RESUMO
The base excision repair glycosylase MUTYH prevents mutations associated with the oxidatively damaged base, 8-oxo-7,8-dihydroguanine (OG), by removing undamaged misincorporated adenines from OG:A mispairs. Defects in OG:A repair in individuals with inherited MUTYH variants are correlated with the colorectal cancer predisposition syndrome known as MUTYH-associated polyposis (MAP). Herein, we reveal key structural features of OG required for efficient repair by human MUTYH using structure-activity relationships (SAR). We developed a GFP-based plasmid reporter assay to define SAR with synthetically generated OG analogs in human cell lines. Cellular repair results were compared with kinetic parameters measured by adenine glycosylase assays in vitro. Our results show substrates lacking the 2-amino group of OG, such as 8OI:A (8OI = 8-oxoinosine), are not repaired in cells, despite being excellent substrates in in vitro adenine glycosylase assays, new evidence that the search and detection steps are critical factors in cellular MUTYH repair functionality. Surprisingly, modification of the O8/N7H of OG, which is the distinguishing feature of OG relative to G, was tolerated in both MUTYH-mediated cellular repair and in vitro adenine glycosylase activity. The lack of sensitivity to alterations at the O8/N7H in the SAR of MUTYH substrates is distinct from previous work with bacterial MutY, indicating that the human enzyme is much less stringent in its lesion verification. Our results imply that the human protein relies almost exclusively on detection of the unique major groove position of the 2-amino group of OG within OGsyn:Aanti mispairs to select contextually incorrect adenines for excision and thereby thwart mutagenesis. These results predict that MUTYH variants that exhibit deficiencies in OG:A detection will be severely compromised in a cellular setting. Moreover, the reliance of MUTYH on the interaction with the OG 2-amino group suggests that disrupting this interaction with small molecules may provide a strategy to develop potent and selective MUTYH inhibitors.
RESUMO
Base excision repair glycosylases locate and remove damaged bases in DNA with remarkable specificity. The MutY glycosylases, unusual for their excision of undamaged adenines mispaired to the oxidized base 8-oxoguanine (OG), must recognize both bases of the mispair in order to prevent promutagenic activity. Moreover, MutY must effectively find OG:A mismatches within the context of highly abundant and structurally similar T:A base pairs. Very little is known about the factors that initiate MutY's interaction with the substrate when it first encounters an intrahelical OG:A mispair, or about the order of recognition checkpoints. Here, we used structure-activity relationships (SAR) to investigate the features that influence the in vitro measured parameters of mismatch affinity and adenine base excision efficiency by E. coli MutY. We also evaluated the impacts of the same substrate alterations on MutY-mediated repair in a cellular context. Our results show that MutY relies strongly on the presence of the OG base and recognizes multiple structural features at different stages of recognition and catalysis to ensure that only inappropriately mispaired adenines are excised. Notably, some OG modifications resulted in more dramatic reductions in cellular repair than in the in vitro kinetic parameters, indicating their importance for initial recognition events needed to locate the mismatch within DNA. Indeed, the initial encounter of MutY with its target base pair may rely on specific interactions with the 2-amino group of OG in the major groove, a feature that distinguishes OG:A from T:A base pairs. These results furthermore suggest that inefficient substrate location in human MutY homologue variants may prove predictive for the early onset colorectal cancer phenotype known as MUTYH-Associated Polyposis, or MAP.
Assuntos
Adenina/metabolismo , Pareamento Incorreto de Bases , DNA Glicosilases/metabolismo , Reparo do DNA , Escherichia coli/enzimologia , Guanina/análogos & derivados , Adenina/análise , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Guanina/análise , Guanina/metabolismo , Modelos Moleculares , Especificidade por SubstratoRESUMO
8-Oxo-2'-deoxyguanosine (OdG) is an abundant and promutagenic damaged nucleotide that has been linked to aging and disease. To gain insight into the alternate base pairings of OdG, 8-chloro- and 8-iodo-2'-deoxyguanosine were incorporated into oligonucleotides and, along with 2'-deoxyguanosine and 8-bromo-2'-deoxyguanosine, were tested for their stability in base pairs opposite dC. We found a strong correlation between increased atomic radius and bond length at C8 and decreased base pair stability. These findings along with NMR studies on the base conformation of the corresponding nucleosides support the theory that the steric bulk of the 8-oxygen plays a role in OdG mutation and disease.
