Fapy Lesions Research Articles

Chemical Biology of N5-Substituted Formamidopyrimidine DNA Adducts.

DNA nucleobases are the prime targets for chemical modifications by endogenous and exogenous electrophiles. Alkylation of the N7 position of guanine and adenine in DNA triggers base-catalyzed imidazole ring opening and the formation of N5-substituted formamidopyrimidine (N5-R-FAPy) lesions. Me-FAPy-dG adducts induced by exposure to methylating agents and AFB-FAPy-dG lesions formed by aflatoxin B1 have been shown to persist in cells and to contribute to toxicity and mutagenicity. In contrast, the biological outcomes of other N5-substituted FAPy lesions have not been fully elucidated. To enable their structural and biological evaluation, N5-R-FAPy adducts must be site-specifically incorporated into synthetic DNA strands using phosphoramidite building blocks, which can be complicated by their unusual structural complexity. N5-R-FAPy exist as a mixture of rotamers and can undergo isomerization between α, β anomers and furanose-pyranose forms. In this Perspective, we will discuss the main types of N5-R-FAPy adducts and summarize the strategies for their synthesis and structural elucidation. We will also summarize the chemical biology studies conducted with N5-R-FAPy-containing DNA to elucidate their effects on DNA replication and to identify the mechanisms of N5-R-FAPy repair.

FaPy lesions and DNA mutations

Formamidopyrimidine residues are damaged purines that retain the same Watson-Crick hydrogen-bonding face found in the parent nucleobase, yet these lesions are mutagenic. Crystallographic evidence suggests molecular mechanisms by which these lesions 'mispair' to generate mutations during DNA replication.

The Formamidopyrimidines: Purine Lesions Formed in Competition With 8-Oxopurines From Oxidative Stress

DNA is constantly exposed to agents that induce structural damage, from sources both internal and external to an organism. Endogenous species, such as oxidizing chemicals, and exogenous agents, such as ultraviolet rays in sunlight, together produce more than 70 distinct chemical modifications of native nucleotides. Of these, about 15 of the lesions have been detected in cellular DNA. This kind of structural DNA damage can be cytotoxic, carcinogenic, or both and is being linked to an increasingly lengthy list of diseases. The formamidopyrimidine (Fapy) lesions are a family of DNA lesions that result after purines undergo oxidative stress. The Fapy lesions are produced in yields comparable to the 8-oxopurines, which, owing in part to a perception of mutagenicity in some quarters, have been subjected to intense research scrutiny. But despite the comparable abundance of the formamidopyrimidines and the 8-oxopurines, until recently very little was known about the effects of Fapy lesions on biochemical processes involving DNA or on the structure and stability of the genomic material. In this Account, we discuss the detection of Fapy lesions in DNA and the mechanism proposed for their formation. We also describe methods for the chemical synthesis of oligonucleotides containing Fapy·dA or Fapy·dG and the outcomes of chemical and biochemical studies utilizing these compounds. These experiments reveal that the formamidopyrimidines decrease the fidelity of polymerases and are substrates for DNA repair enzymes. The mutation frequency of Fapy·dG in mammals is even greater than that of 8-oxodGuo (8-oxo-7,8-dihydro-2'-deoxyguanosine, one of the 8-oxopurines), suggesting that this lesion could be a useful biomarker and biologically significant. Despite clear similarities, the formamidopyrimidines have lived in the shadow of the corresponding 8-oxopurine lesions. But the recent development of methods for synthesizing oligonucleotides containing Fapy·dA or Fapy·dG has accelerated research on these lesions, revealing that the formamidopyrimidines are repaired as efficiently and, in some cases, more rapidly than the 8-oxopurines. Fapy·dG appears to be a lesion of biochemical consequence, and further study of its mutagenicity, repair, and interactions with DNA structure will better define the cellular details involving this important product of DNA stress.

