantified prior to exposure to H(2)O(2) and at 0, 15, 30, 60, and 120-minutes post exposure. We found that there is significant variability in DNA repair efficiency among individuals. When subdivided into quartiles by DNA repair efficiency, we found that the average t(1/2) is 81 minutes for the slowest group and 24 minutes for the fastest group. This work shows that the CometChip can be used to uncover significant differences in repair kinetics among people, pointing to its utility in future epidemiological and clinical studies.

DNA damage can be cytotoxic and mutagenic and is directly linked to aging, cancer, and heritable diseases. To counteract the deleterious effects of DNA damage, cells have evolved highly conserved DNA repair pathways. Many commonly used DNA repair assays are relatively low throughput and are limited to analysis of one protein or one pathway. Here, we have explored the capacity of the CometChip platform for parallel analysis of multiple DNA repair activities. Taking advantage of the versatility of the traditional comet assay and leveraging micropatterning techniques, the CometChip platform offers increased throughput and sensitivity compared to the traditional comet assay. By exposing cells to DNA damaging agents that create substrates of Base Excision Repair, Nucleotide Excision Repair, and Non-Homologous End Joining, we show that the CometChip is an effective method for assessing repair deficiencies in all three pathways. With these advanced applications of the CometChip platform, we expand the efficacy of the comet assay for precise, high-throughput, parallel analysis of multiple DNA repair activities.

Base excision repair (BER) initiated by alkyladenine DNA glycosylase (AAG) is essential for removal of aberrantly methylated DNA bases. Genome instability and accumulation of aberrant bases accompany multiple diseases, including cancer and neurological disorders. While BER is well studied on naked DNA, it remains unclear how BER efficiently operates on chromatin. Here, we show that AAG binds to chromatin and forms complex with RNA polymerase (pol) II. This occurs through direct interaction with Elongator and results in transcriptional co-regulation. Importantly, at co-regulated genes, aberrantly methylated bases accumulate towards the 3′end in regions enriched for BER enzymes AAG and APE1, Elongator and active RNA pol II. Active transcription and functional Elongator are further crucial to ensure efficient BER, by promoting AAG and APE1 chromatin recruitment. Our findings provide insights into genome stability maintenance in actively transcribing chromatin and reveal roles of aberrantly methylated bases in regulation of gene expression., How genome stability is maintained at regions of active transcription is currently not entirely clear. Here, the authors reveal an association between base excision repair factors and transcription elongation to modulate DNA repair.

Base excision repair (BER) initiated by alkyladenine DNA glycosylase (AAG; aka MPG) is essential for removal of aberrantly methylated DNA bases. Genome instability and accumulation of aberrant bases accompany multiple diseases including cancer and neurological disorders. While BER is well studied on naked DNA, it remains unclear how BER efficiently operates on chromatin. Here we show that AAG binds to chromatin and forms complex with active RNA polymerase (pol) II. This occurs through direct interaction with Elongator and results in transcriptional co-regulation. Importantly, at co-regulated genes aberrantly methylated bases accumulate towards 3’end, in regions enriched for BER enzymes AAG and APE1, Elongator and active RNA pol II. Active transcription and functional Elongator are further crucial to ensure efficient BER, by promoting AAG and APE1 chromatin recruitment. Our findings provide novel insights to maintaining genome stability in actively transcribing chromatin, and reveal roles of aberrantly methylated bases in regulation of gene expression.

The relationships between inflammation and cancer are varied and complex. An important connection linking inflammation to cancer development is DNA damage. During inflammation reactive oxygen and nitrogen species (RONS) are created to combat pathogens and to stimulate tissue repair and regeneration, but these chemicals can also damage DNA, which in turn can promote mutations that initiate and promote cancer. DNA repair pathways are essential for preventing DNA damage from causing mutations and cytotoxicity, but RONS can interfere with repair mechanisms, reducing their efficacy. Further, cellular responses to DNA damage, such as damage signaling and cytotoxicity, can promote inflammation, creating a positive feedback loop. Despite coordination of DNA repair and oxidative stress responses, there are nevertheless examples whereby inflammation has been shown to promote mutagenesis, tissue damage, and ultimately carcinogenesis. Here, we discuss the DNA damage-mediated associations between inflammation, mutagenesis and cancer., EPA Superfund Research Program (Grant P42ES027707), NIEHS (Grant T32-ES007020), NCI (Grant P01-CA026731)

The integrity of our DNA is challenged with at least 100,000 lesions per cell on a daily basis. Failure to repair DNA damage efficiently can lead to cancer, immunodeficiency, and neurodegenerative disease. Base excision repair (BER) recognizes and repairs minimally helix-distorting DNA base lesions induced by both endogenous and exogenous DNA damaging agents. Levels of BER-initiating DNA glycosylases can vary between individuals, suggesting that quantitating and understanding interindividual differences in DNA repair capacity (DRC) may enable us to predict and prevent disease in a personalized manner. However, population studies of BER capacity have been limited because most methods used to measure BER activity are cumbersome, time consuming and, for the most part, only allow for the analysis of one DNA glycosylase at a time. We have developed a fluorescence-based multiplex flow-cytometric host cell reactivation assay wherein the activity of several enzymes [four BER-initiating DNA glycosylases and the downstream processing apurinic/apyrimidinic endonuclease 1 (APE1)] can be tested simultaneously, at single-cell resolution, in vivo. Taking advantage of the transcriptional properties of several DNA lesions, we have engineered specific fluorescent reporter plasmids for quantitative measurements of 8-oxoguanine DNA glycosylase, alkyl-adenine DNA glycosylase, MutY DNA glycosylase, uracil DNA glycosylase, and APE1 activity. We have used these reporters to measure differences in BER capacity across a panel of cell lines collected from healthy individuals, and to generate mathematical models that predict cellular sensitivity to methylmethane sulfonate, H2O2, and 5-FU from DRC. Moreover, we demonstrate the suitability of these reporters to measure differences in DRC in multiple pathways using primary lymphocytes from two individuals.

Genotoxicity testing is critical for predicting adverse effects of pharmaceutical, industrial, and environmental chemicals. The alkaline comet assay is an established method for detecting DNA strand breaks, however, the assay does not detect potentially carcinogenic bulky adducts that can arise when metabolic enzymes convert pro-carcinogens into a highly DNA reactive products. To overcome this, we use DNA synthesis inhibitors (hydroxyurea and 1-β-d-arabinofuranosyl cytosine) to trap single strand breaks that are formed during nucleotide excision repair, which primarily removes bulky lesions. In this way, comet-undetectable bulky lesions are converted into comet-detectable single strand breaks. Moreover, we use HepaRG™ cells to recapitulate in vivo metabolic capacity, and leverage the CometChip platform (a higher throughput more sensitive comet assay) to create the ‘HepaCometChip’, enabling the detection of bulky genotoxic lesions that are missed by current genotoxicity screens. The HepaCometChip thus provides a broadly effective approach for detection of bulky DNA adducts.

