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.     

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analogs of the DNA double-helical structure d(CGCGAATTCGCG)₂, in which the base pairs CG at the 3 and 10 positions have been replaced by CA, AG, TC, and TG base pairs, as well as an insertion analog in which an extra adenine has been incorporated into one strand of the above structure between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.     

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.     

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.     

Nuclear magnetic resonance investigation of primer--template models: formation of a pyrimidine bulge upon misincorporation

Our previous studies have shown that misaligned structures can occur upon misincorporation of a dNTP opposite thymine templates. The formation of misaligned structures during DNA replication, if not repaired properly, can be bypassed and extended by low-fidelity polymerases and ultimately lead to mutations. In this study, the base pair structures at the replicating sites of a set of primer-template models which mimic the situation upon misincorporation of a dNTP opposite cytosine templates have been determined. High-resolution NMR structural results show that misaligned structures with a C-bulge can be formed upon incorporation of dCTP, dTTP, and dATP opposite 5'-GC, 5'-AC, and 5'-TC templates, respectively. The stabilities of misaligned structures depend on the types of terminal base pairs at the replicating sites. Together with the structural findings in thymine templates, we conclude that terminal G.C and C.G base pairs always contribute a larger stabilizing effect to the misaligned structures containing a pyrimidine bulge than terminal A.T and T.A base pairs. Misalignment and thus deletion mutation are more likely to occur if misincorporation of a nucleotide opposite a pyrimidine template can cause template slippage to form a terminal G.C or C.G base pair. Although misalignment also occurs when the newly formed terminal base pair is an A.T base pair or a T.A base pair, both misaligned and mismatched conformers coexist, which can lead to deletion and substitution mutations, respectively.     

Structure of the 2-aminopurine-cytosine base pair formed in the polymerase active site of the RB69 Y567A-DNA polymerase

The adenine base analogue 2-aminopurine (2AP) is a potent base substitution mutagen in prokaryotes because of its enhanceed ability to form a mutagenic base pair with an incoming dCTP. Despite more than 50 years of research, the structure of the 2AP-C base pair remains unclear. We report the structure of the 2AP-dCTP base pair formed within the polymerase active site of the RB69 Y567A-DNA polymerase. A modified wobble 2AP-C base pair was detected with one H-bond between N1 of 2AP and a proton from the C4 amino group of cytosine and an apparent bifurcated H-bond between a proton on the 2-amino group of 2-aminopurine and the ring N3 and O2 atoms of cytosine. Interestingly, a primer-terminal region rich in AT base pairs, compared to GC base pairs, facilitated dCTP binding opposite template 2AP. We propose that the increased flexibility of the nucleotide binding pocket formed in the Y567A-DNA polymerase and increased "breathing" at the primer-terminal junction of A+T-rich DNA facilitate dCTP binding opposite template 2AP. Thus, interactions between DNA polymerase residues with a dynamic primer-terminal junction play a role in determining base selectivity within the polymerase active site of RB69 DNA polymerase.     

Minor groove functional groups are critical for the B-form conformation of duplex DNA

Two analogue bases are described: 3-deazaadenine is a derivative of adenine from which N3 has been deleted and 3-methyl-2-pyridone is a C-nucleoside that mimics thymine but lacks the O2 carbonyl. The dc(3)A-dm(3)2P base pair is similar to dA-dT but eliminates the polar functional groups in the minor groove. The presence of this base pair in dA-dT rich sequences results in destabilized duplexes or conformational preferences for monomolecular hairpins rather than bimolecular duplexes. When present in dG-dC rich sequences, no significant differences in helix stability are observed. These differences are explained on the basis of hydration effects, most notably, the elimination of the minor groove spine of hydration normally present in dA-dT rich sequences. CD spectra suggest that sequences with a fully modified core (four analogue base pairs) are more A-like helices than B-like helices. Sequences containing two analogue base pairs can be transformed to A-like helices under conditions of high salt, or 65% trifluoroethanol. These conformational changes are also explained in terms of a loss of hydration in the minor groove that normally stabilizes the B-form conformation. In the absence of such hydration, the helices are conformationally mobile and adopt a more A-like helix form.     

Studies on gene control regions XII. The functional significance of a lac operator constitutive mutation.