Selective Incision of the α‐N5‐Methyl‐Formamidopyrimidine Anomer by Escherichia coli Endonuclease IV

Formamidopyrimidines (Fapy) lesions result from ring opening of the imidazole portion of purines. Fapy lesions can isomerize from the natural β-anomeric stereochemistry to the α-configuration. We have unambiguously demonstrated that the α-methyl-Fapy-dG (MeFapy-dG) lesion is a substrate for Escherichia coli Endonuclease IV (Endo IV). Treatment of a MeFapy-dG-containing 24 mer duplex with Endo IV resulted in 36–40% incision. The catalytic efficiency of the incision was comparable to that of α-dG in the same duplex sequence. The α- and β-MeFapy-dG anomers equilibrate to ~21 : 79 ratio over ~3 days. Related studies with a duplex containing the α-Fapy-dG lesion derived from aflatoxin B1 epoxide (α-AFB-Fapy-dG) showed only low levels of incision. It is hypothesized that the steric bulk of the aflatoxin moiety interferes with the binding of the substrate to Endo IV and the incision chemistry.

Characterization of the major formamidopyrimidine-DNA glycosylase homolog inMycobacterium tuberculosisand its linkage to variable tandem repeats

The ability to repair DNA damage is likely to play an important role in the survival of facultative intracellular parasites because they are exposed to high levels of reactive oxygen species and nitrogen intermediates inside phagocytes. Correcting oxidative damage in purines and pyrimidines is the primary function of the enzymes formamidopyrimidine (faPy)–DNA glycosylase (Fpg) and endonuclease VIII (Nei) of the base excision repair pathway, respectively. Four gene homologs, belonging to the fpg/nei family, have been identified in Mycobacterium tuberculosis H37Rv. The recombinant protein encoded by M. tuberculosis Rv2924c, termed Mtb-Fpg1, was overexpressed, purified and biochemically characterized. The enzyme removed faPy and 5-hydroxycytosine lesions, as well as 8-oxo-7,8-dihydroguanine (8oxoG) opposite to C, T and G. Mtb-Fpg1 thus exhibited substrate specificities typical for Fpg enzymes. Although Mtb-fpg1 showed nearly complete nucleotide sequence conservation in 32 M. tuberculosis isolates, the region upstream of Mtb-fpg1 in these strains contained tandem repeat motifs of variable length. A relationship between repeat length and Mtb-fpg1 expression level was demonstrated in M. tuberculosis strains, indicating that an increased length of the tandem repeats positively influenced the expression levels of Mtb-fpg1. This is the first example of such a tandem repeat region of variable length being linked to the expression level of a bacterial gene.

Characterization of the meningococcal DNA glycosylase Fpg involved in base excision repair

BackgroundNeisseria meningitidis, the causative agent of meningococcal disease, is exposed to high levels of reactive oxygen species inside its exclusive human host. The DNA glycosylase Fpg of the base excision repair pathway (BER) is a central player in the correction of oxidative DNA damage. This study aimed at characterizing the meningococcal Fpg and its role in DNA repair.ResultsThe deduced N. meningitidis Fpg amino acid sequence was highly homologous to other Fpg orthologues, with particularly high conservation of functional domains. As for most N. meningitidis DNA repair genes, the fpg gene contained a DNA uptake sequence mediating efficient transformation of DNA. The recombinant N. meningitidis Fpg protein was over-expressed, purified to homogeneity and assessed for enzymatic activity. N. meningitidis Fpg was found to remove 2,6-diamino-4-hydroxy-5-formamidopyrimidine (faPy) lesions and 7,8-dihydro-8-oxo-2'-deoxyguanosine (8oxoG) opposite of C, T and G and to a lesser extent opposite of A. Moreover, the N. meningitidis fpg single mutant was only slightly affected in terms of an increase in the frequency of phase variation as compared to a mismatch repair mutant.ConclusionCollectively, these findings show that meningococcal Fpg functions are similar to those of prototype Fpg orthologues in other bacterial species.