Due to a harsh environment mitochondrial genomes accumulate high levels of DNA damage, in particular oxidation, hydrolytic deamination, and alkylation adducts. While repair of alkylated bases in nuclear DNA has been explored in detail, much less is known about the repair of DNA alkylation damage in mitochondria. Alkyladenine DNA glycosylase (AAG) recognizes and removes numerous alkylated bases, but to date AAG has only been detected in the nucleus, even though mammalian mitochondria are known to repair DNA lesions that are specific substrates of AAG. Here we use immunofluorescence to show that AAG localizes to mitochondria, and we find that native AAG is present in purified human mitochondrial extracts, as well as that exposure to alkylating agent promotes AAG accumulation in the mitochondria. We identify mitochondrial single-stranded binding protein (mtSSB) as a novel interacting partner of AAG; interaction between mtSSB and AAG is direct and increases upon methyl methanesulfonate (MMS) treatment. The consequence of this interaction is specific inhibition of AAG glycosylase activity in the context of a single-stranded DNA (ssDNA), but not a double-stranded DNA (dsDNA) substrate. By inhibiting AAG-initiated processing of damaged bases, mtSSB potentially prevents formation of DNA breaks in ssDNA, ensuring that base removal primarily occurs in dsDNA. In summary, our findings suggest the existence of AAG-initiated BER in mitochondria and further support a role for mtSSB in DNA repair., National Institutes of Health (U.S.) (Grant CA055042), National Institutes of Health (U.S.) (Grant ES002109), University of Zurich

The ribonucleotide reductase (RNR) enzyme catalyzes an essential step in the production of deoxyribonucleotide triphosphates (dNTPs) in cells. Bulk biochemical measurements in synchronized Saccharomyces cerevisiae cells suggest that RNR mRNA production is maximal in late G(1) and S phases; however, damaged DNA induces RNR transcription throughout the cell cycle. But such en masse measurements reveal neither cell-to-cell heterogeneity in responses nor direct correlations between transcript and protein expression or localization in single cells which may be central to function. We overcame these limitations by simultaneous detection of single RNR transcripts and also Rnr proteins in the same individual asynchronous S. cerevisiae cells, with and without DNA damage by methyl methanesulfonate (MMS). Surprisingly, RNR subunit mRNA levels were comparably low in both damaged and undamaged G(1) cells and highly induced in damaged S/G(2) cells. Transcript numbers became correlated with both protein levels and localization only upon DNA damage in a cell cycle-dependent manner. Further, we showed that the differential RNR response to DNA damage correlated with variable Mec1 kinase activity in the cell cycle in single cells. The transcription of RNR genes was found to be noisy and non-Poissonian in nature. Our results provide vital insight into cell cycle-dependent RNR regulation under conditions of genotoxic stress.

Cancer cells can resist the effects of DNA-damaging therapeutic agents via utilization of DNA repair pathways, suggesting that DNA repair capacity (DRC) measurements in cancer cells could be used to identify patients most likely to respond to treatment. However, the limitations of available technologies have so far precluded adoption of this approach in the clinic. We recently developed fluorescence-based multiplexed host cell reactivation (FM-HCR) assays to measure DRC in multiple pathways. Here we apply a mathematical model that uses DRC in multiple pathways to predict cellular resistance to killing by DNA-damaging agents. This model, developed using FM-HCR and drug sensitivity measurements in 24 human lymphoblastoid cell lines, was applied to a panel of 12 patient-derived xenograft (PDX) models of glioblastoma (GBM) to predict GBM response to treatment with the chemotherapeutic DNA damaging agent temozolomide (TMZ). This work showed that, in addition to changes in O6-methylguanine DNA methyltransferase (MGMT) activity, small changes in mismatch repair (MMR), nucleotide excision repair (NER), and homologous recombination (HR) capacity contributed to acquired TMZ resistance in PDX models, and lead to reduced relative survival prolongation following TMZ treatment of orthotopic mouse models in vivo. Our data indicate that measuring the combined status of MMR, HR, NER, and MGMT provided a more robust prediction of TMZ resistance than assessments of MGMT activity alone., National Institutes of Health (U.S.) (DP1-ES022576), National Institutes of Health (U.S.) (U54-CA112967)

Background: In the event of a nuclear accident, people are exposed to elevated levels of continuous low dose-rate radiation. Nevertheless, most of the literature describes the biological effects of acute radiation. Objectives: DNA damage and mutations are well established for their carcinogenic effects. We assessed several key markers of DNA damage and DNA damage responses in mice exposed to low dose-rate radiation to reveal potential genotoxic effects associated with low dose-rate radiation. Methods: We studied low dose-rate radiation using a variable low dose-rate irradiator consisting of flood phantoms filled with 125Iodine-containing buffer. Mice were exposed to 0.0002 cGy/min (~ 400-fold background radiation) continuously over 5 weeks. We assessed base lesions, micronuclei, homologous recombination (HR; using fluorescent yellow direct repeat mice), and transcript levels for several radiation-sensitive genes. Results: We did not observe any changes in the levels of the DNA nucleobase damage products hypoxanthine, 8-oxo-7,8-dihydroguanine, 1,N6-ethenoadenine, or 3,N4-ethenocytosine above background levels under low dose-rate conditions. The micronucleus assay revealed no evidence that low dose-rate radiation induced DNA fragmentation, and there was no evidence of double strand break–induced HR. Furthermore, low dose-rate radiation did not induce Cdkn1a, Gadd45a, Mdm2, Atm, or Dbd2. Importantly, the same total dose, when delivered acutely, induced micronuclei and transcriptional responses. Conclusions: These results demonstrate in an in vivo animal model that lowering the dose-rate suppresses the potentially deleterious impact of radiation and calls attention to the need for a deeper understanding of the biological impact of low dose-rate radiation.

To efficiently repair DNA, human alkyladenine DNA glycosylase (AAG) must search the million-fold excess of unmodified DNA bases to find a handful of DNA lesions. Such a search can be facilitated by the ability of glycosylases, like AAG, to interact with DNA using two affinities: a lower-affinity interaction in a searching process and a higher-affinity interaction for catalytic repair. Here, we present crystal structures of AAG trapped in two DNA-bound states. The lower-affinity depiction allows us to investigate, for the first time, the conformation of this protein in the absence of a tightly bound DNA adduct. We find that active site residues of AAG involved in binding lesion bases are in a disordered state. Furthermore, two loops that contribute significantly to the positive electrostatic surface of AAG are disordered. Additionally, a higher-affinity state of AAG captured here provides a fortuitous snapshot of how this enzyme interacts with a DNA adduct that resembles a one-base loop.

