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John Cairns was a magnet for scientists (and non-scientists) of all types, from undergraduates to Nobel laureates. For him science was fun, and discussing science with others even more fun. Soon after finishing medical school (Oxford) at the end of the Second World War he gravitated towards research, his first publication linking the incidence of penicillin-resistant bacteria with long hospital stays. He then studied virology with Macfarlane Burnet FRS in Melbourne, Australia, after which he repaid his two years of National Service from the Second World War at the Virus Research Institute in Entebbe, Uganda. He returned to Australia (Canberra), continuing influenza virus research. However, while visiting Caltech in the 1950s, he was exposed to the emerging field of molecular biology, of which he became a pioneer. After returning to Canberra from a sabbatical with Alfred Hershey at Cold Spring Harbor in 1961, he showed that the Escherichia coli genome is circular, and demonstrated the replication fork. In 1963 he was appointed director of the Cold Spring Harbor Laboratory (CSHL). He successfully saved CSHL from bankruptcy before handing over directorship to Jim Watson (ForMemRS 1981). At CSHL he proved that the so-called Kornberg DNA polymerase was not required for replication of the E. coli genome. In the 1970s Cairns turned his attention to understanding cancer and moved to North London to direct a small research institute in Mill Hill, a satellite of the Imperial Cancer Research Fund based in central London. Cairns wrote two influential reviews on cancer plus the book Cancer: science and society , seamlessly weaving together information across disciplines, from human population levels to molecular levels, plus everything in between. He fostered multi-disciplinary basic research at the Mill Hill labs, recruiting scientists who used model organisms such as bacteria, fruit flies, slime moulds, amphibians and mice as models to explore the determinants of cell fate during development; such pathways seemed likely to be involved in the transformation of normal cells into cancer cells. Cairns created and fostered an exceptionally stimulating, interactive, nurturing and cutting-edge research environment that launched the careers of many scientists. In his own lab at Mill Hill, Cairns discovered new pathways for DNA alkylation repair in E. coli , and subsequently as professor of cancer biology at the Harvard School of Public Health he discovered what is now known as stress-induced mutagenesis, also in E. coli .

Table S1. Relative sensitivity of lymphoblastoid cell lines to alkylating agents; Table S2. DNA repair capacity data for 24 cell lines. Table S3. Correlation coefficients for pairwise linear relationships between DNA repair capacity in multiple pathways in 24 lymphoblastoid cell lines. Table S4. Statistical significance of linear relationships between DNA repair capacity in multiple pathways in 24 lymphoblastoid cell lines. Table S5. Correlation coefficients for pairwise linear relationships between DNA repair capacity and sensitivity to killing with DNA damaging agents in 24 lymphoblastoid cell lines. Table S6. Statistical significance of linear relationships between DNA repair capacity and sensitivity to killing with DNA damaging agents in 24 lymphoblastoid cell lines. Table S7. DNA Repair capacity for indicated repair pathways in PDX models of GBM. Figure S1. Computational Workflow. Figure S2. Diagram of a linear model relating MGMT activity to sensitivity to MNNG. Figure S3. Correlation between MGMT activity (as measured by FM-HCR) and sensitivity to a DNA damaging agent. Figure S4. Scatter plots of observed sensitivity (% Control Growth) versus predicted sensitivity calculated from leave-one-out analysis of the models listed in Table 1. Figure S5. Predicted and observed sensitivity of paired GBM xenograft lines with differential TMZ sensitivities. Figure S6. Correlation between MGMT activity and promoter methylation status.

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 to predict glioblastoma response to treatment with the chemotherapeutic DNA-damaging agent temozolomide. 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 temozolomide resistance in PDX models and led to reduced relative survival prolongation following temozolomide 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 temozolomide resistance than assessments of MGMT activity alone. Cancer Res; 77(1); 198–206. ©2016 AACR.

