Induction of theEscherichia coli UVM response by oxidative stress

UVM (ultravioletmodulation of mutagenesis) is a recently describedrecA-independent, inducible mutagenic phenomenon in which prior UV irradiation ofEscherichia coli cells strongly enhances mutation fixation at a site-specific 3-N⁴-ethenocytosine (C) lesion borne on a transfected single-stranded M13 DNA vector. Subsequent studies demonstrated that UVM is also induced by alkylating agents, and is distinct from both the SOS response and the adaptive response to alkylation damage. Because of the increasing significance being attributed to oxidative DNA damage, it is interesting to ask whether this class of DNA damage can also induce UVM. By transfecting M13 vector DNA bearing a site-specificC lesion into cells pretreated with inducing agents, we show here that the oxidative agent H₂O₂ is a potent inducer of UVM, and that the induction of UVM by H₂O₂ does not requireoxyR-regulated gene expression. UVM induction by H₂O₂ appears to be mediated by DNA damage, as indicated by the observation of a concomitant reduction in cellular toxicity and UVM response in OxyR^c cells. Available evidence suggests that UVM represents a generalized cellular response to a broad range of chemical and physical genotoxicants, and that DNA damage constitutes the most likely signal for its induction.     

Reversible covalent modification of DNA

The reaction of bromomethylbenzoyl esters of choline and dimethylaminoethanol with DNA and model compounds led predominantly to phosphotriester formation. In model compounds the phosphotriester formation was verified by uv spectrometry. The bromomethylbenzoyl cationic esters reacted with DNA at room temperature at neutral pH values. The amount of the reagent chromophores was assessed semiquantitatively by spectrophotometry. The maximum binding appeared to be stoichiometric, i.e., one residue per phosphorus. The binding of one mole of reagent per phosphorus was confirmed by electron spectroscopic measurements of the phosphorus atom electron emission of maximally modified DNA. The modified DNA showed altered CD spectra indicating that the reagent chromophores are arranged in an orderly fashion affording a strong (Δ_? > 4), positive, apparently extrinsic CD band at ~240 nm; a double helical array is proposed. The introduced chromophores were readily removed by heat treatment or by treatment with nucleophiles at neutral pH values at moderate temperatures (<37 °C); no measurable fraction of the DNA became dialyzable. A decrease in viscosity accompanied the reversal, indicative of some chain breaking. The modified DNA's show higher T_m values than the native DNA and some display a higher and some a lower degree of cooperativity in their melting curves. No chemically detectable amounts of base alkylation, depurination, or depyrimidination were found when dialyzates of treated DNA and hydrolyzed samples of modified DNA were examined. However, presumptive evidence for some base alkylation by these novel alkylating agents was found utilizing Salmonella typhimurium tester strains sensitive to reversion by alkylation. No comparable binding of benzoylcholine, a nonalkylating analogue, by DNA was seen under conditions utilized here.     

Aag DNA Glycosylase Promotes Alkylation-Induced Tissue Damage Mediated by Parp1

Alkylating agents comprise a major class of front-line cancer chemotherapeutic compounds, and while these agents effectively kill tumor cells, they also damage healthy tissues. Although base excision repair (BER) is essential in repairing DNA alkylation damage, under certain conditions, initiation of BER can be detrimental. Here we illustrate that the alkyladenine DNA glycosylase (AAG) mediates alkylation-induced tissue damage and whole-animal lethality following exposure to alkylating agents. Aag-dependent tissue damage, as observed in cerebellar granule cells, splenocytes, thymocytes, bone marrow cells, pancreatic β-cells, and retinal photoreceptor cells, was detected in wild-type mice, exacerbated in Aag transgenic mice, and completely suppressed in Aag ^−/− mice. Additional genetic experiments dissected the effects of modulating both BER and Parp1 on alkylation sensitivity in mice and determined that Aag acts upstream of Parp1 in alkylation-induced tissue damage; in fact, cytotoxicity in WT and Aag transgenic mice was abrogated in the absence of Parp1. These results provide in vivo evidence that Aag-initiated BER may play a critical role in determining the side-effects of alkylating agent chemotherapies and that Parp1 plays a crucial role in Aag-mediated tissue damage.     