Western Pacific amyotrophic lateral sclerosis (ALS) and parkinsonism-dementia complex (PDC), a prototypical neurodegenerative disease (tauopathy) affecting distinct genetic groups with common exposure to neurotoxic chemicals in cycad seed, has many features of Parkinson's and Alzheimer's diseases (AD), including early olfactory dysfunction. Guam ALS-PDC incidence correlates with cycad flour content of cycasin and its aglycone methylazoxymethanol (MAM), which produces persistent DNA damage (O(6)-methylguanine) in the brains of mice lacking O(6)-methylguanine methyltransferase (Mgmt(-/-)). We described in Mgmt(-/-)mice up to 7 days post-MAM treatment that brain DNA damage was linked to brain gene expression changes found in human neurological disease, cancer, and skin and hair development. This addendum reports 6 months post-MAM treatment- related brain transcriptional changes as well as elevated mitogen activated protein kinases and increased caspase-3 activity, both of which are involved in tau aggregation and neurofibrillary tangle formation typical of ALS-PDC and AD, plus transcriptional changes in olfactory receptors. Does cycasin act as a "slow (geno)toxin" in ALS-PDC?

Reactive oxygen and nitrogen species, generated by neutrophils and macrophages in chronically inflamed tissues, readily damage DNA, producing a variety of potentially genotoxic etheno base lesions; such inflammation-related DNA damage is now known to contribute to carcinogenesis. Although the human alkyladenine DNA glycosylase (AAG) can specifically bind DNA containing either 1,N(6)-ethenoadenine (εA) lesions or 3,N(4)-ethenocytosine (εC) lesions, it can only excise εA lesions. AAG binds very tightly to DNA containing εC lesions, forming an abortive protein-DNA complex; such binding not only shields εC from repair by other enzymes but also inhibits AAG from acting on other DNA lesions. To understand the structural basis for inhibition, we have characterized the binding of AAG to DNA containing εC lesions and have solved a crystal structure of AAG bound to a DNA duplex containing the εC lesion. This study provides the first structure of a DNA glycosylase in complex with an inhibitory base lesion that is induced endogenously and that is also induced upon exposure to environmental agents such as vinyl chloride. We identify the primary cause of inhibition as a failure to activate the nucleotide base as an efficient leaving group and demonstrate that the higher binding affinity of AAG for εC versus εA is achieved through formation of an additional hydrogen bond between Asn-169 in the active site pocket and the O(2) of εC. This structure provides the basis for the design of AAG inhibitors currently being sought as an adjuvant for cancer chemotherapy.

Methylating agents are widely distributed environmental carcinogens. Moreover, they are being used in cancer chemotherapy. The primary target of methylating agents is DNA, and therefore, DNA repair is the first-line barrier in defense against their toxic and carcinogenic effects. Methylating agents induce in the DNA O[superscript 6]-methylguanine (O[superscript 6]MeG) and methylations of the ring nitrogens of purines. The lesions are repaired by O[superscript 6]-methylguanine-DNA methyltransferase (Mgmt) and by enzymes of the base excision repair (BER) pathway, respectively. Whereas O[superscript 6]MeG is well established as a pre-carcinogenic lesion, little is known about the carcinogenic potency of base N-alkylation products such as N3-methyladenine and N3-methylguanine. To determine their role in cancer formation and the role of BER in cancer protection, we checked the response of mice with a targeted gene disruption of Mgmt or N-alkylpurine-DNA glycosylase (Aag) or both Mgmt and Aag, to azoxymethane (AOM)-induced colon carcinogenesis, using non-invasive mini-colonoscopy. We demonstrate that both Mgmt- and Aag-null mice show a higher colon cancer frequency than the wild-type. With a single low dose of AOM (3 mg/kg) Aag-null mice showed an even stronger tumor response than Mgmt-null mice. The data provide evidence that both BER initiated by Aag and O[superscript 6]MeG reversal by Mgmt are required for protection against alkylation-induced colon carcinogenesis. Further, the data indicate that non-repaired N-methylpurines are not only pre-toxic but also pre-carcinogenic DNA lesions., Deutsche Forschungsgemeinschaft (DFG) (FOR 527), Deutsche Forschungsgemeinschaft (DFG) (DFG KA 724/13-3), Deutsche Forschungsgemeinschaft (DFG) (WI 3304/1-1)

Neurons of the developing brain are especially vulnerable to environmental agents that damage DNA (i.e., genotoxicants), but the mechanism is poorly understood. The focus of the present study is to demonstrate that DNA damage plays a key role in disrupting neurodevelopment. To examine this hypothesis, we compared the cytotoxic and DNA damaging properties of the methylating agents methylazoxymethanol (MAM) and dimethyl sulfate (DMS) and the mono- and bifunctional alkylating agents chloroethylamine (CEA) and nitrogen mustard (HN2), in granule cell neurons derived from the cerebellum of neonatal wild type mice and three transgenic DNA repair strains. Wild type cerebellar neurons were significantly more sensitive to the alkylating agents DMS and HN2 than neuronal cultures treated with MAM or the half-mustard CEA. Parallel studies with neuronal cultures from mice deficient in alkylguanine DNA glycosylase (Aag[superscript −/−]) or O6-methylguanine methyltransferase (Mgmt[superscript −/−]), revealed significant differences in the sensitivity of neurons to all four genotoxicants. Mgmt−/− neurons were more sensitive to MAM and HN2 than the other genotoxicants and wild type neurons treated with either alkylating agent. In contrast, Aag[superscript −/−] neurons were for the most part significantly less sensitive than wild type or Mgmt[superscript −/−] neurons to MAM and HN2. Aag[superscript −/−] neurons were also significantly less sensitive than wild type neurons treated with either DMS or CEA. Granule cell development and motor function were also more severely disturbed by MAM and HN2 in Mgmt[superscript −/−] mice than in comparably treated wild type mice. In contrast, cerebellar development and motor function were well preserved in MAM-treated Aag[superscript −/−] or MGMT-overexpressing (Mgmt[superscript Tg+]) mice, even as compared with wild type mice suggesting that AAG protein increases MAM toxicity, whereas MGMT protein decreases toxicity. Surprisingly, neuronal development and motor function were severely disturbed in Mgmt[superscript Tg+] mice treated with HN2. Collectively, these in vitro and in vivo studies demonstrate that the type of DNA lesion and the efficiency of DNA repair are two important factors that determine the vulnerability of the developing brain to long-term injury by a genotoxicant., United States. Army Medical Research and Materiel Command (Contract/Grant/Intergovernmental Project Order DAMD 17-98-1-8625), United States. National Institutes of Health (grants CA075576), United States. National Institutes of Health (RO1 C63193), United States. National Institutes of Health (P30 CA043703)

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.