Supplementary Figures S1-S14. p53 levels in p53 knockdown GBM cells (S1); TMZR3 cells obtained from a p53 deficient background display increased ploidy (S2); H2AX activation in temozolomide treated parental and TMZR3 GBM cells (S3); In-cell Host cell reactivation (HCR) assays used to assess the MGMT and MMR repair capacity of GBM cells (S4); MSH6 and MSH2 levels in MSH6 and MSH2 knockdown GBM cells (S5); Cell cycle profiles and quantitation of cell cycle changes in TMZ-treated MSH6 knockdown cells two cell cycle times post-TMZ treatment (S6); Cell cycle profiles and quantitation of cell cycle changes in TMZ-treated MSH2 knockdown cells two cell cycle times post-TMZ treatment (S7); Minor decreases in MSH2 levels correlate with acquired TMZ resistance in LN229 and A172 GBM cells (S8); Effects of MSH6 or MSH2 knockdown on the protein level of its dimerization partner (S9); Msh2 knockdown in GL261 glioblastoma cells (S10); Moderate decreases in Msh2 confer a growth advantage to GL261 GBM cells after TMZ, but not BCNU, exposure in vitro (S11); Distribution of patient survival in TMZ treated TCGA GBM patients and effects of MSH2 transcript levels on the survival of TMZ treated patients in the 95th percentile for patient survival after TMZ therapy (S12); Overall survival of GBM patients stratified by MSH3, MLH1 and PMS2 tumor transcript levels (S13); PMS2 transcript levels correlate with increased chromosome 7 copy number (S14).

Supplementary Tables S1-S2. O6-methylguanine adducts levels in parental and TMZR3 GBM cells 3 hours post-TMZ exposure (S1); shRNA constructs used in this study (S2).

Supplementary Methods and Discussion. Description of additional methods and procedures used in the study. Also includes Supplementary References.

DNA-methylating environmental carcinogens such as N-nitrosodimethylamine (NDMA) and certain alkylators used in chemotherapy form O6-methylguanine (m6G) as a functionally critical intermediate. NDMA is a multi-organ carcinogen found in contaminated water, polluted air, preserved foods, tobacco products, and many pharmaceuticals. Only ten weeks after exposure to NDMA, neonatally-treated mice experienced elevated mutation frequencies in liver, lung and kidney of ∼35-fold, 4-fold and 2-fold, respectively. High-resolution mutational spectra (HRMS) of liver and lung revealed distinctive patterns dominated by GC→AT mutations in 5’-Pu-G-3’ contexts, very similar to human COSMIC mutational signature SBS11. Commonly associated with alkylation damage, SBS11 appears in cancers treated with the DNA alkylator temozolomide (TMZ). When cells derived from the mice were treated with TMZ, N-methyl-N-nitrosourea, and streptozotocin (two other therapeutic methylating agents), all displayed NDMA-like HRMS, indicating mechanistically convergent mutational processes. The role of m6G in shaping the mutational spectrum of NDMA was probed by removing MGMT, the main cellular defense against m6G. MGMT-deficient mice displayed a strikingly enhanced mutant frequency, but identical HRMS, indicating that the mutational properties of these alkylators is likely owed to sequence-specific DNA binding. In sum, the HRMS of m6G-forming agents constitute an early-onset biomarker of exposure to DNA methylating carcinogens and drugs.

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.

Alkylating agents damage DNA and proteins and are widely used in cancer chemotherapy. While cellular responses to alkylation-induced DNA damage have been explored, knowledge of how alkylation affects global cellular stress responses is sparse. Here, we examined the effects of the alkylating agent methylmethane sulfonate (MMS) on gene expression in mouse liver, using mice deficient in alkyladenine DNA glycosylase (Aag), the enzyme that initiates the repair of alkylated DNA bases. MMS induced a robust transcriptional response in wild-type liver that included markers of the endoplasmic reticulum (ER) stress/unfolded protein response (UPR) known to be controlled by XBP1, a key UPR effector. Importantly, this response is significantly reduced in the Aag knockout. To investigate how AAG affects alkylation-induced UPR, the expression of UPR markers after MMS treatment was interrogated in human glioblastoma cells expressing different AAG levels. Alkylation induced the UPR in cells expressing AAG; conversely, AAG knockdown compromised UPR induction and led to a defect in XBP1 activation. To verify the requirements for the DNA repair activity of AAG in this response, AAG knockdown cells were complemented with wild-type Aag or with an Aag variant producing a glycosylase-deficient AAG protein. As expected, the glycosylase-defective Aag does not fully protect AAG knockdown cells against MMS-induced cytotoxicity. Remarkably, however, alkylation-induced XBP1 activation is fully complemented by the catalytically inactive AAG enzyme. This work establishes that, besides its enzymatic activity, AAG has noncanonical functions in alkylation-induced UPR that contribute to cellular responses to alkylation.

