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Alkylated bases in DNA created in the presence of endogenous and exogenous alkylating agents are either cytotoxic or mutagenic, or both to a cell. Currently, cells have evolved several strategies for repairing alkylated base. One strategy is a base excision repair process triggered by a specific DNA glycosylase that is used for the repair of the cytotoxic 3-methyladenine. Additionally, the cytotoxic and mutagenic O6-methylguanine (O6-meG) is corrected by O6-methylguanine methyltransferase (MGMT) via directly transferring the methyl group in the lesion to a specific cysteine in this protein. Furthermore, oxidative DNA demethylation catalyzed by DNA dioxygenase is utilized for repairing the cytotoxic 3-methylcytosine (3-meC) and 1-methyladenine (1-meA) in a direct reversal manner. As the third domain of life, Archaea possess 3-methyladenine DNA glycosylase II (AlkA) and MGMT, but no DNA dioxygenase homologue responsible for oxidative demethylation. Herein, we summarize recent progress in structural and biochemical properties of archaeal AlkA and MGMT to gain a better understanding of archaeal DNA alkylation repair, focusing on similarities and differences between the proteins from different archaeal species and between these archaeal proteins and their bacterial and eukaryotic relatives. To our knowledge, it is the first review on archaeal DNA alkylation repair conducted by DNA glycosylase and methyltransferase. Key points: • Archaeal MGMT plays an essential role in the repair of O6-meG • Archaeal AlkA can repair 3-meC and 1-meA [ABSTRACT FROM AUTHOR]

DNA alkylation damage induced by environmental carcinogens, chemotherapy drugs, or endogenous metabolites plays a central role in mutagenesis, carcinogenesis, and cancer therapy. Base excision repair (BER) is a conserved, front line DNA repair pathway that removes alkylation damage from DNA. The capacity of BER to repair DNA alkylation varies markedly between different cell types and tissues, which correlates with cancer risk and cellular responses to alkylation chemotherapy. The ability to measure cellular rates of alkylation damage repair by the BER pathway is critically important for better understanding of the fundamental processes involved in carcinogenesis, and also to advance development of new therapeutic strategies. Methods for assessing the rates of alkylation damage and repair, especially in human cells, are limited, prone to significant variability due to the unstable nature of some of the alkyl adducts, and often rely on indirect measurements of BER activity. Here, we report a highly reproducible and quantitative, cell-based assay, named alk-BER (alkylation Base Excision Repair) for measuring rates of BER following alkylation DNA damage. The alk-BER assay involves specific detection of methyl DNA adducts (7-methyl guanine and 3-methyl adenine) directly in genomic DNA. The assay has been developed and adapted to measure the activity of BER in fungal model systems and human cell lines. Considering the specificity and conserved nature of BER enzymes, the assay can be adapted to virtually any type of cultured cells. Alk-BER offers a cost efficient and reliable method that can effectively complement existing approaches to advance integrative research on mechanisms of alkylation DNA damage and repair. [ABSTRACT FROM AUTHOR]

In order to increase the contribution of donor HSC cells, irradiation and DNA alkylating agents have been commonly used as experimental methods to eliminate HSCs for adult mice. But a technique of HSC deletion for mouse embryo for increase contribution of donor cells has not been published. Here, we established for the first time a procedure for placental HSC transplantation into E11.5 Runx1-deficient mice mated with G1-HRD-Runx1 transgenic mice (Runx1-/-::Tg mice) that have no HSCs in the fetal liver. Following the transplantation of fetal liver cells from mice (allogeneic) or rats (xenogeneic), high donor cell chimerism was observed in Runx1-/-::Tg embryos. Furthermore, chimerism analysis and colony assay data showed that donor fetal liver hematopoietic cells contributed to both white blood cells and red blood cells. Moreover, secondary transplantation into adult recipient mice indicated that the HSCs in rescued Runx1-/-::Tg embryos had normal abilities. These results suggest that mice lacking fetal liver HSCs are a powerful tool for hematopoiesis reconstruction during the embryonic stage and can potentially be used in basic research on HSCs or xenograft models. [ABSTRACT FROM AUTHOR]

