RESUMO
Human alkyladenine DNA glycosylase (hAAG) excises alkylated purines, hypoxanthine, and etheno bases from DNA to form abasic (AP) sites. Surprisingly, elevated expression of hAAG increases spontaneous frameshift mutagenesis. By random mutagenesis of eight active site residues, we isolated hAAG-Y127I/H136L double mutant that induces even higher rates of frameshift mutation than does the wild-type hAAG; the Y127I mutation accounts for the majority of the hAAG-Y127I/H136L-induced mutator phenotype. The hAAG-Y127I/H136L and hAAG-Y127I mutants increased the rate of spontaneous frameshifts by up to 120-fold in S. cerevisiae and also induced high rates of microsatellite instability (MSI) in human cells. hAAG and its mutants bind DNA containing one and two base-pair loops with significant affinity, thus shielding them from mismatch repair; the strength of such binding correlates with their ability to induce the mutator phenotype. This study provides important insights into the mechanism of hAAG-induced genomic instability.
Assuntos
DNA Glicosilases/metabolismo , Mutação da Fase de Leitura , Instabilidade de Microssatélites , Domínio Catalítico , DNA Glicosilases/química , DNA Glicosilases/genética , Reparo de Erro de Pareamento de DNA , Regulação Enzimológica da Expressão Gênica , Humanos , Células K562 , Modelos Moleculares , Fenótipo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
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 ß-cells, and retinal photoreceptor cells, was detected in wild-type mice, exacerbated in Aag transgenic mice, and completely suppressed in Aagâ»/â» 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.
Assuntos
Antineoplásicos Alquilantes , DNA Glicosilases , Neoplasias/tratamento farmacológico , Poli(ADP-Ribose) Polimerases , Alquilação/efeitos dos fármacos , Alquilação/genética , Animais , Antineoplásicos Alquilantes/efeitos adversos , Antineoplásicos Alquilantes/uso terapêutico , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Humanos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Camundongos , Camundongos Transgênicos/genética , Camundongos Transgênicos/lesões , Neoplasias/genética , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Timócitos/citologia , Timócitos/efeitos dos fármacosRESUMO
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.
Assuntos
Alquilantes/toxicidade , DNA Glicosilases/fisiologia , Reparo do DNA , Degeneração Retiniana/induzido quimicamente , Animais , Apoptose , Metilases de Modificação do DNA/fisiologia , Enzimas Reparadoras do DNA/fisiologia , Metanossulfonato de Metila/toxicidade , Metilnitrosoureia/toxicidade , Camundongos , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Proteínas Supressoras de Tumor/fisiologiaRESUMO
Human alkyladenine-DNA glycosylase (AAG) initiates base excision repair (BER) of alkylated and deaminated bases in DNA. Here, we assessed the mutability of the AAG substrate binding pocket, and the essentiality of individual binding pocket amino acids for survival of methylation damage. We used oligonucleotide-directed mutagenesis to randomize 19 amino acids, 8 of which interact with substrate bases, and created more than 4.5 million variants. We expressed the mutant AAGs in repair-deficient Escherichia coli and selected for protection against the cytotoxicity of either methylmethane sulfonate (MMS) or methyl-lexitropsin (Me-lex), an agent that produces 3-methyladenine as the predominant base lesion. Sequence analysis of 116 methylation-resistant mutants revealed no substitutions for highly conserved Tyr(127)and His(136). In contrast, one mutation, L180F, was greatly enriched in both the MMS- and Me-lex-resistant libraries. Expression of the L180F single mutant conferred 4.4-fold enhanced survival at the high dose of MMS used for selection. The homogeneous L180F mutant enzyme exhibited 2.2-fold reduced excision of 3-methyladenine and 7.3-fold reduced excision of 7-methylguanine from methylated calf thymus DNA. Decreased excision of methylated bases by the mutant glycosylase could promote survival at high MMS concentrations, where the capacity of downstream enzymes to process toxic BER intermediates may be saturated. The mutant also displayed 6.6- and 3.0-fold reduced excision of 1,N(6)-ethenoadenine and hypoxanthine from oligonucleotide substrates, respectively, and a 1.7-fold increase in binding to abasic site-containing DNA. Our work provides in vivo evidence for the substrate binding mechanism deduced from crystal structures, illuminates the function of Leu(180) in wild-type human AAG, and is consistent with a role for balanced expression of BER enzymes in damage survival.
