Mutagenesis by N4-aminocytidine: induction of AT to GC transition and its molecular mechanism

N4-Aminocytidine is a potent mutagen toward Escherichia coli and Salmonella typhimurium. It induced reversion of an amber mutant of phi X174 phage (am3) to the wild type. This reversion was shown to be exclusively due to the AT to GC transition. It is likely that N4-aminocytidine is metabolized within the bacterial cells into N4-aminodeoxycytidine 5'-triphosphate and this nucleotide is incorporated into DNA during the multiplication of the cells and the phages, thereby causing base-pair transitions. The molecular basis for this erroneous replication was obtained in studies of in vitro incorporation of N4-aminodeoxycytidine 5'-triphosphate into polynucleotides catalyzed by the E. coli DNA polymerase I large fragment. The results have shown that this cytosine analogue can be efficiently incorporated as a substitute of cytosine and that it can also be incorporated as a substitute of thymine. The ratio in the rate of the N4-aminocytosine nucleotide incorporation to that of natural nucleotide incorporation was 1/2 to cytosine and 1/30 to thymine. Furthermore, the N4-aminocytosine residues in the polynucleotide templates can be read by the enzyme as efficiently as cytosines, and guanines were incorporated opposite to them.     

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.     

Induction of mutation in mouse FM3A cells by N4-aminocytidine-mediated replicational errors.

To explore the potential use of a nucleoside analog, N4-aminocytidine, in studies of cellular biology, the mechanism of mutation induced by this compound in mouse FM3A cells in culture was studied. On treatment of cells in suspension with N4-aminocytidine, the mutation to ouabain resistance was induced. The major DNA-replicating enzyme in mammalian cells, DNA polymerase alpha, was used to investigate whether the possible cellular metabolite of N4-aminocytidine, N4-aminodeoxycytidine 5'-triphosphate (dCamTP), can be incorporated into the DNA during replication. Using [3H]dCamTP in an in vitro DNA-synthesizing system, we were able to show that this nucleotide analog can be incorporated into newly formed DNA and that it can serve as a substitute for either dCTP or dTTP. dCamTP in the absence of dCTP maintained the activated calf thymus DNA-directed polymerization of deoxynucleoside triphosphates as efficiently as in its presence. Even in the presence of dCTP, dCamTP was incorporated into the polynucleotide. When dCamTP was used as a single substrate in the poly(dA)-oligo(dT)-directed polymerase reaction, it was incorporated into the polynucleotide fraction. The extent of incorporation was 4% of that of dTTP incorporation when dTTP was used as a single substrate. Even in the presence of dTTP, dCamTP incorporation was observed. A copolymer containing N4-aminocytosine residues was shown to incorporate guanine residues opposite the N4-aminocytosines. However, we were unable to observe adenine incorporation opposite N4-aminocytosine in templates. These cell-free experiments show that an AT-to-GC transition can take place in the presence of dCamTP during DNA synthesis, strongly suggesting that the mutation induced in the FM3A cells by N4-aminocytidine is due to replicational errors.     

Ab initio molecular orbital study of the mispairing ability of a nucleotide base analogue, N4-aminocytosine

The intrinsic properties of N4-aminocytosine, a base analogue of cytosine, are analyzed by an ab initio molecular orbital method. Relative stabilities of four possible isomeric structures of N4-aminocytosine are shown. The more stable isomer has the smaller dipole moment, so the relative stabilities of the isomers in solutions are subject to solvent polarity. The mutagenicity of this base analogue must arise because it can behave like either cytosine or thymine. It can form a guanine-cytosine-like base pair more easily than cytosine, and an adenine-thymine-like base pair less easily than thymine.     

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.     

Cross-linking between 16S ribosomal RNA and protein S4 in Escherichia coli ribosomal 30S subunits effected by treatment with bisulfite/hydrazine and bromopyruvate