O6-methylguanine DNA methyl transferase (MGMT) is a metalloenzyme participating in the repair of alkylated DNA. In this research, we performed a comparative study for evaluating the impact of zinc metal ion on the behavior and interactions of MGMT in the both enzymatic forms of apo MGMT and holo MGMT. DNA and proliferating cell nuclear antigen (PCNA), as partners of MGMT, were utilized to evaluate molecular interactions by virtual microscopy of molecular dynamics simulation. The stability and conformational alterations of each forms (apo and holo) MGMT-PCNA, and (apo and holo) MGMT-DNA complexes were calculated by MM/PBSA method. A total of seven systems including apo MGMT, holo MGMT, free PCNA, apo MGMT-PCNA, holo MGMT-PCNA, apo MGMT–DNA, and holo MGMT-DNA complexes were simulated. In this study, we found that holo MGMT was more stable and had better folding and functional properties than that of apo MGMT. Simulation analysis of (apo and holo) MGMT-PCNA complexes displayed that the sequences of the amino acids involved in the interactions were different in the two forms of MGMT. The important amino acids of holo MGMT involved in its interaction with PCNA included E92, K101, A119, G122, N123, P124, and K125, whereas the important amino acids of apo MGMT included R128, R135, S152, N157, Y158, and L162. Virtual microscopy of molecular dynamics simulation showed that the R128 and its surrounding residues were important amino acids involved in the interaction of holo MGMT with DNA that was exactly consistent with X-ray crystallography structure. In the apo form of the protein, the N157 and its surrounding residues were important amino acids involved in the interaction with DNA. The binding free energies of − 387.976, − 396.226, − 622.227, and − 617.333 kcal/mol were obtained for holo MGMT-PCNA, apo MGMT-PCNA, holo MGMT-DNA, and apo MGMT-DNA complexes, respectively. The principle result of this research was that the area of molecular interactions differed between the two states of MGMT. Therefore, in investigations of metalloproteins, the metal ion must be preserved in their structures. Finally, it is recommended to use the holo form of metalloproteins in in vitro and in silico researches. [ABSTRACT FROM AUTHOR]

DNA alkylation damage is repaired by base excision repair (BER) initiated by alkyladenine DNA glycosylase (AAG). Despite its role in DNA repair, AAG-initiated BER promotes cytotoxicity in a process dependent on poly (ADP-ribose) polymerase-1 (PARP-1); a NAD+-consuming enzyme activated by strand break intermediates of the AAG-initiated repair process. Importantly, PARP-1 activation has been previously linked to impaired glycolysis and mitochondrial dysfunction. However, whether alkylation affects cellular metabolism in the absence of AAG-mediated BER initiation is unclear. To address this question, we temporally profiled repair and metabolism in wild-type and Aag−/− cells treated with the alkylating agent methyl methanesulfonate (MMS). We show that, although Aag−/− cells display similar levels of alkylation-induced DNA breaks as wild type, PARP-1 activation is undetectable in AAG-deficient cells. Accordingly, Aag−/− cells are protected from MMS-induced NAD+ depletion and glycolysis inhibition. MMS-induced mitochondrial dysfunction, however, is AAG-independent. Furthermore, treatment with FK866, a selective inhibitor of the NAD+ salvage pathway enzyme nicotinamide phosphoribosyltransferase (NAMPT), synergizes with MMS to induce cytotoxicity and Aag−/− cells are resistant to this combination FK866 and MMS treatment. Thus, AAG plays an important role in the metabolic response to alkylation that could be exploited in the treatment of conditions associated with NAD+ dysregulation. [ABSTRACT FROM AUTHOR]

Fe2+/2-ketoglutarate-dependent DNA-dioxygenase AlkB from Escherichia coli is able to restore the native structure of alkylated DNA bases. The enzymatic process utilizes the molecular oxygen, and proceeds through a mechanism of oxidative dealkylation. Here, the kinetics of conformational changes of AlkB and DNA substrates in the course of binding steps were studied. Nickel and cobalt divalent ions were used instead of Fe2+ as metal cofactors in order to inhibit the catalytic activity of AlkB and to study certain stages leading to the formation of a catalytically active enzyme–substrate complex. [ABSTRACT FROM AUTHOR]

