Incorporation of Zebularine from its 2′-Deoxyribonucleoside Triphosphate Derivative and Activity as a Template-Coding Nucleobase

Zebularine (1-(β-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one) was studied as both a 2 ′-deoxyribosyl 5 ′-triphosphate derivative and as a template incorporated into an oligonucleotide. Using a novel pyrosequencing assay, zebularine acted as cytosine analog and was incorporated into DNA with a template pairing profile most similar to cytosine, pairing with greatest efficiency opposite guanine in the template strand. Guanine was incorporated with greater affinity than adenine opposite a zebularine in the template strand. Computer modeling of base-pairing structures supported a better fit of zebularine opposite guanine than adenine. Zebularine acts as a cytosine analog, which supports its activity as an inhibitor of cytosine methyltransferase.     

Efficient and erroneous incorporation of oxidized DNA precursors by human DNA polymerase eta

Altered oxidative metabolism is a property of many tumor cells. Oxidation of DNA precursors, i.e., dNTP pool, as well as DNA is a major source of mutagenesis and carcinogenesis. Here, we report the remarkable nature of human DNA polymerase eta that incorporates oxidized dNTPs into a nascent DNA strand in an efficient and erroneous manner. The polymerase almost exclusively incorporated 8-hydroxy-dGTP (8-OH-dGTP) opposite template adenine (A) at 60% efficiency of normal dTTP incorporation, and incorporated 2-hydroxy-dATP (2-OH-dATP) opposite template thymine (T), guanine (G), or cytosine (C) at substantial rates. The synthetic primers having 8-hydroxy-G paired with template A or 2-hydroxy-A paired with template T, G, or C at the termini were efficiently extended. In contrast, human DNA polymerase iota incorporated 8-OH-dGTP opposite template A with much lower efficiency and did not incorporate 2-OH-dATP opposite any of the template bases. It did not extend the primers having the oxidized bases at the termini either. We propose that human DNA polymerase eta may participate in oxidative mutagenesis through the efficient and erroneous incorporation of oxidized dNTPs during DNA synthesis.     

Mechanistic Insights on the Inhibition of C5 DNA Methyltransferases by Zebularine

In mammals DNA methylation occurs at position 5 of cytosine in a CpG context and regulates gene expression. It plays an important role in diseases and inhibitors of DNA methyltransferases (DNMTs)—the enzymes responsible for DNA methylation—are used in clinics for cancer therapy. The most potent inhibitors are 5-azacytidine and 5-azadeoxycytidine. Zebularine (1-(β-D-ribofuranosyl)-2(1H)- pyrimidinone) is another cytidine analog described as a potent inhibitor that acts by forming a covalent complex with DNMT when incorporated into DNA. Here we bring additional experiments to explain its mechanism of action. First, we observe an increase in the DNA binding when zebularine is incorporated into the DNA, compared to deoxycytidine and 5-fluorodeoxycytidine, together with a strong decrease in the dissociation rate. Second, we show by denaturing gel analysis that the intermediate covalent complex between the enzyme and the DNA is reversible, differing thus from 5-fluorodeoxycytidine. Third, no methylation reaction occurs when zebularine is present in the DNA. We confirm that zebularine exerts its demethylation activity by stabilizing the binding of DNMTs to DNA, hindering the methylation and decreasing the dissociation, thereby trapping the enzyme and preventing turnover even at other sites.     

Pyrosequencing for the quantitative assessment of 8-oxodG bypass DNA synthesis

Translesion synthesis (TLS) with specialized DNA polymerases allows dealing with a base lesion on the template strand during DNA replication; a base is inserted opposite the lesion, correctly or incorrectly, depending on the lesion, the involved DNA polymerase(s) and the sequence context. The major oxidized DNA base 8-oxo-7, 8-dihydro-2′-deoxyguanosine (8-oxodG) is highly mutagenic due to its ability to pair with either cytosine or adenine during DNA synthesis, depending on its conformation and involved DNA polymerases. To measure the correct or mutagenic outcome of lesion bypass, an original quantitative pyrosequencing method was developed and analytically validated. The method was applied to the study of DNA synthesis fidelity through an 8-oxodG or an undamaged guanine. After an in vitro primer-extension through 8-oxodG in the presence of the four deoxynucleotides triphosphates and a total nuclear protein extract, obtained from normal human intestinal epithelial cells (FHs 74 Int cell line), the reaction products were amplified by polymerase chain reaction and analyzed by pyrosequencing to measure nucleotides inserted opposite the lesion. The 8-oxodG bypass fidelity of FHs 74 Int cells nuclear extract is about 85.3%. We calculated within-day and total precisions for both 8-oxodG (2.8% and 2.8%, respectively) and undamaged templates (1.0% and 1.1%, respectively). We also demonstrated that only cytosine is incorporated opposite a normal guanine and that both cytosine and adenine can be incorporated opposite an 8-oxodG lesion. The proposed method is straightforward, fast, reproducible and easily adaptable to other sequences and lesions. It thus has a wide range of applications in the biological field, notably to elucidate TLS mechanisms and modulators.     