Molecular Mechanisms of DNA Damage and Repair: Progress in Plants

Dedicated to Prof. Jan H. J. Hoeijmakers.

Referee: Dr. Nawin C. Mishra, Professor of Genetics, University of South Carolina, Department of Biological Sciences, Columbia, SC 29208

Despite stable genomes of all living organisms, they are subject to damage by chemical and physical agents in the environment (e.g., UV and ionizing radiations, chemical mutagens, fungal and bacterial toxins, etc.) and by free radicals or alkylating agents endogenously generated in metabolism. DNA is also damaged because of errors during its replication. The DNA lesions produced by these damaging agents could be altered base, missing base, mismatch base, deletion or insertion, linked pyrimidines, strand breaks, intra- and inter-strand cross-links.     

On the integrity of sperm DNA

Ejaculated rabbit spermatozoa washed with buffer prior to decondensation by Triton X-100 and dithiothreitol were good templates for DNA synthesis by Escherichia coli DNA polymerase. This result agrees with the observations of Zirkin and Chang [1977], and implies that the sperm DNA is nicked. Template activity, however, was reduced if spermatozoa were extensively washed before decondensation, and if DNase inhibitors EDTA or Na₂SO₄ were present during decondensation. Template activity was also low if decondensation was induced with DNase inhibitors thioglycollic acid, Na₂SO₃ or sodium dodecylsulphate and dithiothreitol instead of with Triton X-100 and dithiothreitol. Calf thymus DNA was completely degraded when incubated with rabbit seminal plasma or buffer-washed spermatozoa, but much less degradation was observed if EDTA, Na₂SO₄, thioglycollic acid, Na₂SO₃ or sodium dodecylsulphate were also present, or if spermatozoa were extensively washed with buffer. Centrifugation of spermatozoa through 2.05 M sucrose completely removed contaminating DNase, and such spermatozoa were inactive as DNA templates after decondensation. The DNA template activity of swollen rabbit sperm nuclei thus parallels the activity of a contaminating seminal plasma DNase. This suggest that the nicks in sperm DNA enabling it to act as a template for DNA synthesis were generated by the DNase during decondensation and thus are not a natural structural feature of the DNA. The presence of breaks in the DNA of decondensed buffer-washed spermatozoa (DNase contaminated) was confirmed by their incorporation of phosphate from [γ^?32 P] ATP in the presence of the enzyme polynucleotide kinase. These spermatozoa were found to contain as few as two breaks/mole of DNA, but sucrose-washed spermatozoa (DNase free) were free of breaks. The possible use of this enzymic procedure for the assessment of sperm genome damage and the evaluation of the quality of a sperm population are discussed.     

Induction of theEscherichia coli UVM response by oxidative stress

UVM (ultravioletmodulation of mutagenesis) is a recently describedrecA-independent, inducible mutagenic phenomenon in which prior UV irradiation ofEscherichia coli cells strongly enhances mutation fixation at a site-specific 3-N⁴-ethenocytosine (?C) lesion borne on a transfected single-stranded M13 DNA vector. Subsequent studies demonstrated that UVM is also induced by alkylating agents, and is distinct from both the SOS response and the adaptive response to alkylation damage. Because of the increasing significance being attributed to oxidative DNA damage, it is interesting to ask whether this class of DNA damage can also induce UVM. By transfecting M13 vector DNA bearing a site-specific?C lesion into cells pretreated with inducing agents, we show here that the oxidative agent H₂O₂ is a potent inducer of UVM, and that the induction of UVM by H₂O₂ does not requireoxyR-regulated gene expression. UVM induction by H₂O₂ appears to be mediated by DNA damage, as indicated by the observation of a concomitant reduction in cellular toxicity and UVM response in OxyR^c cells. Available evidence suggests that UVM represents a generalized cellular response to a broad range of chemical and physical genotoxicants, and that DNA damage constitutes the most likely signal for its induction.     