It is assumed that intracellular pathogenic bacteria have to cope with DNA alkylating stress within host cells. Here we use single-cell reporter systems to show that the pathogen Brucella abortus does encounter alkylating stress during the first hours of macrophage infection. Genes encoding direct repair and base-excision repair pathways are required by B. abortus to face this stress in vitro and in a mouse infection model. Among these genes, ogt is found to be under the control of the conserved cell-cycle transcription factor GcrA. Our results highlight that the control of DNA repair in B. abortus displays distinct features that are not present in model organisms such as Escherichia coli. It is assumed that intracellular pathogenic bacteria must cope with DNA alkylating stress within host cells. Here, Poncin et al. show that the pathogen Brucella abortus does encounter alkylating stress within macrophages, and shed light into the pathways required for DNA repair in this organism. [ABSTRACT FROM AUTHOR]

Eukaryotic genomic DNA is packed into chromatin, whose fundamental structural unit is the nucleosome. As DNA-histone protein complexes, nucleosomes show different properties toward exogenous and endogenous DNA-damaging agents. This review summarizes nucleosome DNA damage due to different sources, including alkylating agents, radicals, UV radiation and reactive DNA damage intermediates. In most cases, the histone core protects the associated DNA against damage via its structure and/or scavenging of damaging agents. In contrast, histones react with damaged DNA and, in some instances, catalyze DNA damage in the nucleosome. The biological consequence of nucleosome DNA damage and future prospects in this field are briefly discussed. [ABSTRACT FROM AUTHOR]

N-nitroso compounds are alkylating agents, which are widespread in our diet and the environment. They induce DNA alkylation adducts such as O6-methylguanine (O6-MeG), which is repaired by O6-methylguanine-DNA methyltransferase (MGMT). Persistent O6-MeG lesions have detrimental biological consequences like mutagenicity and cytotoxicity. Due to its pivotal role in the etiology of cancer and in cytotoxic cancer therapy, it is important to detect and quantify O6-MeG in biological specimens in a sensitive and accurate manner. Here, we used immunological approaches and established an ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to monitor O6-MeG adducts. First, colorectal cancer (CRC) cells were treated with the methylating anticancer drug temozolomide (TMZ). Immunofluorescence microscopy and an immuno-slot blot assay, both based on an adduct-specific antibody, allowed for the semi-quantitative, dose-dependent assessment of O6-MeG in CRC cells. Using the highly sensitive and specific UPLC-MS/MS, TMZ-induced O6-MeG adducts were quantified in CRC cells and even in peripheral blood mononuclear cells exposed to clinically relevant TMZ doses. Furthermore, all methodologies were used to detect O6-MeG in wildtype (WT) and MGMT-deficient mice challenged with the carcinogen azoxymethane. UPLC-MS/MS measurements and dose-response modeling revealed a non-linear formation of hepatic and colonic O6-MeG adducts in WT, whereas linear O6-MeG formation without a threshold was observed in MGMT-deficient mice. Collectively, the UPLC-MS/MS analysis is highly sensitive and specific for O6-MeG, thereby allowing for the first time for the determination of a genotoxic threshold upon exposure to O6-methylating agents. We envision that this method will be instrumental to monitor the efficacy of methylating chemotherapy and to assess dietary exposures. [ABSTRACT FROM AUTHOR]

This study has shown for the first time that deuterium oxide (D2O) enhances expression of the E. coli ada-regulon induced by alkylating compounds, N-nitroso-N-methylurea (NMU) and methyl methanesulfonate (MMS). To compare the induction of ada-regulon in deuterated and in non-deuterated (control) cultures of E. coli, a biosensor based on the E. coli strain K12 MG1655 (pAlkA-lux) luminescing as a result of activating the alkA gene promoter in response to DNA alkylation was used. Depending on the D2O concentration from 5 to 10% in the pre-deuterium medium, these alkylating compounds at a concentration of 0.005 M induced expression of the alkA gene from 1.3 to 5 times higher as compared to induction in nondeuterated culture. It is suggested that deuterium can be one of the main factors in stabilizing the bond between the promoter and the alkylated Ada protein, leading to increased transcription of the ada-regulon. [ABSTRACT FROM AUTHOR]