Following publication of the original article [1], the author reported that incorrect version of Tables 1, 3, 5 and 6 were published.

Alkylating agents damage DNA and proteins and are widely used in cancer chemotherapy. While the cellular responses to alkylation-induced DNA damage have been explored, knowledge of how alkylation damage affects global cellular stress responses is still sparse. Here, we examined the effects of the alkylating agent methylmethane sulfonate (MMS) on gene expression in mouse liver taking advantage of mice deficient in alkyladenine DNA glycosylase (Aag), the enzyme that initiates the repair of alkylated DNA bases. MMS induced a robust transcriptional response in wild-type liver that included markers of the endoplasmic reticulum (ER) stress/unfolded protein response (UPR) known to be controlled by the transcription factor XBP1, a key UPR effector. Importantly, this response is significantly reduced in the Aag knockout. To investigate a potential role for AAG in alkylation-induced UPR, the expression of UPR markers after MMS treatment was interrogated in human glioblastoma cell lines expressing different AAG levels. Alkylation induced the UPR in cells expressing AAG; conversely, AAG knock-down compromised UPR induction and led to a defect in XBP1 activation plus a decrease in the expression of the ER chaperone BiP. To verify that the DNA repair activity of AAG is required for this response, AAG knockdown cells were complemented with wild-type Aag or with a mutant version of the Aag gene producing a glycosylase-deficient AAG protein. As expected, the glycosylase-defective mutant Aag does not fully protect AAG knockdown cells against MMS-induced cytotoxicity. Remarkably, however, alkylation-induced XBP1 activation is fully complemented by the catalytically inactive AAG enzyme. This work establishes that, in addition to its enzymatic activity, AAG has non-canonical functions in alkylation-induced UPR that contribute to the overall cellular response to alkylation.Significance StatementStress response pathways, such as the DNA damage response (DDR) and the UPR, are critical in both the etiology and treatment of cancer and other chronic diseases. Knowledge of an interplay between ER stress and genome damage repair is emerging, but evidence linking defective DNA repair and impaired ER stress response is lacking. Here, we show that AAG is necessary for UPR activation in response to alkylating agents. AAG-deficient mice and human cancer cells are impaired in alkylation-induced UPR. Strikingly, this defect can be complemented by an AAG variant defective in glycosylase activity. Our studies suggest AAG has non-canonical functions and identify AAG as a point of convergence for stress response pathways. This knowledge could be explored to improve cancer treatment.

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.

SummaryN-nitrosodimethylamine (NDMA) is a DNA methylating agent that has been discovered to contaminate water, food and drugs. The alkyladenine glycosylase (AAG) removes methylated bases to initiate the base excision repair (BER) pathway. To understand how gene-environment interactions impact disease susceptibility, we studied Aag−/− and Aag-overexpressing mice that harbor increased levels of either replication-blocking lesions (3-methyladenine, or 3MeA) or strand breaks (BER intermediates), respectively. Remarkably, the disease outcome switched from cancer to lethality simply by changing AAG levels. To understand the underlying basis for this observation, we integrated a suite of molecular, cellular and physiological analyses. We found that unrepaired 3MeA is somewhat toxic but highly mutagenic (promoting cancer), whereas excess strand breaks are poorly mutagenic and highly toxic (suppressing cancer and promoting lethality). We demonstrate that the levels of a single DNA repair protein tips the balance between blocks and breaks, and thus dictates the disease consequences of DNA damage.