Alkylation-induced O 6 -methylguanine ( O 6 MeG) DNA lesions can be mutagenic or cytotoxic if unrepaired by the O 6 MeG-DNA methyltransferase (Mgmt) protein. O 6 MeG pairs with T during DNA replication, and if the O 6 MeG:T mismatch persists, a G:C to A:T transition mutation is fixed at the next replication cycle. O 6 MeG:T mismatch detection by MutSα and MutLα leads to apoptotic cell death, but the mechanism by which this occurs has been elusive. To explore how mismatch repair mediates O 6 MeG-dependent apoptosis, we used an Mgmt -null mouse model combined with either the Msh6 -null mutant (defective in mismatch recognition) or the Exo1 -null mutant (impaired in the excision step of mismatch repair). Mouse embryonic fibroblasts and bone marrow cells derived from Mgmt -null mice were much more alkylation-sensitive than wild type, as expected. However, ablation of either Msh6 or Exo1 function rendered these Mgmt -null cells just as resistant to alkylation-induced cytotoxicity as wild-type cells. Rapidly proliferating tissues in Mgmt -null mice (bone marrow, thymus, and spleen) are extremely sensitive to apoptosis induced by O 6 MeG-producing agents. Here, we show that ablation of either Msh6 or Exo1 function in the Mgmt -null mouse renders these rapidly proliferating tissues alkylation-resistant. However, whereas the Msh6 defect confers total alkylation resistance, the Exo1 defect leads to a variable tissue-specific alkylation resistance phenotype. Our results indicate that Exo1 plays an important role in the induction of apoptosis by unrepaired O 6 MeGs.

Human alkyladenine-DNA glycosylase (AAG) initiates base excision repair (BER) of alkylated and deaminated bases in DNA. Here, we assessed the mutability of the AAG substrate binding pocket, and the essentiality of individual binding pocket amino acids for survival of methylation damage. We used oligonucleotide-directed mutagenesis to randomize 19 amino acids, 8 of which interact with substrate bases, and created more than 4.5 million variants. We expressed the mutant AAGs in repair-deficient Escherichia coli and selected for protection against the cytotoxicity of either methylmethane sulfonate (MMS) or methyl-lexitropsin (Me-lex), an agent that produces 3-methyladenine as the predominant base lesion. Sequence analysis of 116 methylation-resistant mutants revealed no substitutions for highly conserved Tyr(127)and His(136). In contrast, one mutation, L180F, was greatly enriched in both the MMS- and Me-lex-resistant libraries. Expression of the L180F single mutant conferred 4.4-fold enhanced survival at the high dose of MMS used for selection. The homogeneous L180F mutant enzyme exhibited 2.2-fold reduced excision of 3-methyladenine and 7.3-fold reduced excision of 7-methylguanine from methylated calf thymus DNA. Decreased excision of methylated bases by the mutant glycosylase could promote survival at high MMS concentrations, where the capacity of downstream enzymes to process toxic BER intermediates may be saturated. The mutant also displayed 6.6- and 3.0-fold reduced excision of 1,N(6)-ethenoadenine and hypoxanthine from oligonucleotide substrates, respectively, and a 1.7-fold increase in binding to abasic site-containing DNA. Our work provides in vivo evidence for the substrate binding mechanism deduced from crystal structures, illuminates the function of Leu(180) in wild-type human AAG, and is consistent with a role for balanced expression of BER enzymes in damage survival.

Human lymphoblastoid cells derived from different healthy individuals display considerable variation in their transcription profiles. Here we show that such variation in gene expression underlies interindividual susceptibility to DNA damaging agents. The results demonstrate the massive differences in sensitivity across a diverse cell line panel exposed to an alkylating agent. Computational models identified 48 genes with basal expression that predicts susceptibility with 94% accuracy. Modulating transcript levels for two member genes, MYH and C21ORF56, confirmed that their expression does indeed influence alkylation sensitivity. Many proteins encoded by these genes are interconnected in cellular networks related to human cancer and tumorigenesis.

DNA interstrand cross-links (ICLs), widely used in chemotherapy, are cytotoxic lesions because they block replication and transcription. Repair of ICLs involves proteins from different repair pathways however the precise mechanism is still not completely understood. Here, we report that the 3-methyladenine DNA glycosylase (Aag), an enzyme that initiates base excision repair at a variety of alkylated bases, is also involved plays a role in the repair of ICLs. Aag−/− mouse embryonic stem cells were shown to be more sensitive to the cross-linking agent 4, 5’, 8-trimethylpsoralen than wild-type cells, but no more sensitive than wild-type to the psoralen derivative Angelicin that forms only monoadducts. We show that γ-H2AX foci formation, a marker for double strand breaks that are formed during ICL repair, is impaired in psoralen treated Aag−/− cells in both quantity and kinetics. However, in our in vitro system, purified human AAG can neither bind to the ICL nor cleave it. Taken together, our results suggest that Aag is important for the resistance of mouse ES cells to psoralen-induced ICLs. has a role in the repair of ICLs in mouse ES cells, but that its involvement may be indirect, perhaps mediated through interaction with other proteins, or by its action on an intermediate of ICL repair.

Endogenous formation of the mutagenic DNA adduct 1,N6-ethenoadenine (εA) originates from lipid peroxidation. Elevated levels of εA in cancer-prone tissues suggest a role for this adduct in the development of some cancers. The base excision repair pathway has been considered the principal repair system for εA lesions until recently, when it was shown that the Escherichia coli AlkB dioxygenase could directly reverse the damage. We report here kinetic analysis of the recombinant human AlkB homologue 2 (hABH2), which is able to repair εA lesions in DNA. Furthermore, cation exchange chromatography of nuclear extracts from wild-type and mABH2−/− mice indicates that mABH2 is the principal dioxygenase for εA repair in vivo. This is further substantiated by experiments showing that hABH2, but not hABH3, is able to complement the E. coli alkB mutant with respect to its defective repair of etheno adducts. We conclude that ABH2 is active in the direct reversal of εA lesions, and that ABH2, together with the alkyl-N-adenine-DNA glycosylase, which is the most effective enzyme for the repair of εA, comprise the cellular defense against εA lesions. [Cancer Res 2008;68(11):4142–9]

DNA glycosylases initiate base excision repair by first binding, then excising aberrant DNA bases. Saccharomyces cerevisiae encodes a 3-methyladenine (3MeA) DNA glycosylase, Mag, that recognizes 3MeA and various other DNA lesions including 1,N6-ethenoadenine (epsilon A), hypoxanthine (Hx) and abasic (AP) sites. In the present study, we explore the relative substrate specificity of Mag for these lesions and in addition, show that Mag also recognizes cisplatin cross-linked adducts, but does not catalyze their excision. Through competition binding and activity studies, we show that in the context of a random DNA sequence Mag binds epsilon A and AP-sites the most tightly, followed by the cross-linked 1,2-d(ApG) cisplatin adduct. While epsilon A binding and excision by Mag was robust in this sequence context, binding and excision of Hx was extremely poor. We further studied the recognition of epsilon A and Hx by Mag, when these lesions are present at different positions within A:T and G:C tracts. Overall, epsilon A was slightly less well excised from each position within the A:T and G:C tracts compared to excision from the random sequence, whereas Hx excision was greatly increased in these sequence contexts (by up to 7-fold) compared to the random sequence. However, given most sequence contexts, Mag had a clear preference for epsilon A relative to Hx, except in the TTXTT (X=epsilon A or Hx) sequence context from which Mag removed both lesions with almost equal efficiency. We discuss how DNA sequence context affects base excision by various 3MeA DNA glycosylases.