Sulfur mustard (SM) is a chemical warfare agent which is cytotoxic in nature, and at the molecular level, SM acts as DNA alkylating agent leading to genotoxic and reproductive effects. Mostly, the exposed areas of the body are the main targets for SM; however, it also adversely affects various tissues of the body and ultimately exhibits long-term complications including genotoxic and reproductive effects, even in the next generations. The effect of SM on reproductive system is the reason behind male infertility. The chronic genotoxic and reproductive complications of SM have been observed in the next generation, such as reproductive hormones disturbances, testicular atrophy, deficiency of sperm cells, retarded growth of sperm and male infertility. SM exerts toxic effects through various mechanisms causing reproductive dysfunction. The key mechanisms include DNA alkylation, production of reactive oxygen species (ROS) and nicotinamide adenine dinucleotide (NAD) depletion. However, the exact molecular mechanism of such long-term effects of SM is still unclear. In general, DNA damage, cell death and defects in the cell membrane are frequently observed in SM-exposed individuals. SM can activate various cellular and molecular mechanisms related to oxidative stress (OS) and inflammatory responses throughout the reproductive system, which can cause decreased spermatogenesis and impaired sperm quality via damage to tissue function and structure. Moreover, the toxic effects of SM on the reproductive system as well as the occurrence of male infertility among exposed war troopers in the late exposure phase is still uncertain. The chronic effects of SM exposure in parents can cause congenital defects in their children. In this review, we aimed to investigate chronic genotoxic and reproductive effects of SM and their molecular mechanisms in the next generations. [ABSTRACT FROM AUTHOR]

Research on DNA binding antitumor agents has been classically steered by either non-covalent (DNA intercalation) or covalent (DNA alkylation) interactions. In this context bi-functional anticancer molecules are particularly attractive since they are capable of sequential DNA intercalation followed by DNA alkylation. Here we describe the synthesis and in vitro anticancer activity of bi-functional 1,8-naphthalimide N(4) and S(4)-derivatives. Cell viability assays indicate that our amonafide- N-mustard chimeras are selective, effective only on certain tumor cell lines, and less toxic toward non-malignant cells than the drug amonafide. The biological activities of the bi-functional derivatives presented here are encouraging and the compounds are suitable for further optimization and in vivo studies. Graphical Abstract: Here we describe the synthesis and in vitro anticancer activity of three bi-functional 1,8-naphthalimide N(4) and S(4)-derivatives presenting both DNA intercalation and alkylation capabilities. Cell viability assays indicate that these amonafide- N-mustard chimeras are effective only on certain tumor cell lines and are less toxic towards non-malignant cells than their parent drug: amonafide.[Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

[Figure not available: see fulltext.] Sodium salt of 3 Н -benzo[ e][1,3]thiazine-2,4-dione is easily alkylated at position 3. In the case of 2-(arylimino)-2,3-dihydrobenzo[ e][1,3]-thiazin-4-ones, the alkylation reaction at this position occurs regioselectively. Alkylation of 2-thioxo-2,3-dihydrobenzo[ e][1,3]thiazin-4-one under the same conditions produced regioisomers, but S-alkylation at the exocyclic sulfur atom occurred in the presence of triethylamine. The reaction of 1-(4-oxo-3,4-dihydrobenzo[ e][1,3]thiazin-2-ylidene)guanidine with methyl anthranilate led to the formation of tetracyclic quinazolinо[2,1- b]quinazolinedione. [ABSTRACT FROM AUTHOR]

[MediaObject not available: see fulltext.]The reaction of imidazole and benzimidazole derivatives with 1-(iodomethyl)-1,1,3,3,3-pentamethyldisiloxane resulted in N-alkylation, cleavage of siloxane bonds by the evolved hydrogen iodide, and the formation of cyclic organosilicon iodides and triiodides. [ABSTRACT FROM AUTHOR]

Telomeres protect chromosome ends from degradation and inappropriate DNA damage response activation through their association with specific factors. Interestingly, these telomeric factors are able to localize outside telomeric regions, where they can regulate the transcription of genes involved in metabolism, immunity and differentiation. These findings delineate a signalling pathway by which telomeric changes control the ability of their associated factors to regulate transcription. This mechanism is expected to enable a greater diversity of cellular responses that are adapted to specific cell types and telomeric changes, and may therefore represent a pivotal aspect of development, ageing and telomere-mediated diseases. [ABSTRACT FROM AUTHOR]