Oxanine DNA glycosylase activity from Mammalian alkyladenine glycosylase

Oxanine (Oxa) is a deaminated base lesion derived from guanine in which the N(1)-nitrogen is substituted by oxygen. This work reports the mutagenicity of oxanine as well as oxanine DNA glycosylase (ODG) activities in mammalian systems. Using human DNA polymerase beta, deoxyoxanosine triphosphate is only incorporated opposite cytosine (Cyt). When an oxanine base is in a DNA template, Cyt is efficiently incorporated opposite the template oxanine; however, adenine and thymine are also incorporated opposite Oxa with an efficiency approximately 80% of a Cyt/Oxa (C/O) base pair. Guanine is incorporated opposite Oxa with the least efficiency, 16% compared with cytosine. ODG activity was detected in several mammalian cell extracts. Among the known human DNA glycosylases tested, human alkyladenine glycosylase (AAG) shows ODG activity, whereas hOGG1, hNEIL1, or hNEIL2 did not. ODG activity was detected in spleen cell extracts of wild type age-matched mice, but little activity was observed in that of Aag knock-out mice, confirming that the ODG activity is intrinsic to AAG. Human AAG can excise Oxa from all four Oxa-containing double-stranded base pairs, Cyt/Oxa, Thy/Oxa, Ade/Oxa, and Gua/Oxa, with no preference to base pairing. Surprisingly, AAG can remove Oxa from single-stranded Oxa-containing DNA as well. Indeed, AAG can also remove 1,N(6)-ethenoadenine from single-stranded DNA. This study extends the deaminated base glycosylase activities of AAG to oxanine; thus, AAG is a mammalian enzyme that can act on all three purine deamination bases, hypoxanthine, xanthine, and oxanine.     

Abasic sites from cytosine as termination signals for DNA synthesis.

DNA with abasic sites has been prepared by deamination of cytosine followed by treatment of the product with uracil N-glycosylase. Termination in vitro on such templates does not occur until treatment with uracil N-glycosylase. DNA terminated one base before abasic sites created from C's has been used as a template in "second stage" reactions. With enzymes devoid or deficient in 3' greater than 5' exonuclease activity purines, particularly adenine, are preferentially added opposite the putative abasic site. 2-Aminopurine behaves more like adenine than like guanine in these experiments. Polymerase beta preferentially incorporates A opposite abasic sites produced from T, and G opposite abasic sites produced from C. We have eliminated an obvious artefact (e.g. strand switching) which might account for this observation.     

ZEBULARINE: A UNIQUE MOLECULE FOR AN EPIGENETICALLY BASED STRATEGY IN CANCER CHEMOTHERAPY. THE MAGIC OF ITS CHEMISTRY AND BIOLOGY

1-(β-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one (zebularine) is structurally 4-deamino cytidine. The increased electrophilic character of this simple aglycon endows the molecule with unique chemical and biological properties, making zebularine a versatile starting material for the synthesis of complex nucleosides and an effective inhibitor of cytidine deaminase and DNA cytosine methyltransferase. Zebularine is a stable, antitumor agent that preferentially targets cancer cells and shows activity both in vitro and in experimental animals, even after oral administration.     

Zebularine: a unique molecule for an epigenetically based strategy in cancer chemotherapy. The magic of its chemistry and biology

1-(beta-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one (zebularine) is structurally 4-deamino cytidine. The increased electrophilic character of this simple aglycon endows the molecule with unique chemical and biological properties, making zebularine a versatile starting material for the synthesis of complex nucleosides and an effective inhibitor of cytidine deaminase and DNA cytosine methyltransferase. Zebularine is a stable, antitumor agent that preferentially targets cancer cells and shows activity both in vitro and in experimental animals, even after oral administration.     