O(6)-methylguanine DNA methyltransferase (MGMT) deficiency is associated with an increased susceptibility to alkylating agent toxicity. To understand the contribution of MGMT in protecting against cyclophosphamide (CP)-induced toxicity, mutagenesis and tumorigenesis, we compared the biological effects of this agent in transgenic Mgmt knockout and wild-type mice. In addition, neurofibromin (Nf1)+/- background was used to increase the likelihood of CP-induced tumorigenesis. Cohorts of Mgmt-proficient or -deficient mice (either Nf1+/+ or Nf1+/-) were given 6 weekly injections of a maximally tolerated dose of CP (250 mg/kg) or vehicle and followed for 15 months. CP-treated mice had more deaths than control mice but there was no difference in the long-term survival between Mgmt+/+ and Mgmt-/- mice (12 of 83 Mgmt+/+ mice died compared to 12 of 80 Mgmt-/- mice, disregarding Nf1 status). Lymphomas and adrenal tumours were the most frequent malignancies. Interestingly, CP-treated, Mgmt-deficient mice developed fewer tumours than controls. Ten of 71 (14%) Mgmt-proficient mice developed tumours after CP treatment compared to only 2 of 68 (3%) Mgmt-deficient mice (P = 0.02). Mgmt-/-, Nf1+/- mice developed fewer tumours (1 of 35, 3%) following CP compared to Mgmt+/+, Nf1+/- mice (7 of 37, 19%) (P = 0.03). Hypoxanthine-guanine phosphoribosyltransferase mutation assays showed no significant increases in mutant frequencies in Mgmt-/- (18.1 x 10(6)) compared to Mgmt+/+ mice (12.9 x 10(6)). These data indicate that MGMT deficiency does not protect against long-term toxicity or mutagenicity from CP and appears to attenuate the occurrence of CP-induced tumours in an Nf1+/- background.

Helicobacter hepaticus causes hepatitis in susceptible strains of mice. Previous studies indicated that A/JCr mice are susceptible and C57BL/6NCr mice are resistant to H. hepaticus -induced hepatitis. We used F1 hybrid mice derived from A/J and C57BL/6 matings to investigate their phenotype and determine their hepatic gene expression profile in response to H. hepaticus infection. F1 hybrid mice, as well as parental A/J and C57BL/6 mice, were divided equally into control and H. hepaticus -infected groups and euthanized at 18 months postinoculation. Hepatic lesions were evaluated histologically and the differential hepatic gene expression in F1 mice was determined by microarray-based global gene expression profiling analysis. H. hepaticus -infected parental strains including A/J and C57BL/6 mice, as well as F1 mice, developed significant hepatitis. Overall, hepatocellular carcinomas or dysplastic liver lesions were observed in 69% of H. hepaticus -infected F1 male mice and H. hepaticus was isolated from hepatic tissues of all F1 mice with liver tumors. Liver tumors, characterized by hepatic steatosis, developed in livers with high hepatitis scores. To identify gene expression specific to H. hepaticus -induced hepatitis and progression to hepatocellular carcinoma in F1 mice, a method using comparative group transcriptome analysis was utilized. The canonical pathway most significantly enriched was immunological disease. Fatty acid synthase and steaoryl-coenzyme A desaturase, the two rate-limiting enzymes in lipogenesis, were upregulated in neoplastic relative to dysplastic livers. This study suggests a synergistic interaction between hepatic steatosis and infectious hepatitis leading to hepatocellular carcinoma. The use of AB6F1 and B6AF1 mice, as well as genetically engineered mice, on a C57BL/6 background will allow studies investigating the role of chronic microbial hepatitis and steatohepatitis in the pathogenesis of liver cancer.

DNA bases carrying an exocyclic amino group, namely adenine (A), guanine (G) and cytosine (C), encounter deamination under nitrosative stress. Oxanine (O), derived from deamination of guanine, is a cytotoxic and potentially mutagenic lesion and studies of its enzymatic repair are limited. Previously, we reported that the murine alkyladenine glycosylase (Aag) acts as an oxanine DNA glycosylase (JBC, (2004), 279: 38177). Here, we report our recent findings on additional oxanine DNA glycosylase (ODG) activities in Aag knockout mouse tissues and other mammalian tissues. Analysis of the partially purified proteins from the mammalian cell extracts indicated the existence of ODG enzymes in addition to Aag. Data obtained from oxanine DNA cleavage assays using purified human glycosylases demonstrated that two known glycosylases, hNEIL1 and hSMUG1, contained weak but detectable ODG activities. ODG activity was the highest in hAAG and lowest in hSMUG1.

Common alkylating antitumor drugs, such as temozolomide, trigger their cytotoxicity by methylating the O6-position of guanosine in DNA. However, the therapeutic effect of these drugs is dampened by elevated levels of the DNA repair enzyme, O6-methylguanine DNA methyltransferase (MGMT), which directly reverses this alkylation. As a result, assessing MGMT levels in patient samples provides an important predictor of therapeutic response; however, current methods available to measure this protein are indirect, complex and slow. Here we describe the design and synthesis of fluorescent chemosensors that report directly on MGMT activity in a single step within minutes. The chemosensors incorporate a fluorophore and quencher pair, which become separated by the MGMT dealkylation reaction, yielding light-up responses of up to 55-fold, directly reflecting repair activity. Experiments show that the best-performing probe retains near-native activity at mid-nanomolar concentrations. A nuclease-protected probe, NR-1, was prepared and tested in tumor cell lysates, demonstrating an ability to evaluate relative levels of MGMT repair activity in twenty minutes. In addition, a probe was employed to evaluate inhibitors of MGMT, suggesting utility for discovering new inhibitors in a high-throughput manner. Probe designs such as that of NR-1 may prove valuable to clinicians in selection of patients for alkylating drug therapies and in assessing resistance that arises during treatment., National Institutes of Health (U.S.) (DP1-ES 022576)

Somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin ( Ig ) genes require the cytosine deaminase AID, which deaminates cytosine to uracil in Ig gene DNA. Paradoxically, proteins involved normally in error-free base excision repair and mismatch repair, seem to be co-opted to facilitate SHM and CSR, by recruiting error-prone translesion polymerases to DNA sequences containing deoxy-uracils created by AID. Major evidence supports at least one mechanism whereby the uracil glycosylase Ung removes AID-generated uracils creating abasic sites which may be used either as uninformative templates for DNA synthesis, or processed to nicks and gaps that prime error-prone DNA synthesis. We investigated the possibility that deamination at adenines also initiates SHM. Adenosine deamination would generate hypoxanthine (Hx), a substrate for the alkyladenine DNA glycosylase (Aag). Aag would generate abasic sites which then are subject to error-prone repair as above for AID-deaminated cytosine processed by Ung. If the action of an adenosine deaminase followed by Aag were responsible for significant numbers of mutations at A, we would find a preponderance of A:T > G:C transition mutations during SHM in an Aag deleted background. However, this was not observed and we found that the frequencies of SHM and CSR were not significantly altered in Aag −/− mice. Paradoxically, we found that Aag is expressed in B lymphocytes undergoing SHM and CSR and that its activity is upregulated in activated B cells . Moreover, we did find a statistically significant, albeit low increase of T:A > C:G transition mutations in Aag −/− animals, suggesting that Aag may be involved in creating the SHM A > T bias seen in wild type mice.