Assuntos
Aminoácidos/metabolismo , DNA Glicosilases/metabolismo , Escherichia coli/citologia , Escherichia coli/efeitos dos fármacos , Metanossulfonato de Metila/farmacologia , Viabilidade Microbiana/efeitos dos fármacos , Netropsina/análogos & derivados , Sequência de Aminoácidos , Substituição de Aminoácidos/efeitos dos fármacos , Domínio Catalítico , Adutos de DNA/metabolismo , DNA Glicosilases/química , Teste de Complementação Genética , Humanos , Dados de Sequência Molecular , Mutagênese/efeitos dos fármacos , Proteínas Mutantes/química , Mutação/genética , Netropsina/farmacologia , Ligação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato/efeitos dos fármacosRESUMO
Somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin (Ig) genes require the cytosine deaminase AID, which deaminates cytosine to uracil in Ig gene DNA. Paradoxically, proteins involved normally in error-free base excision repair and mismatch repair, seem to be co-opted to facilitate SHM and CSR, by recruiting error-prone translesion polymerases to DNA sequences containing deoxy-uracils created by AID. Major evidence supports at least one mechanism whereby the uracil glycosylase Ung removes AID-generated uracils creating abasic sites which may be used either as uninformative templates for DNA synthesis, or processed to nicks and gaps that prime error-prone DNA synthesis. We investigated the possibility that deamination at adenines also initiates SHM. Adenosine deamination would generate hypoxanthine (Hx), a substrate for the alkyladenine DNA glycosylase (Aag). Aag would generate abasic sites which then are subject to error-prone repair as above for AID-deaminated cytosine processed by Ung. If the action of an adenosine deaminase followed by Aag were responsible for significant numbers of mutations at A, we would find a preponderance of A:T>G:C transition mutations during SHM in an Aag deleted background. However, this was not observed and we found that the frequencies of SHM and CSR were not significantly altered in Aag-/- mice. Paradoxically, we found that Aag is expressed in B lymphocytes undergoing SHM and CSR and that its activity is upregulated in activated B cells. Moreover, we did find a statistically significant, albeit low increase of T:A>C:G transition mutations in Aag-/- animals, suggesting that Aag may be involved in creating the SHM A>T bias seen in wild type mice.
Assuntos
Mutação , N-Glicosil Hidrolases/metabolismo , Recombinação Genética , Animais , Sequência de Bases , Primers do DNA , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , N-Glicosil Hidrolases/genéticaRESUMO
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 O(6)-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.
Assuntos
DNA Glicosilases/genética , Reparo do DNA , Longevidade/genética , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , DNA Glicosilases/metabolismo , Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
The O(6)-methylguanine (O(6)MeG) DNA lesion is well known for its mutagenic, carcinogenic, and cytotoxic properties, and understanding how a cell processes such damage is of critical importance for improving current cancer therapy. Here we use human cells differing only in their O(6)MeG DNA methyltransferase (MGMT) or mismatch repair (MMR) status to explore the O(6)MeG/MMR-dependent molecular and cellular responses to treatment with the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). We find that O(6)MeG triggers MMR-dependent cell cycle perturbations in both the first and second cell cycle post treatment. At lower levels of damage, we show that a transient arrest in the second S-phase precedes survival and progression into subsequent cell cycles. However, at higher levels of damage, arrest in the second S-phase coincides with a cessation of DNA replication followed by initiation of apoptotic cell death. Further, we show that entry into apoptotic cell death is specifically from S-phase of the second cell cycle. Finally, we demonstrate the key role of an O(6)MeG/MMR-dependent multi-pathway, multi-time-scale signaling network activation, led by early ATM, H2AX, CHK1, and p53 phosphorylation and followed by greatly amplified late phosphorylation of the early pathway nodes along with activation of the CHK2 kinase and the stress-activated JNK kinase.