Noncanonical regulation of alkylation damage resistance by the OTUD4 deubiquitinase

Repair of DNA alkylation damage is critical for genomic stability and involves multiple conserved enzymatic pathways. Alkylation damage resistance, which is critical in cancer chemotherapy, depends on the overexpression of alkylation repair proteins. However, the mechanisms responsible for this upregulation are unknown. Here, we show that an OTU domain deubiquitinase, OTUD4, is a positive regulator of ALKBH2 and ALKBH3, two DNA demethylases critical for alkylation repair. Remarkably, we find that OTUD4 catalytic activity is completely dispensable for this function. Rather, OTUD4 is a scaffold for USP7 and USP9X, two deubiquitinases that act directly on the AlkB proteins. Moreover, we show that loss of OTUD4, USP7, or USP9X in tumor cells makes them significantly more sensitive to alkylating agents. Taken together, this work reveals a novel, noncanonical mechanism by which an OTU family deubiquitinase regulates its substrates, and provides multiple new targets for alkylation chemotherapy sensitization of tumors.     

Methylated DNA Causes a Physical Block to Replication Forks Independently of Damage Signalling, O-Methylguanine or DNA Single-Strand Breaks and Results in DNA Damage

Even though DNA alkylating agents have been used for many decades in the treatment of cancer, it remains unclear what happens when replication forks encounter alkylated DNA. Here, we used the DNA fibre assay to study the impact of alkylating agents on replication fork progression. We found that the alkylator methyl methanesulfonate (MMS) inhibits replication elongation in a manner that is dose dependent and related to the overall alkylation grade. Replication forks seem to be completely blocked as no nucleotide incorporation can be detected following 1 h of MMS treatment. A high dose of 5 mM caffeine, inhibiting most DNA damage signalling, decreases replication rates overall but does not reverse MMS-induced replication inhibition, showing that the replication block is independent of DNA damage signalling. Furthermore, the block of replication fork progression does not correlate with the level of DNA single-strand breaks. Overexpression of O⁶-methylguanine (O6meG)-DNA methyltransferase protein, responsible for removing the most toxic alkylation, O6meG, did not affect replication elongation following exposure to N-methyl-N′-nitro-N-nitrosoguanidine. This demonstrates that O6meG lesions are efficiently bypassed in mammalian cells. In addition, we find that MMS-induced γH2AX foci co-localise with 53BP1 foci and newly replicated areas, suggesting that DNA double-strand breaks are formed at MMS-blocked replication forks. Altogether, our data suggest that N-alkylations formed during exposure to alkylating agents physically block replication fork elongation in mammalian cells, causing formation of replication-associated DNA lesions, likely double-strand breaks.     

Assessment of DNA damage in sperm after repeated non‐invasive sampling in zebrafish Danio rerio

Repeated non‐invasive sampling of zebrafish Danio rerio sperm was conducted, sperm counts were obtained and a method for measurement of DNA damage in sperm was developed and validated (single‐cell gel electrophoresis, comet, assay). DNA damage in sperm increased with concentration of hydrogen peroxide (H₂O₂, 0–200 µM), and in vitro exposure of sperm to 200 µM H₂O₂ produced 88·7 ± 3·9% tail DNA compared to unexposed controls [12 ± 0·7% tail DNA (mean ± s.e ., n = 3)]. Frequency of sperm sampling (sampled every 2, 4 or 7 days) did not affect DNA damage in sperm, but sperm counts decreased 57 and 22% for fish sampled every 2 or 4 days, respectively.     