Genome wide experiments indicate both proteasome- and vacuole-mediated protein degradation modulate sensitivity to classical DNA-damaging agents. Here, we show that global protein degradation is significantly increased upon methyl methanesulfonate (MMS) exposure. In addition, global protein degradation is similarly increased upon exposure to 4-nitroquinoline-N-oxide (4NQO) and UV and, to a lesser extent, tert-butyl hydroperoxide. The proteasomal inhibitor MG132 decreases both MMS-induced and 4NQO-induced protein degradation, while addition of the vacuolar inhibitor phenylmethanesulfonyl fluoride does not. The addition of both inhibitors grossly inhibits cell growth upon MMS exposure over and above the growth inhibition induced by MMS alone. The MMS-induced protein degradation response remains unchanged in several ubiquitin-proteasome and vacuolar mutants, presumably because these mutants are not totally deficient in either essential pathway. Furthermore, MMS-induced protein degradation is independent of Mec1, Mag1, Rad23, and Rad6, suggesting that the protein degradation response is not transduced through the classical Mec1 DNA damage response pathway or through repair intermediates generated by the base excision, nucleotide excision, or postreplication-DNA repair pathways. These results identify the regulation of protein degradation as an important factor in the recovery of cells from toxicity induced by classical DNA-damaging agents.

Objective Endogenous and exogenous estrogens influence breast cancer risk by interacting with estrogen receptor (ER). The O6-methylguanine DNA methyltransferase (MGMT) gene has a dual role in repairing alkylation damage and in inhibiting ER-mediated cell proliferation. We assessed the two MGMT polymorphisms, Leu84Phe and Ile143Val, with breast cancer risk. We also evaluated the potential interactions between the two polymorphisms and estrogen-related risk factors and cigarette smoking on breast cancer risk. Methods We conducted a nested case–control study within the Nurses’ Health Study (1311 cases, 1760 controls). Results Compared with the 84Leu/Leu genotype, the Phe/Phe genotype had a multivariate odds ratio (OR) of 1.68 (95% confidence interval (CI), 0.98–2.88). This positive association was magnified among postmenopausal women with body mass index>25 (OR, 3.01; 95% CI, 1.30–6.94), those in the highest tertile of pre-diagnostic plasma endogenous estradiol levels (Phe carriers versus non-carriers, OR, 2.42; 95% CI, 1.49–3.94), non-current postmenopausal hormone users (OR, 2.60; 95% CI, 1.19–5.64), and possibly estrogen receptor-positive cases (OR, 1.82; 95% CI, 0.99–3.35). We did not observe a main effect of the Ile143Val polymorphism or its interactions with these factors. No interaction was observed between either of the polymorphisms and cigarette smoking on breast cancer risk. Conclusions These data suggest that the Leu84Phe polymorphism affect the capacity of MGMT to inhibit estrogen receptor-mediated cell proliferation and is associated with breast cancer risk.

Failure of cells to respond to DNA damage is a primary event associated with mutagenesis and environmental toxicity. To map the transcriptional network controlling the damage response, we measured genomewide binding locations for 30 damage-related transcription factors (TFs) after exposure of yeast to methyl-methanesulfonate (MMS). The resulting 5272 TF-target interactions revealed extensive changes in the pattern of promoter binding and identified damage-specific binding motifs. As systematic functional validation, we identified interactions for which the target changed expression in wild-type cells in response to MMS but was nonresponsive in cells lacking the TF. Validated interactions were assembled into causal pathway models that provide global hypotheses of how signaling, transcription, and phenotype are integrated after damage.

Oxidative stress converts lipids into DNA-damaging agents. The genomic lesions formed include 1,N(6)-ethenoadenine (epsilonA) and 3,N(4)-ethenocytosine (epsilonC), in which two carbons of the lipid alkyl chain form an exocyclic adduct with a DNA base. Here we show that the newly characterized enzyme AlkB repairs epsilonA and epsilonC. The potent toxicity and mutagenicity of epsilonA in Escherichia coli lacking AlkB was reversed in AlkB(+) cells; AlkB also mitigated the effects of epsilonC. In vitro, AlkB cleaved the lipid-derived alkyl chain from DNA, causing epsilonA and epsilonC to revert to adenine and cytosine, respectively. Biochemically, epsilonA is epoxidized at the etheno bond. The epoxide is putatively hydrolyzed to a glycol, and the glycol moiety is released as glyoxal. These reactions show a previously unrecognized chemical versatility of AlkB. In mammals, the corresponding AlkB homologs may defend against aging, cancer and oxidative stress.

Using genome-wide information to understand holistically how cells function is a major challenge of the postgenomic era. Recent efforts to understand molecular pathway operation from a global perspective have lacked experimental data on phenotypic context, so insights concerning biologically relevant network characteristics of key genes or proteins have remained largely speculative. Here, we present a global network investigation of the genotype/phenotype data set we developed for the recovery of the yeast Saccharomyces cerevisiae from exposure to DNA-damaging agents, enabling explicit study of how protein–protein interaction network characteristics may be associated with phenotypic functional effects. We show that toxicity-modulating proteins have similar topological properties as essential proteins, suggesting that cells initiate highly coordinated responses to damage similar to those needed for vital cellular functions. We also identify toxicologically important protein complexes, pathways, and modules. These results have potential implications for understanding toxicity-modulating processes relevant to a number of human diseases, including cancer and aging.

DNA repair and checkpoint pathways protect against carcinogen-induced toxicity. Here, we describe additional, equally protective pathways discovered by interrogating 4,733 yeast proteins for their ability to diminish toxicity induced by four known carcinogens. A computational mapping strategy for global phenotypic data was developed to build a systems toxicology model detailing recovery from carcinogen exposure and identifying protein complexes that modulate toxicity. Global phenotypic data were merged with global subcellular localization and protein interactome data to generate an integrated picture of cellular recovery after carcinogen exposure. Statistically validated results from this systems-wide integration demonstrate that, in addition to the nucleus, subnetworks of toxicity-modulating proteins were overrepresented in the vacuolar membrane, endosome, endoplasmic reticulum, and mitochondrion. In addition, we show that many proteins associated with RNA polymerase II, macromolecular trafficking, and vacuole function can now be counted among the many proteins that modulate carcinogen-induced toxicity.