Cytosine in nucleic acids can be modified by treatment with a mixture of bisulfite and hydrazine. The reaction is specific for single-stranded regions of nucleic acids and the product is N4-aminocytosine. Bromopyruvate has been used for alkylation of protein SH groups and through its 2-oxo group it can form a hydrazone with N4-aminocytosine. Escherichia coli ribosomal 30S subunits were treated with 1 M sodium bisulfite + 2 M hydrazine in the presence of 10 mM MgCl2 at pH 7.0 and 37 degrees C for 30 min. By this treatment, 2.4 cytosine residues/molecule 16S rRNA were derivatized into N4-aminocytosines. 35S-labeled 30S subunits were modified in this way and then treated with 10 mM bromopyruvate at pH 8.0 and 37 degrees C for 5 min. Analysis in sodium dodecyl sulfate/sucrose density gradient centrifugation showed co-sedimentation of a part of the 35S radioactivity with the RNA. The co-sedimentation was dependent on both the bisulfite/hydrazine and the bromopyruvate treatments. The RNA-protein complex was prepared from unlabeled 30S subunits. The protein portion was labeled with 125I, the RNA portion was digested with nucleases, and then the hydrazone linkage between the protein and oligonucleotides was cleaved by treatment with 0.2 M HCl. The oligonucleotides formed were removed by dialysis and the protein was identified as S4 by two-dimensional electrophoresis and by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The results indicate that the cysteinyl residue of protein S4 at position 31 from the N-terminus is located close to a cytosine residue which is non-base-paired and easily accessible by the externally present bisulfite/hydrazine reagent.     

Synthesis and Stability of Oligonucleotide Duplexes Containing N4 Aralkyl-Substituted Cytosine Bases

Abstract

N4 aralkyl-substituted cytosine nucleosides, available dxectly by displacement of the PfpO group at C4 of 5′-O-DMT-protected nucleoside 4, were efficiently incorporated into short oligonucleotides. Aralkyl substitution at the N4 of cytosine was entropically stabilising but offset by loss in enthalpy resulting overall in duplex destabilisation.     

Mutagenic bypass of the butadiene-derived 2'-deoxyuridine adducts by polymerases eta and zeta

Butadiene is a ubiquitous environmental chemical carcinogen that when activated to its monoepoxide intermediate can react with the N3 position of cytosine, resulting in two stereoisomeric adducted bases that rapidly deaminate to N3 2′-deoxyuridine lesions. We have previously shown that replication of DNAs containing these adducts through mammalian cells resulted in 97% mutagenicity, predominantly C to T transitions. Since replicative DNA polymerases were blocked by these lesions in vitro, translesional polymerases were assessed for their ability to bypass these adducts. While polymerases ι, κ and ζ were significantly blocked one nucleotide prior to the lesion, pol η incorporated nucleotides opposite the adducts with a preference for insertion of a G or A. Following polymerase dissociation and reassociation, pol η was also able to extend primers with mispaired termini opposite the lesions, with extensions from the A and T mismatched primer termini being the most efficient. Pol ζ was also able to extend primers containing all mismatched nucleotides opposite the lesions, with the most efficient extension occurring off of the A mismatched primer.     

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.     

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.     

Incorporation of zebularine from its 2'-deoxyribonucleoside triphosphate derivative and activity as a template-coding nucleobase

Zebularine (1-(beta-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.     

An allylic/acyclic adenosine nucleoside triphosphate for termination of DNA synthesis by DNA template-dependent polymerases.

An allylic adenosine triphosphate analog (AATP) was tested as a substrate for commercially available DNA polymerases. All but one of the enzymes assayed incorporated AATP opposite thymidine (T) with concomitant termination of the elongation reaction. A concentration of only 1 microM was sufficient for complete termination of the polymerization reaction for a short template mediated by Ampli Taq DNA polymerase FS (Taq FS). This result suggests that AATP could be used as a 2',3'-dideoxyadenosine-5'-triphosphate (ddA) surrogate. Kinetics of incorporation revealed that AATP was 48 times less efficiently incorporated than ddA. Furthermore, AATP was used in dye-primer sequencing as a substitute for ddA.     

A new reaction useful for chemical cross-linking between nucleic acids and proteins

Cytosine in nucleic acids can be converted into N4-aminocytosine by treatment with a mixture of hydrazine and bisulfite. The hydrazino group thus formed at position 4 of the pyrimidine ring can be linked to a sulhydryl group in proteins by the use of bromopyruvate as a linker. Successful use of this scheme of chemical cross-linking between nucleic acid and protein was demonstrated in the linking of poly(C) with glutathione, and of RNA with protein in the E. coli 30 S ribosomal subunit.     

Difference between deoxyribose- and tetrahydrofuran-type abasic sites in the in vivo mutagenic responses in yeast

We have analyzed the mutagenic specificity of an abasic site in DNA using the yeast oligonucleotide transformation assay. Oligonucleotides containing an abasic site or its analog were introduced into B7528 or its derivatives, and nucleotide incorporation opposite abasic sites was analyzed. Cytosine was most frequently incorporated opposite a natural abasic site (O) (‘C-rule’), followed by thymine. Deletion of REV1 decreased the transformation efficiency and the incorporation of cytosine nearly to a background level. In contrast, deletion of RAD30 did not affect them. We compared the mutagenic specificity with that of a tetrahydrofuran abasic site (F), an abasic analog used widely. Its mutation spectrum was clearly different from that of O. Adenine, not cytosine, was most favorably incorporated. However, deletion of REV1 decreased the transformation efficiency with F-containing oligonucleotide as in the case of O. These results suggest that the bypass mechanism of F is different from that of O, although the bypasses in both cases are dependent on REV1. We also found that the mutagenic specificity of F can be affected by not only the adjacent bases, but also a base located two positions away from F.     