Assuntos
Antineoplásicos/farmacologia , Apoptose , DNA/efeitos dos fármacos , Guanina/análogos & derivados , Neoplasias/tratamento farmacológico , Pareamento Incorreto de Bases , Ciclo Celular , Morte Celular , Linhagem Celular , DNA/metabolismo , Dano ao DNA , Reparo do DNA , Replicação do DNA , Relação Dose-Resposta a Droga , Regulação Neoplásica da Expressão Gênica , Guanina/farmacologia , Humanos , Fase S , Transdução de SinaisRESUMO
The use of DNA equilibrium binding molecules to transfer alkyl groups to specific positions on DNA is an approach to generating cytotoxic DNA damage while avoiding the formation of promutagenic lesions that increase the risk for the development of secondary cancer. We have previously reported that in vitro a neutral DNA equilibrium binding agent based on an N-methylpyrrolecarboxamide dipeptide (lex) and modified with an O-methyl sulfonate ester functionality (Me-lex) selectively affords N3-methyladenine lesions in >90% yield relative to the formation of other adducts. While in vitro interactions between the lex dipeptide and DNA have been thoroughly studied, in vivo interactions are more difficult to elucidate. We report herein the relationship between the in vivo formation of N3-methyladenine and toxicity in wild-type and base excision repair defective mutant Escherichia coli. In addition, it is demonstrated that both N3-methyladenine adduction and cytotoxicity can be inhibited in vivo with netropsin, a potent competitive inhibitor of binding of lex to DNA. The results show a clear relationship between the levels of N3-methyladenine and toxicity in an alkA/tag glycosylase mutant that cannot remove the adduct from its genome. For methyl methanesulfonate, which does not sequence selectively methylate DNA, a relationship between the formation of N3-methyladenine and toxicity is also observed. However, netropsin affects neither the level of N3-methyladenine nor the toxicity of methyl methanesulfonate in E. coli.
Assuntos
Adenina/metabolismo , Escherichia coli/metabolismo , Mesilatos/farmacologia , Netropsina/farmacologia , Adenina/análogos & derivados , Animais , Bovinos , Metilação de DNA , Ensaio de Imunoadsorção Enzimática , Ésteres , Mesilatos/químicaRESUMO
Using a yeast shuttle vector system, we have previously reported on the toxicity and mutagenicity of Me-lex, [1-methyl-4-[1-methyl-4-[3-(methoxysulfonyl)propanamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]propane, a compound that selectively generates 3-methyladenine (3-MeA). We observed that a mutant strain defective in Mag1, the glycosylase that excises 3-MeA in the initial step of base excision repair (BER) to generate an abasic site, is significantly more sensitive to the toxicity of Me-lex with respect to wild type but shows only a marginal increase in mutagenicity. A strain defective in AP endonuclease activity (Deltaapn1apn2), also required for functional BER, is equally sensitive to the toxicity as the Deltamag1 mutant but showed a significantly higher mutation frequency. In the present work, we have explored the role of nucleotide excision repair (NER) in Me-lex-induced toxicity and mutagenicity since it is known that NER acts on abasic sites in vivo in yeast and in vitro assays. To accomplish this, we have deleted one of the genes essential for NER in yeast, namely, RAD14, both in the context of an otherwise DNA repair-proficient strain (Deltarad14) and in a BER-defective isogenic derivative lacking the MAG1 gene (Deltamag1rad14). Interestingly, no sensitivity to the treatment with Me-lex was conferred by the simple deletion of RAD14. However, a significant enhancement in toxicity and mutagenicity was observed when cells lacked both Rad14 and Mag1. The mutation spectrum induced by Me-lex in the Deltamag1rad14 strain is indistinguishable from that observed in the Deltaapn1/Deltaapn2 or in the Deltamag1 strains. The results indicate that in yeast NER can play a protective role against 3-MeA-mediated toxicity and mutagenicity; however, the role of NER is appreciable only in a BER-defective background.