The mouse alkyladenine DNA glycosylase (Aag) initiates base excision repair with a broad substrate range that includes the highly mutagenic exocyclic etheno DNA base adduct 1,N6-ethenodeoxyadenosine ((epsilon)dA). Previous attempts to determine the in vivo role of Aag in (epsilon)dA repair were complicated by technological difficulties in measuring low levels of (epsilon)dA in genomic DNA. Here we describe the development of a new method for (epsilon)dA detection in genomic DNA that couples an immunoaffinity purification with LC-MS/MS analysis and that utilizes an isotopically labeled internal standard. We go on to describe the application of this method to measuring the in vivo repair of (epsilon)dA base lesions in liver and lung tissue of wild type and Aag null mice. Our results demonstrate that while Aag clearly represents the major DNA repair enzyme for the in vivo removal (epsilon)dA bases, these lesions can also be eliminated from the genome via an alternative mechanism.

The Meeting consisted of 9 Symposia, 4 Keynote Lectures, 3 Platform Sessions and 4 Poster Sessions. In addition there were Breakfast Meetings for Special Interest Groups designed to inform attendees about the latest advances in environmental mutagenesis research. Several of the topics to be covered at this broad meeting will be of interest to the Department of Energy, Office of Science. The relevance of this meeting to the DOE derives from the fact that low dose radiation may represent one of the most significant sources of human mutations that are attributable to the environment. The EMS membership, and those who attended the EMS Annual Meeting were interested in both chemical and radiation induced biological effects, such as cell death, mutation, teratogenesis, carcinogenesis and aging. These topics thate were presented at the 2004 EMS Annual meeting that were of clear interest to DOE include: human variation in cancer susceptibility, unusual mechanisms of mutation, germ and stem cell mutagenesis, recombination and the maintenance of genomic stability, multiple roles for DNA mismatch repair, DNA helicases, mutation, cancer and aging, Genome-wide transcriptional responses to environmental change, Telomeres and genomic stability: when ends don?t meet, systems biology approach to cell phenotypic decision processes, and themore » surprising biology of short RNAs. Poster and platform sessions addressed topics related to environmental mutagen exposure, DNA repair, mechanisms of mutagenesis, epidemiology, genomic and proteomics and bioinformatics. These sessions were designed to give student, postdocs and more junior scientists a chance to present their workl.« less

Chronic infection and associated inflammation are key contributors to human carcinogenesis. Ulcerative colitis (UC) is an oxyradical overload disease and is characterized by free radical stress and colon cancer proneness. Here we examined tissues from noncancerous colons of ulcerative colitis patients to determine (a) the activity of two base excision–repair enzymes , AAG, the major 3-methyladenine DNA glycosylase, and APE1, the major apurinic site endonuclease; and (b) the prevalence of microsatellite instability (MSI). AAG and APE1 were significantly increased in UC colon epithelium undergoing elevated inflammation and MSI was positively correlated with their imbalanced enzymatic activities. These latter results were supported by mechanistic studies using yeast and human cell models in which overexpression of AAG and/or APE1 was associated with frameshift mutations and MSI. Our results are consistent with the hypothesis that the adaptive and imbalanced increase in AAG and APE1 is a novel mechanism contributing to MSI in patients with UC and may extend to chronic inflammatory or other diseases with MSI of unknown etiology.

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.

Regulated necrosis has emerged as a major cell death mechanism in response to different forms of physiological and pharmacological stress. The AlkB homolog 7 (ALKBH7) protein is required for regulated cellular necrosis in response to chemotherapeutic alkylating agents but its role within a whole organism is unknown. Here, we show that ALKBH7 modulates alkylation-induced cellular death through a tissue and sex-specific mechanism. At the whole-animal level, we find that ALKBH7 deficiency confers increased resistance to MMS-induced toxicity in male but not female mice. Moreover, ALKBH7-deficient mice exhibit protection against alkylation-mediated cytotoxicity in retinal photoreceptor and cerebellar granule cells, two cell types that undergo necrotic death through the initiation of the base excision repair pathway and hyperactivation of the PARP1/ARTD1 enzyme. Notably, the protection against alkylation-induced cerebellar degeneration is specific to ALKBH7-deficient male but not female mice. Our results uncover an in vivo role for ALKBH7 in mediating a sexually dimorphic tissue response to alkylation damage that could influence individual responses to chemotherapies based upon alkylating agents.

Inflammation is accompanied by the release of highly reactive oxygen and nitrogen species (RONS) that damage DNA, among other cellular molecules. Base excision repair (BER) is initiated by DNA glycosylases and is crucial in repairing RONS-induced DNA damage; the alkyladenine DNA glycosylase (Aag/Mpg) excises several DNA base lesions induced by the inflammation-associated RONS release that accompanies ischemia reperfusion (I/R). Using mouse I/R models we demonstrate that Aag(-/-) mice are significantly protected against, rather than sensitized to, I/R injury, and that such protection is observed across three different organs. Following I/R in liver, kidney, and brain, Aag(-/-) mice display decreased hepatocyte death, cerebral infarction, and renal injury relative to wild-type. We infer that in wild-type mice, Aag excises damaged DNA bases to generate potentially toxic abasic sites that in turn generate highly toxic DNA strand breaks that trigger poly(ADP-ribose) polymerase (Parp) hyperactivation, cellular bioenergetics failure, and necrosis; indeed, steady-state levels of abasic sites and nuclear PAR polymers were significantly more elevated in wild-type vs. Aag(-/-) liver after I/R. This increase in PAR polymers was accompanied by depletion of intracellular NAD and ATP levels plus the translocation and extracellular release of the high-mobility group box 1 (Hmgb1) nuclear protein, activating the sterile inflammatory response. We thus demonstrate the detrimental effects of Aag-initiated BER during I/R and sterile inflammation, and present a novel target for controlling I/R-induced injury.

Cancer susceptibility varies between people, affected by genotoxic exposures, genetic makeup and physiological state. Yet, how these factors interact among each other to define cancer risk is largely unknown. Here, we uncover the interactive effects of genetical, environmental and physiological factors on genome rearrangements driven by homologous recombination (HR). Using FYDR mice to quantify HR-driven rearrangements in pancreas tissue, we show that DNA methylation damage (induced by methylnitrosourea) and cell proliferation (induced by thyroid hormone) each induce HR and together act synergistically to induce HR-driven rearrangements in vivo. These results imply that developmental or regenerative proliferation as well as mitogenic exposures may sensitize tissues to DNA damaging exposures. We exploited mice genetically deficient in alkyl-adenine DNA glycosylase (Aag) to analyse the relative contributions of unrepaired DNA base lesions versus intermediates formed during base excision repair (BER). Remarkably, results show that, in the pancreas, Aag is a major driver of spontaneous HR, indicating that BER intermediates (including abasic sites and single strand breaks) are more recombinogenic than the spontaneous base lesions removed by Aag. Given that mammals have about a dozen DNA glycosylases, these results point to BER as a major source of pressure on the HR pathway in vivo. Taken together, methylation damage, cell proliferation and Aag interact to define the risk of HR-driven sequence rearrangements in vivo. These data identify important sources of sequence changes in a cancer-relevant organ, and advance the effort to identify populations at high-risk for cancer.