// Mariacarmela Allocca 1, 3 , Joshua J. Corrigan 1, 3 , Kimberly R. Fake 1, 3 , Jennifer A. Calvo 1, 3 and Leona D. Samson 1, 2, 3, 4 1 Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA 2 Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA 3 Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA 4 David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA Correspondence to: Leona D. Samson, email: lsamson@mit.edu Keywords: alkylating agents, PARP1, PARP inhibitors, AAG/MPG, retinal and cerebellar degeneration Received: April 19, 2017 Accepted: June 08, 2017 Published: August 03, 2017 ABSTRACT Alkylating agents are commonly used to treat cancer. Although base excision repair (BER) is a major pathway for repairing DNA alkylation damage, under certain conditions, the initiation of BER produces toxic repair intermediates that damage healthy tissues. The initiation of BER by the alkyladenine DNA glycosylase (AAG, a.k.a. MPG) can mediate alkylation-induced cytotoxicity in specific cells in the retina and cerebellum of male mice. Cytotoxicity in both wild-type and Aag -transgenic ( AagTg ) mice is abrogated in the absence of Poly(ADP-ribose) polymerase-1 (PARP1). Here, we tested whether PARP inhibitors can also prevent alkylation-induced retinal and cerebellar degeneration in male and female WT and AagTg mice. Importantly, we found that WT mice display sex-dependent alkylation-induced retinal damage (but not cerebellar damage), with WT males being more sensitive than females. Accordingly, estradiol treatment protects males against alkylation-induced retinal degeneration. In AagTg male and female mice, the alkylation-induced tissue damage in both the retina and cerebellum is exacerbated and the sex difference in the retina is abolished. PARP inhibitors, much like Parp1 gene deletion, protect against alkylation-induced AAG-dependent neuronal degeneration in WT and AagTg mice, regardless of the gender, but their efficacy in preventing alkylation-induced neuronal degeneration depends on PARP inhibitor characteristics and doses. The recent surge in the use of PARP inhibitors in combination with cancer chemotherapeutic alkylating agents might represent a powerful tool for obtaining increased therapeutic efficacy while avoiding the collateral effects of alkylating agents in healthy tissues.

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.

Background Growing evidence indicates that in utero arsenic exposures in humans may increase the risk of adverse health effects and development of diseases later in life. This study aimed to evaluate potential health risks of in utero arsenic exposure on genetic damage in newborns in relation to maternal arsenic exposure. Methods A total of 205 pregnant women residing in arsenic-contaminated areas in Hanam province, Vietnam, were recruited. Prenatal arsenic exposure was determined by arsenic concentration in mother’s toenails and urine during pregnancy and in umbilical cord blood collected at delivery. Genetic damage in newborns was assessed by various biomarkers of early genetic effects including oxidative/nitrative DNA damage (8-hydroxydeoxyguanosine, 8-OHdG, and 8-nitroguanine), DNA strand breaks and micronuclei (MN) in cord blood. Results Maternal arsenic exposure, measured by arsenic levels in toenails and urine, was significantly increased (p

Certain Escherichia coli strains residing in the human gut produce colibactin, a small-molecule genotoxin implicated in colorectal cancer pathogenesis. However, colibactin’s chemical structure and the molecular mechanism underlying its genotoxic effects have remained unknown for more than a decade. Here we combine an untargeted DNA adductomics approach with chemical synthesis to identify and characterize a covalent DNA modification from human cell lines treated with colibactin-producing E. coli. Our data establish that colibactin alkylates DNA with an unusual electrophilic cyclopropane. We show that this metabolite is formed in mice colonized by colibactin-producing E. coli and is likely derived from an initially formed, unstable colibactin-DNA adduct. Our findings reveal a potential biomarker for colibactin exposure and provide mechanistic insights into how a gut microbe may contribute to colorectal carcinogenesis., National Institutes of Health (U.S.) (Grant R01 ES022872), National Institute of Environmental Health Sciences (Grant R44 ES024698), National Institute of Environmental Health Sciences (Grant P30 ES002109), National Cancer Institute (U.S.) (Cancer Center Support Grant CA-77598)