Mutagenicity of the cytidine analog zebularine in Escherichia coli

We have examined the mutagenic properties of zebularine, a cytidine analog lacking the amino group at C-4 that has potential use in chemotherapy. Because the hydrate is a strong inhibitor of cytidine deaminase, its use can enhance the potency of other cytosine based compounds such as 5-azacytidine (5AzaC) and cytosine arabinoside (ara-C) that are inactivated by cytidine deaminase. Using the newly developed rpoB/Rifr system in Escherichia coli, we examined base substitution mutations caused by zebularine in the chromosomal rpoB gene. Zebularine is a potent mutagen that causes mainly G : C --> A : T transitions and favors certain hotspots. Mutations are not specific to the rpoB gene, since there is also a strong induction of mutations in the thyA gene. In the absence of mismatch repair, zebularine induces both base substitutions and frame shifts at rates well above those seen in wild-type strains treated with zebularine or in mismatch repair deficient strains without treatment. The nature of these induced mutations indicates that zebularine is stimulating the induction of increased replication errors, in addition to the targeted G : C --> A : T mutations, and that these errors are normally repaired by the mismatch repair system.     

Molecular-Mechanical studies of the mismatched base analogs of d(CGCGAATTCGCG)2:d(CGTGAATTCGCG)2, d(CGAGAATTCGCG)2, d(CGCGAATTCACG)2, d(CGCGAATTCTCG)2, and d(CGCAGAATTCGCG)·d(CGCGAATTCGCG)

Molecular-mechanical simulations have been carried out on “mismatched base” analogs of the DNA double-helical structure d(CGCGAATTCGCG)₂, in which the base pairs CG at the 3 and 10 positions have been replaced by CA, AG, TC, and TG base pairs, as well as an insertion analog in which an extra adenine has been incorporated into one strand of the above structure between bases 3 and 4. The results of these simulations (calculated relative stabilities, structures, and nmr ring-current shifts) have been compared with calorimetric and nmr data. The calculated relative stabilities of the double-helical parent dodecamer and the various “wobble” base pairs qualitatively correlate with the experimental melting temperatures. The base-pairing structure for the GT wobble pair is in agreement with that previously determined from nmr experiments. For the GA base pair, the structure with both bases anti has a slightly more favorable energy from base pairing and stacking than a structure with non-Watson-Crick H-bonding with adenine syn, in agreement with nmr experiments. The CA wobble base is calculated to favor an adenine 6NH₂ …? cytosine N3 H-bond over cytosine 4NH₂ …? adenine N1, again, in agreement with nmr experiments. There is no definitive experimental data on the TC base pair, but the existence of (somewhat long and weak) H-bonds involving cytosine 4NH₂ …? thymine 4CO and cytosine N3 …? thymine HN3 seems reasonable. We find a structure in which the extra adenine base of the insertion analogs sits “inside” the double helix.     

Mechanisms of dCMP transferase reactions catalyzed by mouse Rev1 protein.

The Rev1 protein, a member of a large family of translesion DNA polymerases, catalyzes a dCMP transfer reaction. Recombinant mouse Rev1 protein was found to insert a dCMP residue opposite guanine, adenine, thymine, cytosine, uracil, and an apurinic/apyrimidinic site and to have weak ability for transfer to a mismatched terminus. The mismatch-extension ability was strongly enhanced by a guanine residue on the template near the mismatched terminus; this was not the case with an apurinic/apyrimidinic site and the other template nucleotides. Kinetic analysis of the dCMP transferase reaction provided evidence for high affinity for dCTP with template G but not the other templates, whereas the template nucleotide did not much affect the V(max) value. Furthermore, it could be established that the mouse Rev1 protein inserts dGMP and dTMP residues opposite template guanine at a V(max) similar to that for dCMP.     