Why does a constant barrage of DNA damage lead to disease in some individuals, while others remain healthy? This article surveys current work addressing the implications of inter-individual variation in DNA repair capacity for human health, and discusses the status of DNA repair assays as potential clinical tools for personalized prevention or treatment of disease. In particular, we highlight research showing that there are significant inter-individual variations in DNA repair capacity (DRC), and that measuring these differences provides important biological insight regarding disease susceptibility and cancer treatment efficacy. We emphasize work showing that it is important to measure repair capacity in multiple pathways, and that functional assays are required to fill a gap left by genome wide association studies, global gene expression and proteomics. Finally, we discuss research that will be needed to overcome barriers that currently limit the use of DNA repair assays in the clinic.

The capacity to repair different types of DNA damage varies among individuals, making them more or less susceptible to the detrimental health consequences of damage exposures. Current methods for measuring DNA repair capacity (DRC) are relatively labor intensive, often indirect, and usually limited to a single repair pathway. Here, we describe a fluorescence-based multiplex flow-cytometric host cell reactivation assay (FM-HCR) that measures the ability of human cells to repair plasmid reporters, each bearing a different type of DNA damage or different doses of the same type of DNA damage. FM-HCR simultaneously measures repair capacity in any four of the following pathways: nucleotide excision repair, mismatch repair, base excision repair, nonhomologous end joining, homologous recombination, and methylguanine methyltransferase. We show that FM-HCR can measure interindividual DRC differences in a panel of 24 cell lines derived from genetically diverse, apparently healthy individuals, and we show that FM-HCR may be used to identify inhibitors or enhancers of DRC. We further develop a next-generation sequencing-based HCR assay (HCR-Seq) that detects rare transcriptional mutagenesis events due to lesion bypass by RNA polymerase, providing an added dimension to DRC measurements. FM-HCR and HCR-Seq provide powerful tools for exploring relationships among global DRC, disease susceptibility, and optimal treatment.

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.

Exposure to carcinogenic alkylating agents, oxidizing agents, and ionizing radiation modulates transcript levels for over one third of Saccharomyces cerevisiae's 6,200 genes. Computational analysis delineates groups of coregulated genes whose upstream regions bear known and novel regulatory sequence motifs. One group of coregulated genes contain a number of DNA excision repair genes (including the MAG1 3-methyladenine DNA glycosylase gene) and a large selection of protein degradation genes. Moreover, transcription of these genes is modulated by the proteasome-associated protein Rpn4, most likely via its binding to MAG1 upstream repressor sequence 2-like elements, that turn out to be almost identical to the recently identified proteasome-associated control element (G. Mannhaupt, R. Schnall, V. Karpov, I. Vetter, and H. Feldmann, FEBS Lett. 450:27–34, 1999). We have identified a large number of genes whose transcription is influenced by Rpn4p.

Base excision repair (BER), as initiated by at least seven different DNA glycosylases or by enzymes that cleave DNA at abasic sites, executes the repair of a wide variety of DNA damages. Many of these damages arise spontaneously because DNA interacts with the cellular milieu, and so BER profoundly influences spontaneous mutation rates. In addition, BER provides significant protection against the toxic and mutagenic effects of DNA damaging agents present in the external environment, and as such is likely to prevent the adverse health effects of such agents. BER pathways have been studied in a wide variety of organisms (including yeasts) and here we review how these varied studies have shaped our current view of human BER.

DNA damage is unavoidable, and organisms across the evolutionary spectrum possess DNA repair pathways that are critical for cell viability and genomic stability. To understand the role of base excision repair (BER) in protecting eukaryotic cells against alkylating agents, we generated Schizosaccharomyces pombe strains mutant for the mag1 3-methyladenine DNA glycosylase gene. We report that S. pombe mag1 mutants have only a slightly increased sensitivity to methylation damage, suggesting that Mag1-initiated BER plays a surprisingly minor role in alkylation resistance in this organism. We go on to show that other DNA repair pathways play a larger role than BER in alkylation resistance. Mutations in genes involved in nucleotide excision repair ( rad13 ) and recombinational repair ( rhp51 ) are much more alkylation sensitive than mag1 mutants. In addition, S. pombe mutant for the flap endonuclease rad2 gene, whose precise function in DNA repair is unclear, were also more alkylation sensitive than mag1 mutants. Further, mag1 and rad13 interact synergistically for alkylation resistance, and mag1 and rhp51 display a surprisingly complex genetic interaction. A model for the role of BER in the generation of alkylation-induced DNA strand breaks in S. pombe is discussed.

Alkylating agents represent a highly cytotoxic class of chemotherapeutic compounds that are extremely effective anti-tumor agents. Unfortunately, alkylating agents damage both malignant and non-malignant tissues. Bone marrow is especially sensitive to damage by alkylating agent chemotherapy, and is a dose-limiting tissue when treating cancer patients. One strategy to overcome bone marrow sensitivity to alkylating agent exposure involves gene transfer of the DNA repair protein O(6)-methylguanine DNA methyltransferase (O(6)MeG DNA MTase) into bone marrow cells. O(6)MeG DNA MTase is of particular interest because it functions to protect against the mutagenic, clastogenic and cytotoxic effects of many chemotherapeutic alkylating agents. By increasing the O(6)MeG DNA MTase repair capacity of bone marrow cells, it is hoped that this tissue will become alkylation resistant, thereby increasing the therapeutic window for the selective destruction of malignant tissue. In this review, the field of O(6)MeG DNA MTase gene transfer into bone marrow cells will be summarized with an emphasis placed on strategies used for suppressing the deleterious side effects of chemotherapeutic alkylating agent treatment.

One important question facing the field of DNA repair is: how does the cell sense and respond to the presence of DNA damage? Many sessions at this meeting discussed the potential signalling cascades that might contribute to this poorly understood process. There are many possible players, including some or all of the following: the DNA lesion itself, ‘naked’ single-stranded DNA, stalled replication and transcription complexes, MMR proteins bound to DNA damage, futile DNA repair cycles, protein kinases including DNA-PK and ATM, and cell-cycle regulatory proteins including p53, p73 and p21. Modrich presented evidence that MMR proteins act at an early step in this process: both hMutSα and hMutLα are required for DNA-damage-dependent phosphorylation of p537xhMutSα- and hMutLα-dependent phosphorylation of p53 in response to DNA methylator damage. Duckett, D.R. et al. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12384–12388Crossref | PubMed | Scopus (146)See all References7, and others have shown that hMutSα is required for the induction of apoptosis in response to certain alkylated DNA bases8xRole of DNA mismatch repair and p53 in signaling induction of apoptosis by alkylating agents. Hickman, M.J. and Samson, L.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10764–10769Crossref | PubMed | Scopus (248)See all References8. Thus, eukaryotic MMR proteins might regulate directly or indirectly at least one branch of the DNA-damage-dependent signalling cascade. This is an important area for future studies and is likely to show significant progress between now and the next Mega-Meeting on DNA Repair and Mutagenesis.