DNA-alkylating agents are commonly used to kill cancer cells, but the base excision repair (BER) pathway they trigger can produce toxic intermediates that damage healthy tissues as well, including retinal degeneration (RD). Apoptosis, a process of programmed cell death, is assumed to be the main mechanism of this alkylation-induced photoreceptor (PR) cell death in RD. Here, we studied the involvement of necroptosis (another process of cell death) and inflammation in alkylation-induced RD. Male mice exposed to a methylating agent exhibited a reduced number of PR cell rows, active gliosis, and cytokine induction and macrophage infiltration in the retina. Dying PRs exhibited a necrotic morphology, increased 8-hydroxyguanosine (a marker of oxidative damage), and overexpression of the necroptosis-associated genes Rip1 and Rip3. The activity of PARP1, an enzyme that mediates BER, cell death and inflammation, was increased in PR cells and associated with the release of proinflammatory chemokine and inflammatory ligand HMGB1 from PR nuclei. Deficiency of the anti-inflammatory cytokine IL-10 resulted in more severe RD, whereas deficiency of RIP3 conferred partial protection. Female mice were partially protected from alkylation-induced RD, showing reduced markers of necroptosis and inflammation compared to those in males. PRs in mice lacking the BER-initiating DNA glycosylase AAG did not exhibit alkylation-induced necroptosis or inflammation. Our findings show that AAG-initiated BER at alkylated DNA bases induces sex-dependent RD primarily by triggering necroptosis and activating an inflammatory response that amplifies the original damage and, further, reveal novel potential targets to prevent this side-effect of chemotherapy.

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 important industrial and environmental carcinogen 1,3-butadiene (BD) forms a range of adenine adducts in DNA, including N6-(2-hydroxy-3-buten-1-yl)-2′-deoxyadenosine (N6-HB-dA), 1,N6-(2-hydroxy-3-hydroxymethylpropan-1,3-diyl)-2′-deoxyadenosine (1,N6-HMHP-dA), and N6,N6-(2,3-dihydroxybutan-1,4-diyl)-2′-deoxyadenosine (N6,N6-DHB-dA). If not removed prior to DNA replication, these lesions can contribute to A → T and A → G mutations commonly observed following exposure to BD and its metabolites. In this study, base excision repair of BD-induced 2′-deoxyadenosine (BD-dA) lesions was investigated. Synthetic DNA duplexes containing site-specific and stereospecific (S)-N6-HB-dA, (R,S)-1,N6-HMHP-dA, and (R,R)-N6,N6-DHB-dA adducts were prepared by a postoligomerization strategy. Incision assays with nuclear extracts from human fibrosarcoma (HT1080) cells have revealed that BD-dA adducts were recognized and cleaved by a BER mechanism, with the relative excision efficiency decreasing in the following order: (S)-N...

ephrotoxicity is a common toxic side-effect of chemotherapeutic alkylating agents. Although the base excision repair (BER) pathway is essential in repairing DNA alkylation damage, under certain conditions the initiation of BER produces toxic repair intermediates that damage healthy tissues. We have shown that the alkyladenine DNA glycosylase, Aag (a.k.a. Mpg), an enzyme that initiates BER, mediates alkylation-induced whole-animal lethality and cytotoxicity in the pancreas, spleen, retina, and cerebellum, but not in the kidney. Cytotoxicity in both wild-type and Aag-transgenic mice (AagTg) was abrogated in the absence of Poly(ADP-ribose) polymerase-1 (Parp1). Here we report that Parp1-deficient mice expressing increased Aag (AagTg/Parp1-/-) develop sex-dependent kidney failure upon exposure to the alkylating agent, methyl methanesulfonate (MMS), and suffer increased whole-animal lethality compared to AagTg and wild-type mice. Macroscopic, histological, electron microscopic and immunohistochemical analyses revealed morphological kidney damage including dilated tubules, proteinaceous casts, vacuolation, collapse of the glomerular tuft, and deterioration of podocyte structure. Moreover, mice exhibited clinical signs of kidney disease indicating functional damage, including elevated blood nitrogen urea and creatinine, hypoproteinemia and proteinuria. Pharmacological Parp inhibition in AagTg mice also resulted in sensitivity to MMS-induced nephrotoxicity. These findings provide in vivo evidence that Parp1 modulates Aag-dependent MMS-induced nephrotoxicity in a sex-dependent manner and highlight the critical roles that Aag-initiated BER and Parp1 may play in determining the side-effects of chemotherapeutic alkylating agents., United States. National Institutes of Health (R01- CA075576), United States. National Institutes of Health (R01-CA055042), United States. National Institutes of Health (R01-CA149261), United States. National Institutes of Health (AGSS- 3046-12), United States. National Institutes of Health (P30-ES02109), United States. National Institutes of Health (P30-CA014051)