Nanogravimetric and voltammetric DNA-hybridization biosensors for studies of DNA damage by common toxicants and pollutants

Electrochemical and nanogravimetric DNA-hybridization biosensors have been developed for sensing single mismatches in the probe-target ssDNA sequences. The voltammetric transduction was achieved by coupling ferrocene moiety to streptavidin linked to biotinylated tDNA. The mass-related frequency transduction was implemented by immobilizing the sensory pDNA on a gold-coated quartz crystal piezoresonators oscillating in the 10 MHz band. The high sensitivity of these sensors enabled us to study DNA damage caused by representative toxicants and environmental pollutants, including Cr(VI) species, common pesticides and herbicides. We have found that the sensor responds rapidly to any damage caused by Cr(VI) species, with more severe DNA damage observed for Cr₂O₇²⁻ and for CrO₄²⁻ in the presence of H₂O₂ as compared to CrO₄²⁻ alone. All herbicides and pesticides examined caused DNA damage or structural alterations leading to the double-helix unwinding. Among these compounds, paraoxon-ethyl and atrazine caused the fastest and most severe damage to DNA. The physico-chemical mechanism of damaging interactions between toxicants and DNA has been proposed. The methodology of testing voltammetric and nanogravimetric DNA-hybridization biosensors developed in this work can be employed as a simple protocol to obtain rapid comparative data concerning DNA damage caused by herbicide, pesticides and other toxic pollutants. The DNA-hybridization biosensor can, therefore, be utilized as a rapid screening device for classifying environmental pollutants and to evaluate DNA damage induced by these compounds.     

Quantitative comparison of carcinogenicity, mutagenicity and electrophilicity of 10 direct-acting alkylating agents and of the initial O: 7-alkylguanine ratio in DNA with carcinogenic potency in rodents

The quantitative relationship between carcinogenicity in rodents and mutagenicity in Salmonella typhimurium was examined, by using 10 monofunctional alkylating agents, including N-nitrosamides, alkyl methanesulfonates, epoxides, β-propiolactone and 1,3-propane sultone. The compounds were assayed for mutagenicity in two S. typhimurium strains (TA1535 and TA100) and in plate and liquid assays. The mutagenic activity of the agents was compared with their alkylating activity towards 4-(4′-nitrobenzyl)pyridine and with their half-lives (solvolysis constants) in an aqueous medium. No correlations between these variables were found, nor was mutagenic activity correlated with estimates of carcinogenicity in rodents.There was a positive relationship between carcinogenicity and the initial ratios of 7-: O⁶-alkylguanine formed or expected after their reaction with double-stranded DNA in vitro. The results suggest that alkylation of guanine at position O⁶ (or at other O atoms of DNA bases) may be a critical DNA-base modification that determines the overall carcinogenicity of these alkylating agents in rodents.     

Some chemical aspects of dose-response relationships in alkylation mutagenesis

Alkylation of DNA can lead to induction of potentially miscoding groups (promutagenic) or potentially template-inactivating groups (lethal). The proportions of these are found to vary with the chemical nature of the alkylating agent. Agents of low Swain and Scott s factor (or those tending to Ingold's S_Ni type) react relatively more extensively at O-atom sites in DNA, and yield relatively more of the miscoding O₆-alkylguanine residues. Phosphotriester formation is also relatively more extensive with S_Ni agents.Inactivation of DNA can result from depurinations, strand breakage, and cross-linkage.Both promutagenic and lethal lesions are subject to repair; 3 principal enzymatic systems appear to exist; one for excision and repair of cross-links or aralkyl groups resembles the uvr system; others for repair of single-strand breaks parallel repair of X-ray-induced breaks (exr, rec systems); another, less well defined at present, recognizes certain methylated bases, and depurinated sites (probably Goldthwait's endonuclease II).These factors can be shown to influence dose-response in alkylation mutagenesis. This, broadly, can be classified as linear with the promutagenic group-inducing or directly miscoding agents, and is independent of cytotoxicity; whereas with other agents non-linear response parallels the occurrence of “shouldered” survival curves, and reflects mutation induction by “repairs errors”.Additionally, alkylation of cellular constituents other than DNA, e.g. repair enzymes, may influence dose response, and will again depend on chemical reactivity of the agent.     