The FANCM/FAAP24 heterodimer has distinct functions in protecting cells from complex DNA lesions such as interstrand crosslinks. These functions rely on the biochemical activity of FANCM/FAAP24 to recognize and bind to damaged DNA or stalled replication forks. However, the DNA-binding activity of this complex was not clearly defined. We investigated how FAAP24 contributes to the DNA-interacting functions of the FANCM/FAAP24 complex by acquiring the N-terminal and C-terminal solution structures of human FAAP24. Modeling of the FAAP24 structure indicates that FAAP24 may possess a high affinity toward single-stranded DNA (ssDNA). Testing of various FAAP24 mutations in vitro and in vivo validated this prediction derived from structural analyses. We found that the DNA-binding and FANCM-interacting functions of FAAP24, although both require the C-terminal (HhH)2 domain, can be distinguished by segregation-of-function mutations. These results demonstrate dual roles of FAAP24 in DNA damage response against crosslinking lesions, one through the formation of FANCM/FAAP24 heterodimer and the other via its ssDNA-binding activity required in optimized checkpoint activation. [ABSTRACT FROM AUTHOR]

Azinomycin B-a well-known antitumor drug-forms cross-links with DNA through alkylation of purine bases and blocks tumor cell growth. This reaction has been modeled using the ONIOM (B3LYP/6-31 + g(d):UFF) method to understand the mechanism and sequence selectivity. ONIOM results have been checked for reliability by comparing them with full quantum mechanics calculations for selected paths. Calculations reveal that, among the purine bases, guanine is more reactive and is alkylated by aziridine ring through the C10 position, followed by alkylation of the epoxide ring through the C21 position of Azinomycin B. While the mono alkylation is controlled kinetically, bis-alkylation is controlled thermodynamically. Solvent effects were included using polarized-continuum-model calculations and no significant change from gas phase results was observed. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

A correction to the article "A novel thermostable protein-tag: optimization of the Sulfolobus solfataricus DNAâ€'alkylâ€'transferase by protein engineering" that was published in the December 14, 2015 issue is presented.

One of the main problems of anti-cancer therapy is an insufficient differentiation between normal and malignant cells by the known anti-proliferant agents. The antibody-directed enzyme prodrug therapy is a promising approach for a selective treatment of cancer, in which a non-toxic prodrug is enzymatically converted into a highly cytotoxic drug at the surface of malignant cells by a targeted antibody–enzyme conjugate. The transformations and the stability of a very promising novel prodrug and its corresponding cytotoxic derivative were now investigated in detail by high-performance liquid chromatography (HPLC)–mass spectrometry (MS). In order to determine the time-dependent DNA alkylation efficiency and the sequence selectivity of the novel compounds, DNA binding studies using direct electrospray–Fourier transform ion cyclotron resonance–MS (ESI–FTICR–MS) have been performed. These measurements were accompanied by HPLC analyses followed by MS of the separated species to confirm the results of the direct ESI–FTICR–MS measurements. The sites of DNA alkylation could be identified unambiguously by the mass spectrometric fragmentation pattern of the alkylated oligodeoxynucleotides as well as by the results of HPLC followed by MS. A combination of all techniques applied led to a better understanding of the mode of action of the new therapeutics and might be used for an estimation of the cytotoxicity of different prodrugs and drugs since the alkylation efficiency correlates with the bioactivity of the compounds in cell culture investigations. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Formation and fragmentation of recognition complexes between trioxacarcin A and various DNA sequences were examined by temperature-dependent UV and CD spectroscopy, HPLC analysis, and ESI mass spectrometry with regard to reaction conditions, intermediates, products, mechanism, and sequence specificity. Cleavage of the trioxacarcin–DNA complexes provided the natural product gutingimycin by guanine abstraction. The resulting DNA with an abasic site was further cleaved into a DNA fragment with a furanyl unit at the 3′-end and an oligonucleotide with a phosphorylated 5′-end. [ABSTRACT FROM AUTHOR]

We previously demonstrated in an immunohistochemical study that reduced expression of O6−methylguanine-DNA methyltransferase (MGMT) correlated with a poorer prognosis in patients with biliary tract cancers. The purpose of this study was to clarify how MGMT deficiency leads to a poor outcome in biliary tract cancer. Thus, we examined epigenetic (promoter methylation) and genetic (gene mutation) alterations in biliary tract cancer. We examined 37 biliary tract cancer specimens from patients who underwent surgical resection. Promoter methylation was determined by one-step or two-step methylation-specific polymerase chain reaction. Gene mutation was identified by direct sequencing. The expression of MGMT protein in paraffin-embedded tissue was examined by immunohistochemistry. Frequencies of promoter methylation were 70% for p16/INK4a, 49% for MGMT, 46% for hMLH1, 41% for E-cadherin, and 32% for DAPK genes. MGMT methylation status was closely correlated with the MGMT protein expression determined by immunohistochemistry ( P < .001). Although this was not statistically significant, biliary tract cancer tumors with MGMT methylation expressed multigene methylation more frequently than tumors without MGMT methylation ( P = .071). A total of 33 mutations were identified in 4 cancer-related genes: p53, K-ras, β -catenin, and p16/INK4a genes. The most common mutation was GC to AT transitions (58%), which were significantly associated with MGMT promoter methylation ( P = .011). These findings suggest that loss of MGMT expression by promoter methylation results in accumulation of GC to AT gene mutations. Reduced MGMT expression may increase the malignant potential of biliary tract cancer through both epigenetic and genetic mechanisms. [ABSTRACT FROM AUTHOR]