Assuntos
Citosina/análogos & derivados , Desoxiguanosina/análogos & derivados , Pareamento de Bases , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Citosina/química , Citosina/metabolismo , Desoxiguanosina/química , Desoxiguanosina/metabolismo , Ligação de Hidrogênio , Modelos Químicos , Conformação de Ácido Nucleico , Temperatura de TransiçãoRESUMO
Aflatoxin B(1) (AFB(1)), the most potent member of the aflatoxin family of hepatocarcinogens, upon metabolic activation reacts with DNA and forms a population of covalent adducts. The most prevalent adduct, 8,9-dihydro-8-(N(7)-guanyl-)-9-hydroxyaflatoxin (AFB(1)-N(7)-dG), as well as the AFB(1) formamidopyrimidine adduct (AFB(1)-FAPY), resulting from imidazole ring opening of the major adduct, are thought to be responsible for mutations caused by AFB(1). The AFB(1)-N(7)-dG lesions are rapidly removed in Escherichia coli and mammals, whereas the AFB(1)-FAPY lesions persist in mammalian cells, which along with the higher stability of this lesion suggests that AFB(1)-FAPY might significantly contribute to the observed toxicity and carcinogenicity of AFB(1) in higher organisms. Other workers have shown in vitro evidence that AFB(1)-FAPY lesions are substrates for both nucleotide excision repair (NER) and base excision repair (BER). The present study, done in vivo, utilized a modified host cell reactivation assay and showed that AFB(1)-FAPY lesions are preferentially repaired in E.coli by NER. Comparisons of repair in wild-type, NER-deficient (uvrA), BER-deficient (mutM) and NER/BER double mutant E.coli strains transformed with plasmids enriched for AFB(1)-N(7)-dG or AFB(1)-FAPY lesions indicate that both lesions are efficiently repaired by NER-proficient cells (both wild-type and BER-deficient strains). We have found that the level of activity of the reporter gene is significantly affected by the presence of either lesion in NER-deficient strains due to the lack of repair. This effect is similar in NER-deficient and NER/BER-deficient strains indicating that BER (specifically in the strains we investigated) does not contribute significantly to the repair of these lesions in vivo. Consistent with this finding, in vitro analysis of AFB(1)-FAPY adduct excision by purified MutM and its functional analog human 8-oxoguanine DNA glycosylase using site-specifically modified oligonucleotides indicates that this lesion is a poor substrate for both proteins compared with canonical substrates for these enzymes, such as 7,8-dihydro-8-oxoguanine and methylformamidopyrimidine.
Assuntos
Aflatoxina B1/toxicidade , Reparo do DNA , Pirimidinas/química , Aflatoxina B1/química , Sequência de Bases , OligonucleotídeosRESUMO
A G to T mutation has been observed at the third position of codon 249 of the p53 tumor-suppressor gene in over 50% of the hepatocellular carcinoma cases associated with high exposure to aflatoxin B(1) (AFB(1)). Hypotheses have been put forth that AFB(1), in concert with hepatitis B virus (HBV), may play a role in the formation of, and/or the selection for, this mutation. The primary DNA adduct of AFB(1) is 8,9-dihydro-8-(N(7)-guanyl)-9-hydroxyaflatoxin B(1) (AFB(1)-N7-Gua), which is converted naturally to two secondary lesions, an apurinic site and an AFB(1)-formamidopyrimidine (AFB(1)-FAPY) adduct. AFB(1)-FAPY is detected at near maximal levels in rat DNA days to weeks after AFB(1) exposure, underscoring its high persistence in vivo. The present study reveals two striking properties of this DNA adduct: (i) AFB(1)-FAPY was found to cause a G to T mutation frequency in Escherichia coli approximately 6 times higher than that of AFB(1)-N7-Gua, and (ii) one proposed rotamer of AFB(1)-FAPY is a block to replication, even when the efficient bypass polymerase MucAB is used by the cell. Taken together, these characteristics make the FAPY adduct the prime candidate for both the genotoxicity of aflatoxin, because mammalian cells also have similar bypass mechanisms for combating DNA damage, and the mutagenicity that ultimately may lead to liver cancer.