N-methylpurine DNA glycosylase overexpression increases alkylation sensitivity by rapidly removing non-toxic 7-methylguanine adducts

Previous studies indicate that overexpression of N-methylpurine DNA glycosylase (MPG) dramatically sensitizes cells to alkylating agent-induced cytotoxicity. We recently demonstrated that this sensitivity is preceded by an increased production of AP sites and strand breaks, confirming that overexpression of MPG disrupts normal base excision repair and causes cell death through overproduction of toxic repair intermediates. Here we establish through site-directed mutagenesis that MPG-induced sensitivity to alkylation is dependent on enzyme glycosylase activity. However, in contrast to the sensitivity seen to heterogeneous alkylating agents, MPG overexpression generates no cellular sensitivity to MeOSO₂(CH₂)₂-lexitropsin, an alkylator which exclusively induces 3-meA lesions. Indeed, MPG overexpression has been shown to increase the toxicity of alkylating agents that produce 7-meG adducts, and here we demonstrate that MPG-overexpressing cells have dramatically increased removal of 7-meG from their DNA. These data suggest that the mechanism of MPG-induced cytotoxicity involves the conversion of non-toxic 7-meG lesions into highly toxic repair intermediates. This study establishes a mechanism by which a benign DNA modification can be made toxic through the overexpression of an otherwise well-tolerated gene product, and the application of this principle could lead to improved chemotherapeutic strategies that reduce the peripheral toxicity of alkylating agents.     

Effects of pretreatment with inducers of hepatic mixed function oxidases on DNA repair elicited by various compounds in hepatocytes from adult and neonatal rats

Studies were conducted to assess the effects of inducers of hepatic mixed function oxidases on DNA repair responses to 13 different genotoxic agents in hepatocytes from adult male mice. Phenobarbital pretreatment increased DNA repair elicited by diethylnitrosamine but had no effect on responses to the other compounds. Pretreatment with p,p-dichlorodiphenyltrichloroethane, 3-methylcholanthrene or -naphthoflavone induced the DNA repair responses to a variety of activation-dependent carcinogens. DNA repair responses to the direct-acting alkylating agents methyl methanesulfonate and N-methyl-N-nitro-N-nitrosoguanidine were not increased by any of the pretreatments, which indicated that the pretreatment-related enhancement of responses to the other compounds was due to induction of their metabolic activation. Taken together, the findings suggest that Aroclor, or other pretreatments, may increase the sensitivity of the hepatocyte DNA repair assay for detecting the genotoxicity of certain compounds; however, the potential benefit may be limited due to specific features of the assay. In contrast, Aroclor pretreatment did not produce any enhancement of in vivo DNA repair elicited by dimethylnitrosamine, diethylnitrosamine, o-aminoazotoluene, 2-acetylaminofluorene, 3-methylcholanthrene or aflatoxin B₁, and thus does not appear to be useful for improving the sensitivity of the in vivo/in vitro assay.Whereas the amount of DNA repair produced by dimethylnitrosamine was not increased by classical inducers of liver microsomal enzymes, pretreatment with pyrazole greatly augmented in vitro and in vivo DNA repair responses to dimethylnitrosamine; responses to diethylnitrosamine were increased to a lesser degree by pyrazole pretreatment. The effects of lactational exposure to enzyme inducing agents on DNA repair in neonatal hepatocytes was also investigated.Abbreviations 2-AAF 2-acetylaminofluorene - 4-AB 4-aminobiphenyl - 6-AC 6-aminochrysene - AFB aflatoxin B₁ - ARO Aroclor 1254 - o-AT o-aminoazotoluene - B(a)P benzo[a]pyrene - B-NF beta-naphthoflavone - BZ benzidine - DDT p,p-dichlorodiphenyltrichloroethane - DDE p,p-dichlorodiphenyldichloroethylene - DEN diethylnitrosamine - DMBA 7,12-dimethylbenzanthracene - DMN dimethylnitrosamine - 3-MC 3-methylcholanthrene - MMS methyl methanesulfonate - MNNG N-methyl-N-nitro-N-nitrosoguanidine - 2-NA 2-naphthylamine - NNG net nuclear grains - PB phenobarbital - PYR pyrazole     