A unique unnatural base pair between a C analogue, pseudoisocytosine, and an A analogue, 6-methoxypurine, in replication

Pseudoisocytidine, a C-nucleoside analogue of cytosine, has two possible isomers of the H1- and H3-forms. Enzymatic incorporation experiments confirmed the existence of the two isomers in solution, and the 2'-deoxyribonucleoside triphosphate of pseudoisocytosine (PIC) was incorporated into DNA opposite both guanine and 6-methoxypurine (M) by the Klenow fragment of Escherichia coli DNA polymerase I. In addition to the PIC*M pairing in replication, M also functioned as an A analogue and T was efficiently incorporated opposite M. Thus, the PIC*M pair is regarded as a base pair between a C analogue and an A analogue, and can mediate the interconversion between the G*C and A*T base pairs. The combination of PIC and M could be used as a G*C<-->A*T transition mutagen.     

Strand-specific processing of 8-oxoguanine by the human mismatch repair pathway: inefficient removal of 8-oxoguanine paired with adenine or cytosine

Genomic DNA and its precursors are susceptible to oxidation during aerobic cellular metabolism, and at least five distinct repair activities target a single common lesion, 7,8-dihydro-8-oxoguanine (8-oxoG). The human mismatch repair (MMR) pathway, which has been implicated in an apoptotic response to covalent DNA damage, is likely to encounter 8-oxoG in both the parental and daughter strand during replication. Here, we show that lesions containing 8-oxoG paired with adenine or cytosine, which are most likely to arise during replication, are not efficiently processed by the mismatch repair system. Lesions containing 8-oxoG paired with thymine or guanine, which are unlikely to arise, are excised in an MSH2/MSH6-dependent manner as effectively as the corresponding mismatches when placed in a context that reflects the daughter strand during replication. Using a newly developed assay based on methylation sensitivity, we characterized strand-excision events opposite 8-oxoG situated to reflect placement in the parental strand. Lesions that efficiently trigger strand excision and resynthesis (8-oxoG paired with thymine or guanine) result in adenine or cytosine insertion opposite 8-oxoG. These latter pairings are poor substrates for further action by mismatch repair, but precursors for alternative pathways with non-mutagenic outcomes. We suggest that the lesions most likely to be encountered by the human mismatch repair pathway during replication, 8-oxoG.A or 8-oxoG.C, are likely to escape processing in either strand by this system. Taken together, these data suggest that the human mismatch repair pathway is not a major contributor to removal of misincorporated 8-oxoG, nor is it likely to trigger repeated attempts at lesion processing.     

Human mitochondrial DNA polymerase γ exhibits potential for bypass and mutagenesis at UV-induced cyclobutane thymine dimers

Cyclobutane thymine dimers (T-T) comprise the majority of DNA damage caused by short wavelength ultraviolet radiation. These lesions generally block replicative DNA polymerases and are repaired by nucleotide excision repair or bypassed by translesion polymerases in the nucleus. Mitochondria lack nucleotide excision repair, and therefore, it is important to understand how the sole mitochondrial DNA polymerase, pol γ, interacts with irreparable lesions such as T-T. We performed in vitro DNA polymerization assays to measure the kinetics of incorporation opposite the lesion and bypass of the lesion by pol γ with a dimer-containing template. Exonuclease-deficient pol γ bypassed thymine dimers with low relative efficiency; bypass was attenuated but still detectable when using exonuclease-proficient pol γ. When bypass did occur, pol γ misincorporated a guanine residue opposite the 3'-thymine of the dimer only 4-fold less efficiently than it incorporated an adenine. Surprisingly, the pol γ exonuclease-proficient enzyme excised the incorrectly incorporated guanine at similar rates irrespective of the nature of the thymines in the template. In the presence of all four dNTPs, pol γ extended the primer after incorporation of two adenines opposite the lesion with relatively higher efficiency compared with extension past either an adenine or a guanine incorporated opposite the 3'-thymine of the T-T. Our results suggest that T-T usually stalls mitochondrial DNA replication but also suggest a mechanism for the introduction of point mutations and deletions in the mitochondrial genomes of chronically UV-exposed cells.     

Proofreading of a mutagenic nucleotide, N4-aminodeoxycytidylic acid, by Escherichia coli DNA polymerase I

N4-Aminodeoxycytidine triphosphate, a putative metabolite of N4-aminocytidine which is a potent mutagen, is incorporated, in vitro, into polynucleotides in place of dCTP and at a much lesser extent, but significantly, in place of dTTP by E. coli DNA polymerase I large fragment. The activity of the polymerase to proofread this unnatural nucleotide has now been investigated. The results indicate that the 3'-5' exonuclease in the polymerase recognizes N4-aminocytosine as an incorrect base when N4-aminocytosine is incorporated opposite adenine but the enzyme cannot distinguish N4-aminocytosine from cytosine when it is incorporated opposite guanine.     