Efficient Removal of Formamidopyrimidines by 8-Oxoguanine Glycosylases

Under conditions of oxidative stress, the formamidopyrimidine lesions (FapyG and FapyA) are formed in competition with the corresponding 8-oxopurines (OG and OA) from a common intermediate. In order to reveal features of the repair of these lesions, and the potential contribution of repair in mitigating or exacerbating the mutagenic properties of Fapy lesions, their excision by three glycosylases, Fpg, hOGG1 and Ntg1, was examined in various base pair contexts under single-turnover conditions. FapyG was removed at least as efficiently as OG by all three glycosylases. In addition, the rates of removal of FapyG by Fpg and hOGG1 were influenced by their base pair partner, with preference for removal when base paired with the correct Watson-Crick partner C. With the FapyA lesion, Fpg and Ntg1 catalyze its removal more readily than OG opposite all four natural bases. In contrast, the removal of FapyA by hOGG1 was not as robust as FapyG or OG, and was only significant when the lesion was paired with C. The discrimination by the various glycosylases with respect to the opposing base was highly dependent on the identity of the lesion. OG induced the greatest selectivity against its removal when part of a promutagenic base pair. The superb activity of the various OG glycosylases toward removal of FapyG and FapyA in vitro suggests that these enzymes may act upon these oxidized lesions in vivo. The differences in the activity of the various glycosylases for removal of FapyG and FapyA compared to OG in nonmutagenic versus promutagenic base pair contexts may serve to alter the mutagenic profiles of these lesions in vivo.

Genetic effects of oxidative DNA damages: comparative mutagenesis of the imidazole ring-opened formamidopyrimidines (Fapy lesions) and 8-oxo-purines in simian kidney cells

Genetic effects of oxidative DNA damages: comparative mutagenesis of the imidazole ring-opened formamidopyrimidines (Fapy lesions) and 8-oxo-purines in simian kidney cells

Genetic effects of oxidative DNA damages: comparative mutagenesis of the imidazole ring-opened formamidopyrimidines (Fapy lesions) and 8-oxo-purines in simian kidney cells

Fapy·dG and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) are formed in DNA by hydroxyl radical damage. In order to study replication past these lesions in cells, we constructed a single-stranded shuttle vector containing the lesion in 5′-TGT and 5′-TGA sequence contexts. Replication of the modified vector in simian kidney (COS-7) cells showed that Fapy·dG is mutagenic inducing primarily targeted Fapy·G→T transversions. In the 5′-TGT sequence mutational frequency of Fapy·dG was ∼30%, whereas in the 5′-TGA sequence it was ∼8%. In parallel studies 8-oxo-dG was found to be slightly less mutagenic than Fapy·dG, though it also exhibited a similar context effect: 4-fold G→T transversions (24% versus 6%) occurred in the 5′-TGT sequence relative to 5′-TGA. To investigate a possible structural basis for the higher G→T mutations induced by both lesions when their 3′ neighbor was T, we carried out a molecular modeling investigation in the active site of DNA polymerase β, which is known to incorporate both dCTP (no mutation) and dATP (G→T substitution) opposite 8-oxo-G. In pol β, the syn-8-oxo-G:dATP pair showed greater stacking with the 3′-T:A base pair in the 5′-TGT sequence compared with the 3′-A:T in the 5′-TGA sequence, whereas stacking for the anti-8-oxo-G:dCTP pair was similar in both 5′-TGT and 5′-TGA sequences. Similarly, syn-Fapy·G:dATP pairing showed greater stacking in the 5′-TGT sequence compared with the 5′-TGA sequence, while stacking for anti-Fapy·G:dCTP pairs was similar in the two sequences. Thus, for both lesions less efficient base stacking between the lesion:dATP pair and the 3′-A:T base pair in the 5′-TGA sequence might cause lower G→T mutational frequencies in the 5′-TGA sequence compared to 5′-TGT. The corresponding lesions derived from 2′-deoxyadenosine, Fapy·dA and 8-oxo-dA, were not detectably mutagenic in the 5′-TAT sequence, and were only weakly mutagenic (<1%) in the 5′-TAA sequence context, where both lesions induced targeted A→C transversions. To our knowledge this is the first investigation using extrachromosomal probes containing a Fapy·dG or Fapy·dA site-specifically incorporated, which showed unequivocally that in simian kidney cells Fapy·G→T substitutions occur at a higher frequency than 8-oxo-G→T and that Fapy·dA is very weakly mutagenic, as is 8-oxo-dA.