No association between N7-methyldeoxyguanosine and 8-oxodeoxyguanosine levels in human lymphocyte DNA

To examine associations between two different classes of DNA damage that can occur through endogenous processes or exogenous exposures such as smoking, N7-methyldeoxyguanosine (N7-MedG) and 8-oxodeoxyguanosine (8-oxodG) levels were measured in lymphocyte DNA from 22 bronchoscopy patients. 8-OxodG and N7-MedG was detected in 100% and 91% of samples, respectively with 8-oxodG levels being approx 20 times higher (mean 8.39 ± 3.57 8-oxodG/10⁶dG versus 0.41 ± 0.33 N7-MedG/10⁶ dG) which provides an indication of the relative importance of the agents that induce oxidative DNA damage or alkylation damage. The sources of these genotoxic lesions remain to be established but N7-MedG and 8-oxodG levels were not correlated (r² < 0.01) suggesting that there is no association between alkylating agent and reactive oxygen species exposure, their metabolism and/or the DNA repair processes that can remove this DNA damage.     

Effect of varying the exposure and 3H-thymidine labeling period upon the outcome of the primary hepatocyte DNA repair assay

The results presented in this report demonstrate that an 18–20 hour exposure/³H-thymidine DNA labeling period is superior to a 4 hour incubation interval for general genotoxicity screening studies in the rat primary hepatocyte DNA repair assay. When DNA damaging agents which give rise to bulky-type DNA base adducts such as 2-acetylaminofluorene, aflatoxin Bi and benzidine were evaluated, little or no difference was observed between the 4 hour or an 18–20 hour exposure/labeling period. Similar results were also noted for the DNA ethylating agent diethylnitrosamine. However, when DNA damaging chemicals which produce a broader spectrum of DNA lesions were studied, differences in the amount of DNA repair as determined by autoradiographic analysis did occur. Methyl methanesulfonate and dimethylnitrosamine induced repairable DNA damage that was detected at lower dose levels with the 18–20 hour exposure/labeling period. Similar results were also observed for the DNA cross-linking agents, mitomycin C and nitrogen mustard. Ethyl methanesulfonate produced only a marginal amount of DNA repair in primary hepatocytes up to a dose level of 10^–3M during the 4 hour incubation period, whereas a substantial amount of DNA repair was detectable at a dose level of 2.5 × 10^–4M when the 18–20 hour exposure/labeling period was employed. The DNA alkylating agent 4-nitroquinoline-1-oxide, which creates DNA base adducts that are slowly removed from mammalian cell DNA, induced no detectable DNA repair in hepatocytes up to a toxic dose level of 2 × 10^–5M with the 4 hour exposure period, whereas a marked DNA repair response was observed at 10^–5M when the 18–20 hour exposure/labeling period was used.Abbreviations 2AAF 2-acetylaminofluorene - AB₁ aflatoxin B₁ - BENZ benzidine - DEB diepoxybutane - DEN diethylnitrosamine - DMN dimethylnitrosamine - EMS ethyl methanesulfonate - MITC mitomycin C - MMS methyl methanesulfonate - NG mean net nuclear grain counts - NM nitrogen mustard - 4NQO 4-nitroquinoline-N-oxide     

Nuclear DNA Ligase and Its Action on Chromatin DNA in Neuronal, Glial and Liver Nuclei Isolated from Adult Guinea Pigs