Zebularine exerts its antiproliferative activity through S phase delay and cell death in human malignant mesothelioma cells

Malignant mesothelioma is an asbestos-related aggressive tumor and current therapy remains ineffective. Zebularine as a DNA methyltransferase (DNMT) inhibitor has an anti-tumor effect in several human cancer cells. The aim of the present study was to investigate whether zebularine could induce antiproliferative effect in human malignant mesothelioma cells. Zebularine induced cell growth inhibition in a dose-dependent manner. In addition, zebularine dose-dependently decreased expression of DNMT1 in all malignant mesothelioma cells tested. Cell cycle analysis indicated that zebularine induced S phase delay. Zebularine also induced cell death in malignant mesothelioma cells. In contrast, zebularine did not induce cell growth inhibition and cell death in human normal fibroblast cells. These results suggest that zebularine has a potential for the treatment of malignant mesothelioma by inhibiting cell growth and inducing cell death.     

Ambiguous incorporation of N4-aminodeoxycytidine 5'-triphosphate to DNA synthesized in vitro

Molecular mechanism of the mutation induced by N4-aminocytidine was studied. The specificity of in vitro incorporation of N4-aminodeoxycytidine 5'-triphophate catalyzed by E. coli DNA polymerase large fragment was analyzed. The results have shown that this cytosine analog can be efficiently incorporated as a substitute of cytosine, and that it can also be incorporated with a low efficiency as a substitute of thymine. We have also shown that the N4-aminocytosine incorporated opposite adenine can be excised as its monophosphate at a high frequency. The N4-aminocytosine residues in the polynucleotide templates can be read by the enzyme as efficiently as cytosines, and guanines were incorporated opposite them.     

The accessibility of DNA to dimethylsulfate in complexes with recA protein.

recA protein coats DNA co-operatively to form filaments approximately 100 A thick, which in the presence of ATP, and more stably so in the presence of the non-hydrolyzable analog ATP gamma S, have a helical appearance with a deep cleft in the protein coat. This protein helix follows the DNA helix, to which it imparts a new helicity of 18.5 bp per turn of 97 A pitch. Here we test the accessibility of the DNA in the complex to modification by dimethylsulfate, and find that the complexed DNA is approximately 2-fold more reactive on the major groove side than it was in B-DNA (methylation of guanine N7), while it is protected approximately 2-fold on the minor groove side (methylation of adenine N3), suggesting that the protein coats the DNA along the minor groove. Furthermore, N3 of cytosine, a residue involved in base pairing, is found exposed in complexes with single strands as it is in naked single-stranded DNA, while it remains inaccessible in complexes with double strands, suggesting that the latter is not melted at this stage of the strand exchange reaction.     

Minor-groove binding models for acetylaminofluorene modified DNA

Minimized potential energy calculations have been employed to locate and evaluate energetically a number of different models for DNA modified at carbon-8 of guanine by acetylaminofluorene (AAF). Three different duplex nonamer sequences were investigated. In addition to syn guanine models which have some denaturation and a Z-DNA model, we have found two new types of structures in which guanine remains syn and the AAF is placed in the minor groove of a B-DNA helix. One type features Hoogsteen base pairing between the modified guanine and protonated cytosine, with a sharply bent helix. The other (here termed the "wedge" model because the aromatic amine is wedged into the minor groove) maintains a single hydrogen bond between O6 of the modified guanine and N3 of protonated cytosine, with much less deformation of the helix, and close Van der Waals contacts between the AAF and the walls of the minor groove. Both types of structures (as well as the related forms produced by deprotonation of cytosine) are energetically important in all three sequences examined. The wedge-type model, which is most favored except in alternating G-C sequences, has been previously observed in a combined NMR and computational characterization of an aminofluorene (AF) modified guanine opposite adenine in a DNA duplex undecamer (D. Norman, P. Abuaf, B.E. Hingerty, D. Live, D. Grunberger, S. Broyde and D.J. Patel, Biochemistry 28, 7462 (1989)).