Human exposure to alkylating agents metabolized from tobacco- and food-borne carcinogens occurs regularly. Dietary inorganic compounds such as selenium and vanadium have been shown previously to provide chemoprotective benefits in rat and human trials. Here, we present biochemical data on the ability of inorganic compounds to protect DNA from alkylation damage. An enzyme cleavage assay is used to observe alkylated DNA. Simple salts (e.g., NaCl or NiCl2) did not prevent DNA alkylation, whereas anionic oxo species (e.g., Na2SeO4 or Na3VO4) did inhibit alkylation. We propose that these oxo species behave as nucleophilic targets for the electrophilic alkylating agents, thereby preventing DNA damage. [ABSTRACT FROM AUTHOR]

Promyelocytic leukemia protein (PML) nuclear bodies (NBs) are present in variable number in most human cell types and have been linked to various cellular functions, including roles as depots for DNA repair proteins. Here, we show that treatment of human cells with DNA methylating agents leads to redistribution of PML from NBs to a diffuse nuclear localization. Biochemically, this correlates with a specific reduction of PML levels in the nuclear matrix fraction without affecting total PML levels. Similar results were obtained for the other major PML NB component, the Sp100 protein, indicating that DNA methylating agents lead to a general disassembly of PML NBs. Similar to the dispersal of PML NBs in response to some viral infections, PML redistribution after DNA damage was inhibited by the proteasome inhibitor MG132. We propose that the regulated dispersal of PML NBs may facilitate the enhanced release of DNA repair proteins from NB depots in order to respond adequately to extensive DNA damage.Oncogene (2004) 23, 307-310. doi:10.1038/sj.onc.1207119 [ABSTRACT FROM AUTHOR]

Adozelesin and carzelesin are synthetic analogues of the extremely potent antitumor antibiotic CC-1065, which alkylates N3 of adenine in a consensus sequence 5′-(A/T)(A/T)A* (A* is the site of alkylation). We have investigated the DNA sequence selectivity of adozelesin and carzelesin by thermally induced DNA strand cleavage assay using radiolabeled restriction DNA fragments. An analysis of alkylation patterns shows that the consensus sequences for carzelesin and adozelesin have been found to be 5′-(A/T)(A/T)A* and 5′-(A/T)(G/C)(A/T)A*. A new consensus sequence, 5′-(A/T)(A/T)CA*, has been observed to display an additional alkylation site for adozelesin but not for carzelesin. These results indicate that the pattern of sequence selectivity induced by carzelesin is similar but not identical to those induced by adozelesin. [ABSTRACT FROM AUTHOR]

The purpose of this study was to determine whether quantitative differences in hepatic DNA methylation or ethylation are sufficient to explain differences in the carcinogenicity of N-nitrosomethylethylamine (NMEA), N-nitrosodimethylamine (NDMA), and N-nitrosodiethylamine (NDEA). Methylation and ethylation of hepatic DNA were determined in male Fischer 344 rats following a single IP dose of NMEA, NDMA, NDEA or an equimolar mixture of NDMA plus NDEA. The total nitrosamine dose ranged from 0.05 to 0.25 mmol/kg. After 5 h survival, hepatic DNA was extracted by adsorption onto hydroxyapatite. Acid hydrolysates were analyzed by cation exchange HPLC with fluorescence detection. DNA methylation by NMEA (170 μmol O-methylguanine/mol guanine at 0.1 mmol/kg) was comparable to that observed in animals given an equimolar mixture of NDMA plus NDEA, indicating that NMEA is one-half as effective a methylating agent as NDMA. In contrast, the amount of ethylation by NMEA (5.9 μmol O-methylguanine/mol guanine at 0.1 mmol/kg) was approximately 4 times less than that observed in animals treated with an equimolar mixture of NDMA and NDEA. The presence of NDEA had little effect on DNA methylation by NDMA, suggesting that neither synergism nor competition occurs in the simultaneous activation of these two nitrosamines. The role of β-hydroxylation of NMEA as a metabolic pathway that reduces the extent of DNA ethylation is discussed. [ABSTRACT FROM AUTHOR]