Abstract: DNA ligase activities were measured in neuron-rich and glial nuclear preparations and liver nuclei isolated from adult guinea pigs. The enzymatic properties of cerebral and liver nuclear DNA ligases were studied with isolated nuclei and nuclear extracts. ATP (K_m= 46–48 μM) and bivalent cation (Mg²⁺ or Mn²⁺) were required for the maximal activities in cerebral and liver nuclei. β-Mercaptoethanol did not affect the activities, but N-ethylmaleimide and p-chloromercuribenzoate completely inhibited the activities. Deoxyadenosine-5′-triphosphate partially inhibited the activities in both cerebral and liver nuclei. An interdependent effect of Na⁺ and Mg²⁺ on the enzyme activities was observed. A high concentration (200 mM) of Na⁺ activated both enzymes and shifted to the acid side the optimal pH for both enzymes. DNA ligase was more easily extracted with lower concentrations of NaCl from liver nuclei than from cerebral nuclei, but the extraction curves from both nuclear species reached a plateau level (92% of total activities of nuclear enzymes) at 200 mM-NaCl. Apparent K_m for the substrate [³²P]phosphoryl DNA was determined according to a modification of the Michaelis-Menten equation, which was applied for the case where an unknown amount of substrate nicks in chromatin DNA coexisted with the nicks in exogenous substrate DNA. Neuronal and glial nuclear enzymes had similar K_m values (about 20 μg of [³²P]phosphoryl DNA/ml), but the liver nuclear enzyme had a higher K_m value (54 μg of [³²P]phosphoryl DNA/ml). The modified Michaelis-Menten equation provided the amounts of nicks available as substrate in chromatin DNA of isolated nuclei. Neuronal and glial nuclei contained 1.5 and 0.29 pmol of nicks/μg of nuclear DNA, respectively, in contrast to an intermediate amount of nicks in liver nuclei (0.63 pmol/μg of nuclear DNA). DNA ligase activity in neuronal nuclei [312 units (fmol of 5′-phosphomonoester converted into a phosphatase-resistant form per min at 37°C) per μg of nuclear DNA] was 11-fold higher than that in glial nuclei [28.7 units/μg of nuclear DNA]. Liver nuclei contained an intermediate activity [54.7 units/μg of nuclear DNA].     

Repair of alkylation damage in the fungus Aspergillus nidulans

The repair of alkylation damage in Aspergillus nidulans was investigated. We have assayed soluble protein fractions for enzymes known to be involved in the repair of this type of damage in DNA. The presence of a glycosylase activity that can remove 3-methyladenine from DNA was demonstrated, as well as a DNA methyltransferase activity that appears to act against O6-methylguanine. In addition to this approach, a series of mutants were isolated which display increased sensitivity to alkylating agents (sag mutants). 5 such mutants were further characterized, and at least 4 are shown to map to genes which have not previously been characterized. The behaviour of double mutant combinations demonstrates the existence of at least 2 pathways for the repair of alkylation damage. The majority of the sag mutants (sagA1, sagB2, sag4 and sagE5) exhibit an increased sensitivity to a range of alkylating agents, but not to UV light, while sagC3, when irradiated at the germling stage, also shows sensitivity to UV. None of the mutants isolated are defective in either the 3-methyladenine DNA glycosylase activity, or the DNA methyltransferase activity, and the nature of the defects in these strains remains to be determined.     

Repair of endogenous DNA base lesions modulate lifespan in mice

The accumulation of DNA damage is thought to contribute to the physiological decay associated with the aging process. Here, we report the results of a large-scale study examining longevity in various mouse models defective in the repair of DNA alkylation damage, or defective in the DNA damage response. We find that the repair of spontaneous DNA damage by alkyladenine DNA glycosylase (Aag/Mpg)-initiated base excision repair and O⁶-methylguanine DNA methyltransferase (Mgmt)-mediated direct reversal contributes to maximum life span in the laboratory mouse. We also uncovered important genetic interactions between Aag, which excises a wide variety of damaged DNA bases, and the DNA damage sensor and signaling protein, Atm. We show that Atm plays a role in mediating survival in the face of both spontaneous and induced DNA damage, and that Aag deficiency not only promotes overall survival, but also alters the tumor spectrum in Atm^−/− mice. Further, the reversal of spontaneous alkylation damage by Mgmt interacts with the DNA mismatch repair pathway to modulate survival and tumor spectrum. Since these aging studies were performed without treatment with DNA damaging agents, our results indicate that the DNA damage that is generated endogenously accumulates with age, and that DNA alkylation repair proteins play a role in influencing longevity.