Assuntos
Adenina/análogos & derivados , Adenina/metabolismo , Metilação de DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Mutagênese , Mutagênicos/toxicidade , Netropsina/análogos & derivados , Netropsina/toxicidade , Adenina/química , Adenina/toxicidade , Sequência de Bases/efeitos dos fármacos , Sequência de Bases/genética , Análise Mutacional de DNA , Enzimas Reparadoras do DNA , Deleção de Genes , Inativação Gênica , Humanos , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genéticaRESUMO
Minor groove specific DNA equilibrium binding peptides (lex) based on N-methylpyrrole-carboxamide and/or N-methylimidazolecarboxamide subunits have been modified with an O-methyl sulfonate ester functionality to target DNA methylation in the minor groove at Ade/Thy- and/or Gua/Cyt-rich sequences. HPLC and sequencing gel analyses show that the Me-lex compounds all selectively react with DNA to afford N3-alkyladenine as a major adduct. The formation of the N3-alkyladenine lesions is sequence-dependent based on the equilibrium binding preferences of the different lex peptides. In addition to the reaction at adenine, the molecules designed to target Gua/Cyt sequences also generate lesions at guanine; however, the methylation is not sequence dependent and takes places in the major groove at the N7-position. To determine if and how the level of the different DNA adducts and the sequence selectivity for their formation affects cytotoxicity, the Me-lex analogues were tested in wild type Escherichia coli and in mutant strains defective in base excision repair (tag and/or alkA or apn). The results demonstrate the importance of 3-methyladenine, and in some cases 3-methylguanine, lesions in cellular toxicity, and the dominant protective role of the DNA glycosylases. There is no evidence that the sequence specificity is related to toxicity.
Assuntos
Adenina/análogos & derivados , Antibacterianos/toxicidade , Dano ao DNA , DNA Bacteriano/metabolismo , Escherichia coli/efeitos dos fármacos , Guanina/análogos & derivados , Mesilatos/toxicidade , Netropsina/análogos & derivados , Netropsina/toxicidade , Adenina/metabolismo , Adenina/toxicidade , Sequência de Bases/efeitos dos fármacos , Adutos de DNA/análise , Adutos de DNA/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , DNA Bacteriano/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Ésteres , Guanina/metabolismo , Guanina/toxicidade , Dados de Sequência Molecular , Netropsina/síntese química , Netropsina/química , Conformação de Ácido Nucleico/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacosRESUMO
Due to its minor groove selectivity, Me-lex preferentially generates N3-methyladenine (3-MeA) adducts in double-stranded DNA. We undertook a genetic approach in yeast to establish the influence of base excision repair (BER) defects on the processing of Me-lex lesions on plasmid DNA that harbors the p53 cDNA as target. We constructed a panel of isogenic strains containing a reporter gene to test p53 function and the following gene deletions: deltamag1, deltaapn1apn2, and deltaapn1apn2mag1. When compared with the wild-type strain, a decrease in survival was observed in deltamag1, deltaapn1apn2, and deltaapn1apn2mag1. The Me-lex-induced mutation frequency increased in the following order: wild type < deltamag1< deltaapn1apn2 = deltaapn1apn2mag1. A total of 77 mutants (23 in wild type, 31 in deltamag1, and 23 in deltaapn1apn2) were sequenced. Eighty-one independent mutations (24 in wild type, 34 in deltamag1, and 23 in deltaapn1apn2) were detected. The majority of base pair substitutions were AT-targeted in all strains (14/23, 61% in wild type; 20/34, 59%, in deltamag1; and 14/23, 61%, in deltaapn1apn2). The Mag1 deletion was associated with a significant decrease of GC > AT transitions when compared with both the wild-type and the AP endonuclease mutants. This is the first time that the impact of Mag1 and/or AP endonuclease defects on the mutational spectra caused by 3-MeA has been determined. The results suggest that 3-MeA is critical for Me-lex cytotoxicity and that its mutagenicity is slightly elevated in the absence of Mag1 glycosylase activity but significantly higher in the absence of AP endonuclease activity.