Repair of Formamidopyrimidines in DNA Involves Different Glycosylases: ROLE OF THE OGG1, NTH1, AND NEIL1 ENZYMES

The oxidatively induced DNA lesions 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) and 4,6-diamino-5-formamidopyrimidine (FapyA) are formed abundantly in DNA of cultured cells or tissues exposed to ionizing radiation or to other free radical-generating systems. In vitro studies indicate that these lesions are miscoding, can block the progression of DNA polymerases, and are substrates for base excision repair. However, no study has yet addressed how these lesions are metabolized in cellular extracts. The synthesis of oligonucleotides containing FapyG and FapyA at defined positions was recently reported. These constructs allowed us to investigate the repair of Fapy lesions in nuclear and mitochondrial extracts from wild type and knock-out mice lacking the two major DNA glycosylases for repair of oxidative DNA damage, OGG1 and NTH1. The background level of FapyG/FapyA in DNA from these mice was also determined. Endogenous FapyG levels in liver DNA from wild type mice were significantly higher than 8-hydroxyguanine levels. FapyG and FapyA were efficiently repaired in nuclear and mitochondrial extracts from wild type animals but not in the glycosylase-deficient mice. Our results indicated that OGG1 and NTH1 are the major DNA glycosylases for the removal of FapyG and FapyA, respectively. Tissue-specific analysis suggested that other DNA glycosylases may contribute to FapyA repair when NTH1 is poorly expressed. We identified NEIL1 in liver mitochondria, which could account for the residual incision activity in the absence of OGG1 and NTH1. FapyG and FapyA levels were significantly elevated in DNA from the knock-out mice, underscoring the biological role of OGG1 and NTH1 in the repair of these lesions.

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.

Imidazole ring-opened DNA purines and their biological significance.

Fragmentation of purine imidazole ring and production of formamidopyrimidines in deoxynucleosides (Fapy lesions) occurs upon DNA oxidation as well as upon spontaneous or alkali-triggered rearrangement of certain alkylated bases. Many chemotherapeutic agents such as cyclophosphamide or thiotepa produce such lesions in DNA. Unsubstituted FapyA and FapyG, formed upon DNA oxidation cause moderate inhibition of DNA synthesis, which is DNA polymerase and sequence dependent. Fapy-7MeG, a methylated counterpart of FapyG-, a efficiently inhibits DNA replication in vitro and in E.coli, however its mutagenic potency is low. This is probably due to preferential incorporation of cytosine opposite Fapy-7MeG and preferential extension of Fapy-7MeG:C pair. In contrast, FapyA and Fapy-7MeA possess miscoding potential. Both lesions in SOS induced E.coli preferentially mispair with cytosine giving rise to A-->G transitions. Fapy lesions substituted with longer chain alkyl groups also show simult aneous lethal and mutagenic properties. Fapy lesions are actively eliminated from DNA by repair glycosylases specific for oxidized purines and pyrimidines both in bacteria and eukaryotic cells. Bacterial enzymes include E.coli formamidopyrimidine-DNA-glycosylase (Fpg protein), endonuclease III (Nth protein) and endonuclease VIII (Nei protein).

Synthesis of oligonucleotides and thermal stability of duplexes containing the beta-C-nucleoside analogue of Fapy*dG.

The formamidopyrimidine lesions (Fapy.dA, Fapy.dG) are formed in significant amounts when DNA is exposed to oxidative stress. These lesions are unusual in that they readily epimerize in solution. The distribution of configurational isomers in DNA is unknown. Nonepimerizable, nonhydrolyzable analogues are useful probes for investigating the configuration of Fapy lesions in DNA and as potential enzyme inhibitors. The beta-C-nucleoside of Fapy.dG has been prepared and introduced sight-specifically into oligonucleotides via its respective beta-cyanoethyl phosphoramidite. The phosphoramidite was prepared via a Wittig reaction between a protected form of deoxyribose and a suitably functionalized pyrimidine. The pyrimidine contained methyl and 2-propyl groups at the O4 and O2 positions, respectively, to differentiate between them following C-nucleoside formation. The formamide was derived from a nitro group at C5. The phosphoramidite coupled in 80% yield via a single 15-min coupling using tetrazole as activator. Oligonucleotides as long as 36 nucleotides were prepared and characterized by ESI-MS.

Antioxidant-induced changes in oxidized DNA.