Much of the global cancer burden is associated with longstanding inflammation accompanied by release of DNA-damaging reactive oxygen and nitrogen species. Here, we report that the Mbd4 DNA glycosylase is protective in the azoxymethane/dextran sodium sulfate (AOM/DSS) mouse model of inflammation-driven colon cancer. Mbd4 excises T and U from T:G and U:G mismatches caused by deamination of 5-methylcytosine and cytosine. Since the rate of deamination is higher in inflamed tissues, we investigated the role of Mbd4 in inflammation-driven tumorigenesis. In the AOM/DSS assay, Mbd4[superscript –/–] mice displayed more severe clinical symptoms, decreased survival, and a greater tumor burden than wild-type (WT) controls. The increased tumor burden in Mbd4[superscript –/–] mice did not arise from impairment of AOM-induced apoptosis in the intestinal crypt. Histopathological analysis indicated that the colonic epithelium of Mbd4[superscript –/–] mice is more vulnerable than WT to DSS-induced tissue damage. We investigated the role of the Mbd4[superscript –/–] immune system in AOM/DSS-mediated carcinogenesis by repeating the assay on WT and Mbd4[superscript –/–] mice transplanted with WT bone marrow. Mbd4[superscript –/–] mice with WT bone marrow behaved similarly to Mbd4[superscript –/–] mice. Together, our results indicate that the colonic epithelium of Mbd4[superscript –/–] mice is more vulnerable to DSS-induced injury, which exacerbates inflammation-driven tissue injury and cancer., National Institutes of Health (U.S.) (Grant R01-CA075576), National Institutes of Health (U.S.) (Grant R01-CA055042), National Institutes of Health (U.S.) (Grant R01-CA149261), National Institutes of Health (U.S.) (Grant P30-ES000002), National Institutes of Health (U.S.) (Grant P30-ES02109), National Institutes of Health (U.S.) (Training Grant in Environamental Toxicology T32-ES007020MIT), American Cancer Society (Postdoctoral Fellowship PF-13-204-01)