A Mutant of Uracil DNA Glycosylase That Distinguishes between Cytosine and 5-Methylcytosine

We demonstrate that a mutant of uracil DNA glycosylase (N123D:L191A) distinguishes between cytosine and methylcytosine. Uracil DNA glycosylase (UDG) efficiently removes uracil from DNA in a reaction in which the base is flipped into the enzyme’s active site. Uracil is selected over cytosine by a pattern of specific hydrogen bonds, and thymine is excluded by steric clash of its 5-methyl group with Y66. The N123D mutation generates an enzyme that excises cytosine. This N123D:L191A mutant excises C when it is mispaired with A or opposite an abasic site, but not when it is paired with G. In contrast no cleavage is observed with any substrates that contain 5-methylcytosine. This enzyme may offer a new approach for discriminating between cytosine and 5-methylcytosine.     

Linking of N4-aminocytosine to glutathione by use of bromopyruvate

N4-Aminocytosine reacted with acetone and acetaldehyde to form hydrazones that were readily revertible to the parent compound. With pyruvate, in contrast, it formed a stable hydrazone. By use of bromopyruvate, N4-aminocytosine was linked to glutathione.     

Synthesis and properties of oligodeoxyribonucleotides containing a mutagenic base, N4-aminocytosine.

Oligodeoxyribonucleotides containing a mutagenic base analog, N4-aminocytosine, 5'-AATTGC(am)AATT-3' and 5'-AATTAC(am)AATT-3' (C(am); N4-aminocytosine) were prepared by chemical modification of 5'-AATTGCAATT-3' and 5'-AATTACAATT-3', respectively. The values of Tm were 29 degrees C for 5'-AATTGC(am)AATT-3' and 32 degrees C for 5'-AATTGCAATT-3'. In contrast, no melting was observed for 5'-AATTAC(am)AATT-3' and 5'-AATTACAATT-3'. These data show that the stability of C(am)-purine paris is C(am)-G > C(am)-A and that C(am)-G is less stable than C-G. This property is consistent with the incorporation specificity of N4-amino-dCTP during DNA synthesis in vitro.     

Structure of the hydrogen bonding complex of O6-methylguanine with cytosine and thymine during DNA replication.

During DNA replication, mutations occur when an incorrect dNTP is incorporated opposite a carcinogen-modified nucleotide. We have probed the structures of the interaction between O 6-methylguanine ( O 6mG) and cytosine and thymine during replication by kinetic means in order to examine the structure during the rate determining step. The kinetics of incorporation of dCTP and dTTP opposite O 6mG and three analogs, S 6-methyl-6-thioguanine, O 6-methyl-1-deazaguanine and O 6-methylhypoxanthine, have been measured with four polymerases, the Klenow fragment of DNA polymerase I, the Klenow fragment with the proof-reading exonuclease inactivated, Taq and Tth polymerases. In the insertion of dTTP opposite O 6mG, a large decrease in V max/ K m was observed only upon modification of the N1 position. This result is consistent with a Watson-Crick type configuration. For the incorporation of dCTP, the V max/ K m was significantly decreased only with removal of the exocyclic amino group at the 2 position. The pH dependence of the ratio of incorporation of dCTP and dTTP was independent of pH at physiological pH. This result suggests that dCTP is incorporated via an uncharged complex such as the wobble configuration.     

Functional interactions of the genomes of Shigella sonnei phages and Escherichia coli phage T4 in mixed infection]

A comparative study of Shigella sonnei phages U and G and Escherichia coli phage T4 has shown that enzymes coded for by the Sh. sonnei phages can functionally substitute for some T4-coded products. This finding in indicative of an evolutionary relationship between T-even phages and disenteric phages U and G. The U phage is uncapable to compensate amber mutants for the genes that control the conversion of cytosine into 5-hydroxymethyl cytosine (5-HMC) and the glucosylation of the latter, which agrees with our earlier finding that the U phage DNA contains no 5-HMC. U and G phages are also found to exclude the T4 phage in the course of mixed infection.