O-Methylguanine DNA transferase activity was investigated in liver proteins obtained at various intervals after partial hepatectomy and/or after hydroxyurea-induced synchronization of the liver cell cycle. Liver proteins were incubated with H-methylated calf thymus DNA as previously described by Pegg et al. (1981). The loss of O-methylguanine was measured by radiochromatography of DNA hydrolysates. The extent of O-methylguanine repair differed during the cell cycle: the activity increased in late G, reached a maximum in early S phase and declined in late S phase and GM. These results indicate that hepatocytes are endowed with an increased DNA repair capacity for this promutagenic lesion during the period of highest transformation sensitivity in the cell cycle. Though increased, however, this repair potential does not, because of its exhaustibility, appear to be sufficient to prevent initiation of transformation after high doses of alkylating carcinogens. [ABSTRACT FROM AUTHOR]

To compare the formation and persistance of alkylated DNA bases in the G- and S-phase compartments in liver in vivo, regnerating rat liver was exposed to [C]dimethylnitrosamine (0.57 mg/kg, IP injection) or N-[methyl C]- N-nitrosourea (3.3 mg/kg, intraportal injection) during the G phase of the cell cyle (12 h after partial hepatectomy), or at 24 h after partial hepatectomy with 30% hepatocytes in DNA synthesis, or at 43 h after partial hepatectomy, 4 h after an hydroxyurea block from 14 to 39 h after operation with 80% hepatocytes in DNA synthesis. At 120 min after dimethylnitrosamine and 90 s, 5, 10, or 60 min after the intraportal pulse of N-methyl- N-nitrosourea the molar fractions of 7-methylguanine (7megua), O-methylguanine (Omegua), and 3-methyladenine (3mead) and of metabolically labeled guanine were determined from DNA hydrolysates by Sephadex-G10 radiochromatography. After dimethylnitrosamine only minor differences were observed for 7megua formation in the three groups; the 3mead/7megua ratio remained constant irrespective of the number of cells in S phase. In contrast, the Omegua/7megua ratio revealed a loss of Omegua, the extent of which appeared proportional to the fraction of DNA-synthesizing cells in the liver. The rapid loss of Omegua in S-phase cells was confirmed after intraportal administration of N-methyl- N-nitrosourea. During the first 10 min after the methylnitrosourea pulse the Omegua/7megua ratio was constant in G cells and dropped from 90 s to 10 min by about 15% in liver containing 30% S-phase cells and by about 40% with 80% cells in DNA synthesis. DNA-synthesizing hepatocytes are apparently endowed with a higher Omegua DNA transferase activity than nonproliferating liver cells. The rapid, though exhaustible elimination of Omegua during S-phase might result in partial protection of DNA-synthesizing cells from base-mispairing and/or from hypomethylation at G-C sites. [ABSTRACT FROM AUTHOR]

Acetoxymethyl-methylnitrosamine, the acetate ester of the presumed reactive metabolite of dimethylnitrosamine, is an effective teratogen in NMRI mice. An unusual phenomenon of application route specificity and a pronounced phase specificity of the teratogenic effects induced are demonstrated. Dose-response relationships are established. Some further arguments are given to support the basic hypothesis that the teratogenicity of alkylating agents is closely correlated to the DNA alkylation rate of embryonic cells. [ABSTRACT FROM AUTHOR]

The directly acting monofunctional alkylating agent ethylmethanesulfonate is a potent teratogen in NMRI mice. Inspection of skeletal abnormalities revealed a clear-cut phase specificity of the teratogenic outcome, especially of the limb malformations, after single intraperitoneal injections on days 9, 10, 11 or 12 of pregnancy. Dose-response studies were performed using single i.p. treatment of day 11. A computer program was developed for probit transformation of the data and maximum-likelihood estimation of the dose-response curves, including standard deviations and comparison of the slopes. Some arguments are presented for the hypothesis that the teratogenicity of simple alkylating agents is quantitatively correlated to a well-defined DNA alkylation rate of embryonic cells at the O-guanine site. [ABSTRACT FROM AUTHOR]