Genotoxicity testing is critical for predicting adverse effects of pharmaceutical, industrial, and environmental chemicals. The comet assay is a commonly used approach for detecting DNA damage that is based on the underlying principle that damaged DNA migrates more readily than undamaged DNA when electrophoresed in agarose. We previously developed a higher-throughput version of the comet assay (Wood DK, PNAS 2010). For the “CometChip” assay, cells are loaded into an array of agarose microwells to create a cell microarray. The CometChip can be fully automated, making the assay more than 1000X faster, while improving reproducibility. While effective for detecting single-strand breaks, abasic sites, and alkali-sensitive sites, the alkaline comet assay is not effective for detection of carcinogenic bulky DNA lesions that do not impact DNA mobility and that require metabolic activation. To overcome this, we use DNA replication inhibitors (hydroxyurea and 1-β-d-arabinofuranosyl cytosine) shown to trap single-strand breaks that are formed during nucleotide excision repair, which normally removes bulky lesions. Thus, comet-undetectable bulky lesions are converted into comet-detectable single-strand breaks. Together with HepaRG™ cells that recapitulate in vivo metabolic capacity, we have thus created the “HepaCometChip,” which provides a more broadly effective approach for detection of DNA damage. In addition to genotoxicity testing, the microwell array has also been harnessed for use in a cell survival assay. For the “MicroColonyChip” (Ngo LP, Cell Reports 2019), cells are loaded into the agarose microwell array and allowed to grow to form microcolonies. Differences in cell survival can be detected by changes in the distribution of colony sizes, a novel metric for cell survival quantitation. The assay is as sensitive as the traditional “gold standard” colony-forming assay, yet it takes days instead of weeks to complete. Parallel studies of genotoxicity and cytotoxicity can thus be performed using a shared microwell platform. These tools have broad utility, including studies of microbe-induced DNA damage. Examples of S. pneumoniae and E. coli microbial product-induced double-strand breaks and interstrand crosslinks contribute to an emerging paradigm wherein pathogen-induced DNA damage has the potential to promote disease. Citation Format: Le P. Ngo, Prashant Rai, Matthew R. Wilson, Carole Swartz, John Winters, Yang Su, Jing Ge, Tze-Khee Chan, Emily P. Balskus, Vincent Chow, Les Recio, Leona D. Samson, Bevin P. Engelward. Novel cell microarray technologies for studies of DNA damage and cell survival and applications for studies of microbe-induced DNA damage [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr IA20.

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.

The DNA repair protein O6-methylguanine DNA methyltransferase (MGMT) strongly influences the effectiveness of cancer treatment with chemotherapeutic alkylating agents, and MGMT status in cancer cells could potentially contribute to tailored therapies for individual patients. However, the promoter methylation and immunohistochemical assays presently used for measuring MGMT in clinical samples are indirect, cumbersome and sometimes do not accurately report MGMT activity. Here we directly compare the accuracy of 6 analytical methods, including two fluorescent reporter assays, against the in vitro MGMT activity assay that is considered the gold standard for measuring MGMT DNA repair capacity. We discuss the relative advantages of each method. Our data indicate that two recently developed fluorescence-based assays measure MGMT activity accurately and efficiently, and could provide a functional dimension to clinical efforts to identify patients who are likely to benefit from alkylating chemotherapy.

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.

Pollution is the largest environmental cause of disease and premature death in the world today. Diseases caused by pollution were responsible for an estimated 9 million premature deaths in 2015—16% of all deaths worldwide—three times more deaths than from AIDS, tuberculosis, and malaria combined and 15 times more than from all wars and other forms of violence. In the most severely affected countries, pollution-related disease is responsible for more than one death in four.

A small-molecule inhibitor of the OGG1 DNA glycosylase has anti-inflammatory effects

Cell survival is a critical and ubiquitous endpoint inbiology. The broadly accepted colony formationassay (CFA) directly measures a cell’s ability to divide;however, it takes weeks to perform and is incompat-ible with high-throughput screening (HTS) technolo-gies. Here, we describe the MicroColonyChip, whichexploits microwell array technology to create an arrayof colonies. Unlike the CFA, where visible coloniesare counted by eye, using fluorescence microscopy,microcolonies can be analyzed in days rather thanweeks. Using automated analysis of microcolonysize distributions, the MicroColonyChip achievescomparable sensitivity to the CFA (and greater sensi-tivity than the 2,3-bis-(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide [XTT] assay).Compared to CellTiter-Glo, the MicroColonyChip isas sensitive and also robust to artifacts caused bydifferences in initial cell seeding density. We demon-strate efficacy via studies of radiosensitivity andchemosensitivity and show that the approach isamenable to multiplexing. We conclude that theMicroColonyChip is a rapid and automated alternativefor cell survival quantitation., National Institute of Environmental Health Sciences (Grant R44ES024698), National Institutes of Health (U.S.). Superfund Basic Research Program (Grant P42 ES027707), National Institutes of Health (U.S.) (Grant R01 ES022872), National Institute of Environmental Health Sciences (Grant DP1 ES022576), Massachusetts Institute of Technology. Center for Environmental Health Sciences (Grant P30-ES002109)

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)

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)

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.