N-acetylcysteine (NAC), a strong antioxidant, has antigenotoxic and anticarcinogenic properties currently being investigated in clinical trials. NAC detoxifies free radicals (e.g., the hydroxyl radical,.OH) that cause DNA changes implicated in disease (e.g., cancer). The.OH reacts with purines to form mutagenic 8-hydroxypurine (8-OH) and putatively nonmutagenic formamidopyrimidine (Fapy) lesions. Fapy lesions inhibit DNA synthesis likely modulating the mutagenic potential of the 8-OH lesions, which would suggest that the ratio of these oxidized bases is biologically important. However, little is known about how NAC modifies oxidized DNA structure or how such modifications may affect cellular processes, such as replication and transcription. By using gas chromatography-mass spectrometry and Fourier transform-infrared spectroscopy, we found that dietary NAC (5% in the diet for 14 days) affected.OH-induced structural changes in DNA of the hind leg of the BALB/c mouse. For example, mutagenic 8-hydroxyguanine (8-OH-Gua) was reduced approximately 50% (P = 0.02) in mice fed NAC compared with controls. NAC reduced the log(10) (8-OH-Gua/FapyGua) ratio from 0.58 +/- 0.15 to essentially zero, a virtually neutral redox status. DNA from control mice had a remarkably high variance compared with mice fed NAC. Moreover, the DNA from treated and control mice was distinct with respect to base structure and vertical base-stacking interactions. The findings showing that NAC lowered the concentration of 8-OH-Gua, the log ratio, and the variance (previously associated with neoplastic changes) suggest that NAC reduces the mutagenic potential of oxidized DNA. These benefits could be offset by the other structural changes found after NAC exposure, which may affect the fidelity of DNA synthesis.

Recognition of Formamidopyrimidine by Escherichia coli and Mammalian Thymine Glycol Glycosylases: DISTINCTIVE PAIRED BASE EFFECTS AND BIOLOGICAL AND MECHANISTIC IMPLICATIONS

The activity of prokaryotic and mammalian thymine glycol (Tg) glycosylases including Escherichia coli endonuclease III (Endo III) and endonuclease VIII (Endo VIII) and mouse Endo III homologue (mNth1) for formamidopyrimidine (Fapy) has been investigated using defined oligonucleotide substrates. 2, 6-Diamino-4-hydroxy-5-N-methylformamidopyrimidine, a methylated Fapy derived from guanine, was site specifically incorporated in the oligonucleotide. The substrates containing Fapy:N pairs (N = A, G, C, T) as well as a Tg:A pair, a physiological substrate of Endo III, Endo VIII, and mNth1, were treated by the enzymes and nicked products were quantified by gel electrophoresis. The activity of Endo III and Endo VIII for Fapy varied markedly depending on the paired base, being the highest with G (activity relative to Tg = 0. 55 (Endo III) and 0.41 (Endo VIII)) and the lowest with C (0.05 (Endo III) and 0.06 (Endo VIII)). In contrast, mNth1 recognized all Fapy pairs equally well and the activity was comparable to Tg. The results obtained in the nicking assay were further substantiated by the analysis of the Schiff base intermediate using NaBH(4) trapping assays. These results indicate that Escherichia coli and mammalian Tg glycosylases have a potential activity to recognize Fapy. However, as demonstrated for Fapy:C pairs, their distinctive activities implicate unequal participation in the repair of Fapy lesions in cells.

Reduction of the Toxicity and Mutagenicity of Aziridine in Mammalian Cells Harboring the Escherichia Coli fpg Gene

Aziridine (ethyleneimine) reacts with DNA in vitro, mainly at the N7 position of guanine and N3 of adenine, then imidazole ring opening of the modified guanine results in formation of formamidopyrimidine (FaPy) residues. The Escherichia coli fpg gene encodes a DNA glycosylase that removes FaPy residues from DNA. To determine whether aziridine produces FaPy lesions in mammalian cells we have expressed the E.coli fpg gene in CHO cells. The transfected cells, expressing high levels of the bacterial protein, are more resistant to the toxic and mutagenic effects of aziridine than the control population. Less DNA damage was measured by quantitative PCR analysis in transfected than in control cells treated with equimolar concentrations of aziridine. The results suggest that aziridine produces in vivo FaPy residues that could account for the deleterious effects of this compound.