The ASCC3 subunit of the activating signal co-integrator complex is a dual-cassette Ski2-like nucleic acid helicase that provides single-stranded DNA for alkylation damage repair by the α-ketoglutarate-dependent dioxygenase AlkBH3. Other ASCC components integrate ASCC3/AlkBH3 into a complex DNA repair pathway. We mapped and structurally analyzed interacting ASCC2 and ASCC3 regions. The ASCC3 fragment comprises a central helical domain and terminal, extended arms that clasp the compact ASCC2 unit. ASCC2–ASCC3 interfaces are evolutionarily highly conserved and comprise a large number of residues affected by somatic cancer mutations. We quantified contributions of protein regions to the ASCC2–ASCC3 interaction, observing that changes found in cancers lead to reduced ASCC2–ASCC3 affinity. Functional dissection of ASCC3 revealed similar organization and regulation as in the spliceosomal RNA helicase Brr2. Our results delineate functional regions in an important DNA repair complex and suggest possible molecular disease principles. The DNA helicase ASCC3 is the largest subunit of the activating signal co-integrator complex (ASCC), and its DNA unwinding activity is required for the AlkBH3/ASCC-dependent DNA de-alkylation repair pathway. Here, the authors identify a minimal stable complex of the two ASCC subunits ASCC2 and ASCC3, determine the complex crystal structure and further show that cancer-related mutations at the interface between both proteins reduce ASCC2–ASCC3 affinity. [ABSTRACT FROM AUTHOR]

The MNNG hyper-resistance of yeast transformants containing multiple copies of the SNQ3/YAP1 yeast gene is not caused by lowered MNNG activation due to depleted pools of glutathione. On the contrary, the SNQ3/YAP1-encoded protein stimulates production of GSH, apparently by promoter activation due to the AP-1 recognition element. Expression of at least one further gene, encoding a protein with a strong detoxifying activity, must also be stimulated to explain the MNNG hyper-resistance phenotype. [ABSTRACT FROM AUTHOR]

Necrosis, unregulated cell death, is characterized by plasma membrane rupture as well as nuclear and cellular swelling. However, it has recently been reported that necrosis is a regulated form of cell death mediated by poly-(ADP-ribose) polymerase 1 (PARP1). PARP1 is thought to mediate necrosis by inducing DNA damage, although this remains unconfirmed. In this study, we examined the mechanisms of PARP1-mediated necrosis following doxorubicin (DOX)-induced DNA damage in human kidney proximal tubular (HK-2) cells. DOX initiated DNA damage response (DDR) and upregulated PARP1 and p53 expression, resulting in morphological changes similar to those observed during necrosis. Additionally, DOX induced mitochondrial hyper-activation, as evidenced by increased mitochondrial respiration and cytosolic ATP (cATP) production. However, DOX affected mitochondrial mass. DOX-induced DNA damage, cytosolic reactive oxygen species (cROS) generation, and mitochondrial hyper-activation decreased in cells with inhibited PARP1 expression, while generation of nitric oxide (NO) and mitochondrial ROS (mROS) remained unaffected. Moreover, DOX-induced DNA damage, cell cycle changes, and oxidative stress were not affected by p53 inhibition. These findings suggest that DNA damage induced necrosis through a PARP1-dependent and p53-independent pathway. [ABSTRACT FROM AUTHOR]

De novo point mutations and chromosomal structural aberrations (CSA) detected in offspring of unaffected parents show a preferential paternal origin with higher risk for older fathers. Studies in rodents suggest that heritable mutations transmitted from the father can arise from either paternal or maternal misrepair of damaged paternal DNA, and that the entire spermatogenic cycle can be at risk after mutagenic exposure. Understanding the susceptibility and mechanisms of transmission of paternal mutations is important in family planning after chemotherapy and donor selection for assisted reproduction. We report that treatment of male mice with melphalan (MLP), a bifunctional alkylating agent widely used in chemotherapy, induces DNA lesions during male mouse meiosis that persist unrepaired as germ cells progress through DNA repair-competent phases of spermatogenic development. After fertilization, unrepaired sperm DNA lesions are mis-repaired into CSA by the egg's DNA repair machinery producing chromosomally abnormal offspring. These findings highlight the importance of both pre- and post-fertilization DNA repair in assuring the genomic integrity of the conceptus. [ABSTRACT FROM AUTHOR]