Cellular Repair of Synthetic Analogs of Oxidative DNA Damage Reveals a Key Structure-Activity Relationship of the Cancer-Associated MUTYH DNA Repair Glycosylase.

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.

The importance of Ile716 toward the mutagenicity of 8-Oxo-2'-deoxyguanosine with Bacillus fragment DNA polymerase.

8-oxo-2'-deoxyguanosine (OdG) is a prominent DNA lesion that can direct the incorporation of dCTP or dATP during replication. As the latter reaction can lead to mutation, the ratio of dCTP/dATP incorporation can significantly affect the mutagenic potential of OdG. Previous work with the A-family polymerase BF and seven analogues of OdG identified a major groove amino acid, Ile716, which likely influences the dCTP/dATP incorporation ratio opposite OdG. To further probe the importance of this amino acid, dCTP and dATP incorporations opposite the same seven analogues were tested with two BF mutants, I716M and I716A. Results from these studies support the presence of clashing interactions between Ile716 and the C8-oxygen and C2-amine during dCTP and dATP incorporations, respectively. Crystallographic analysis suggests that residue 716 alters the conformation of the template base prior to insertion into the active site, thereby affecting enzymatic efficiency. These results are also consistent with previous work with A-family polymerases, which indicate they have tight, rigid active sites that are sensitive to template perturbations.

Structure-Activity Relationships Reveal Key Features of 8-Oxoguanine: A Mismatch Detection by the MutY Glycosylase.

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.

Insights into the substrate specificity of the MutT pyrophosphohydrolase using structural analogues of 8-oxo-2'-deoxyguanosine nucleotide.

The bacterial repair enzyme MutT hydrolyzes the damaged nucleotide OdGTP (the 5'-triphosphate derivative of 8-oxo-2'-deoxyguanosine; OdG), which is a known mutagen and has been linked to antibacterial action. Previous work has indicated important roles for the C8-oxygen, N7-hydrogen, and C2-exocyclic amine during OdGTP recognition by MutT. In order to gain a more nuanced understanding of the contribution of these three sites to the overall activity of MutT, we determined the reaction parameters for dGTP, OdGTP, and nine of their analogues using steady state kinetics. Our results indicate that overall high reaction efficiencies can be achieved despite altering any one of these sites. However, altering two or more sites leads to a significant decrease in efficiency. The data also suggest that, similar to another bacterial OdG repair enzyme, MutM, a specific carbonyl in the enzyme can not only promote activity by forming an active site hydrogen bond with the N7-hydrogen of OdGTP, but can also hinder activity through electrostatic repulsion with the N7-lone pair of dGTP.

Biochemical investigations into the mutagenic potential of 8-oxo-2'-deoxyguanosine using nucleotide analogues.

8-Oxo-2'-deoxyguanosine (OdG) is an abundant DNA lesion produced during oxidative damage to DNA. It can form relatively stable base pairs with both dC and dA that mimic natural dG:dC and dT:dA base pairs, respectively. Thus, when in the template strand, OdG can direct the insertion of either dCTP or dATP during replication, the latter of which can lead to a dG → T transversion. The potential for OdG to cause mutation is dependent on the preference for dCTP or dATP insertion opposite OdG, as well as the ability to extend past the resulting base pairs. The C2-amine and C8-oxygen could play major roles during these reactions since both would lie outside the Watson-Crick cognate base pairs shape in the major groove when OdG base pairs to dA and dC, respectively, and both have the ability to form strong interactions, like hydrogen bonds. To gain a more generalized understanding of how the C2-amine and C8-oxygen of OdG affect its mutagenic potential, the incorporation opposite and extension past seven analogues of dG/OdG that vary at C2 and/or C8 were characterized for three DNA polymerases, including an exonuclease-deficient version of the replicative polymerase from RB69 (RB69), human polymerase (pol) ß, and polymerase IV from Sulfolobus solfataricus P2 (Dpo4). Based on the results from these studies, as well as those from previous studies with RB69, pol ß, Dpo4, and two A-family polymerases, the influence of the C2-amine and C8-oxygen during each incorporation and extension reaction with each polymerase is discussed. In general, it appears that when the C2-amine and the C8-oxygen are in the minor groove, they allow OdG to retain interactions that are normally present during insertion and extension. However, when the two groups are in the major groove, they each tend to form novel active site interactions, both stabilizing and destabilizing, that are not present during insertion and extension with natural DNA.

Importance of the C2, N7, and C8 positions to the mutagenic potential of 8-Oxo-2'-deoxyguanosine with two A family polymerases.

8-Oxo-2'-deoxyguanosine (OdG) is a prominent DNA lesion produced from the reaction of 2'-deoxyguanosine (dG) with reactive oxygen species. While dG directs the insertion of only dCTP during replication, OdG can direct the insertion of either dCTP or dATP, allowing for the production of dG → dT transversions. When replicated by Klenow fragment-exo (KF-exo), OdG preferentially directs the incorporation of dCTP over dATP, thus decreasing its mutagenic potential. However, when replicated by a highly related polymerase, the large fragment of polymerase I from Bacillus stearothermophilus (BF), dATP incorporation is preferred, and a higher mutagenic potential results. To gain insight into the reasons for this opposite preference and the effects of the C2, N7, and C8 positions on OdG mutagenicity, single-nucleotide insertions of dCTP and/or dATP opposite dG, OdG, and seven of their analogues were examined by steady state kinetics with both KF-exo and BF. Results from these studies suggest that the two enzymes behave similarly and are both sensitive not only to steric and electronic changes within the imidazole ring during both dCTP and dATP incorporation but also to the presence of the C2-exocyclic amine during dATP incorporation. The difference in incorporation preference opposite OdG appears to be due to a somewhat increased sensitivity to structural perturbations during dCTP incorporation with BF. Single-nucleotide extensions past the resulting base pairs were also studied and were not only similar between the two enzymes but also consistent with published ternary crystallographic studies with BF. These results are analyzed in the context of previous biochemical and structural studies, as well as stability studies with the resulting base pairs.

Oligonucleotide incorporation and base pair stability of 9-deaza-2'-deoxyguanosine, an analogue of 8-oxo-2'-deoxyguanosine.

9-Deaza-2'-deoxyguanosine (CdG) is a C-nucleoside and an analogue of the abundant promutagen 8-oxo-2'-deoxyguanosine (OdG). Like 2'-deoxyguanosine (dG), CdG should form a stable base pair with dC, but similar to OdG, CdG contains an N7-hydrogen that should allow it to also form a relatively stable base pair with dA. In order to further investigate the base pairing of CdG, it was incorporated into DNA and paired with either dC or dA. Melting studies revealed CdG:dC base pairs are less stable than dG:dC base pairs, while CdG:dA base pairs are less stable than OdG:dA base pairs. In order to gain a deeper understanding of these results, quantum studies on model structures of nucleoside monomers and base pairs were performed, the results of which indicate that (i) CdG:dC base pairs are likely destabilized relative to dG:dC as a result of structural constraints imposed by the C-nucleotide character of CdG, and (ii) CdG:dA base pairs may be less stable than OdG:dA base pairs, at least in part, because of a third long-range interaction that is possible in OdG:dA but not in CdG:dA.

Synthesis, oligonucleotide incorporation and base pair stability of 7-methyl-8-oxo-2'-deoxyguanosine.

7-Methyl-8-oxo-2'-deoxyguanosine, an analogue of the abundant promutagen 8-oxo-2'-deoxyguanosine, was incorporated into oligonucleotides and tested for its stability in various base pairs.

Base pair stability of 8-chloro- and 8-iodo-2'-deoxyguanosine opposite 2'-deoxycytidine: implications regarding the bioactivity of 8-oxo-2'-deoxyguanosine.

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.

Oligonucleotide incorporation of 8-thio-2'-deoxyguanosine.

[reaction: see text] 8-Thio-2'-deoxyguanosine (SdG) is a useful analogue of the abundant promutagen 8-oxo-2'-deoxyguanosine (OdG). Its synthesis and DNA incorporation using standard phosphoramidite chemistry is reported. To prevent oxidation during DNA synthesis, the sulfur was protected as a 2-(trimethylsilyl)ethyl sulfide. Subsequent treatment with TBAF yielded the desired 8-thiocarbonyl functionality. Melting studies with SdG revealed almost equal stabilities of SdG:dC and SdG:dA base pairs, lending insight into the base-pairing preferences of OdG.

Aflatoxin B1 formamidopyrimidine adducts are preferentially repaired by the nucleotide excision repair pathway in vivo.

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.

The aflatoxin B(1) formamidopyrimidine adduct plays a major role in causing the types of mutations observed in human hepatocellular carcinoma.

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.