The functional significance of a lac operator constitutive mutation has been determined. The transition adenine-thymine to guanine-cytosine was shown to be a constitutive mutation simply because thymine contains the functionally important 5-methyl group whereas cytosine does not. The remainder of the base pair is of no consequence. The experimental approach was to synthesize various modified operators containing cytosine, 5-methyl-cytosine, and 5-bromocytosine. The synthetic operator containing a guanine-cytosine base pair displays an eightfold reduction in stability with lac repressor whereas the operator containing 5-methylcytosine binds repressor at least as tightly as does the wild type sequence. Results published previously have shown that a similar decrease in stability of the repressor-operator complex can be obtained simply by substituting uracil for thymine or by inverting the base pair to thymine-adenine. All these results taken together implicate the thymine 5-methyl as the only important functional group recognized by the lac repressor at this base pair. Further confirmation of this conclusion was obtained by substitution of 5-bromocytosine and 5-bromouracil at this base pair. Both altered the stability of the repressor-operator complex by about the same percent suggesting that the bromine atom was the important determinant of complex stability for 5-bromopyrimidine analogs.     

The main role of human thymine-DNA glycosylase is removal of thymine produced by deamination of 5-methylcytosine and not removal of ethenocytosine

Metabolites of vinyl chloride react with cytosine in DNA to form 3,N(4)-ethenocytosine. Recent studies suggest that ethenocytosine is repaired by the base excision repair pathway with the ethenobase being removed by thymine-DNA glycosylase. Here single turnover kinetics have been used to compare the excision of ethenocytosine by thymine-DNA glycosylase with the excision of thymine. The effect of flanking DNA sequence on the excision of ethenocytosine was also investigated. The 34-bp duplexes studied here fall into three categories. Ethenocytosine base-paired with guanine within a CpG site (i.e. CpG.(epsilon)C-DNA) was by far the best substrate having a specificity constant (k(2)/K(d)) of 25.1 x 10(6) m(-1) s(-1). The next best substrates were DNA duplexes containing TpG.(epsilon)C, GpG.(epsilon)C, and CpG.T. These had specificity constants 45-130 times smaller than CpG.(epsilon)C-DNA. The worst substrates were DNA duplexes containing ApG.(epsilon)C and TpG.T, which had specificity constants, respectively, 1,600 and 7,400 times lower than CpG.(epsilon)C-DNA. DNA containing ethenocytosine was bound much more tightly than DNA containing a G.T mismatch. This is probably because thymine-DNA glycosylase can flip out ethenocytosine from a G.(epsilon)C base pair more easily than it can flip out thymine from a G.T mismatch. Because thymine-DNA glycosylase has a larger specificity constant for the removal of ethenocytosine, it has been suggested its primary purpose is to deal with ethenocytosine. However, these results showing that thymine-DNA glycosylase has a strong sequence preference for CpG sites in the excision of both thymine and ethenocytosine suggest that the main role of thymine-DNA glycosylase in vivo is the removal of thymine produced by deamination of 5-methylcytosine at CpG sites.     

Perturbation of hydrogen bonds in the adenine ... thymine base pair by Na+, Mg2+, Ca2+ and NH4+ cations

Perturbation of the hydrogen bonds in the adenine ... thymine base pair by Na+, Mg2+, Ca2+ and NH4+ cations has been investigated by means of ab initio SCF calculations with the STO-3G basis set. The geometry of adenine...thymine, as well as those of the perturbed pairs were optimized. Approach of any cation to thymine at O6 leads to destabilization of the adenine...thymine pair; divalent cations (Mg2+, Ca2+) have a profound effect on the structure of the base pair. The approach of a cation to other available sites (thymine: O2, adenine N1 and N3) leads, on the other hand, to stabilization of the base pair. If a water molecule is placed between the cation and the base pair, the structure and stability of the base pair are changed only negligibly.     

Comparison of gas phase intrinsic properties of cytosine and thymine nucleobases with their O-alkyl adducts: different hydrogen bonding preferences for thymine versus O-alkyl thymine

In recent years, there has been increasing interest in damaged DNA and RNA nucleobases. These damaged nucleobases can cause DNA mutation, resulting in various diseases such as cancer. Alkylating agents are mutagenic and carcinogenic in a variety of prokaryotic and eukaryotic organisms. The present study employs density functional theory (DFT/B3LYP) with the 6-311++G(d,p) basis set to investigate the effect of chemical damage in O-alkyl pyrimidines such as O⁴-methylthymine, O²-methylcytosine and O²-methylthymine. We compared the intrinsic properties, such as proton affinities, gas phase acidities, equilibrium tautomerization and nucleobase pair’s hydrogen bonding properties, of these molecules with those in the normal nucleobases thymine and cytosine. The results are of interest for chemical reasons and also possibly for biological purposes since biological media can be quite non-polar. Furthermore, we found that N1-H of O⁴-methylthymine is less acidic than N1-H of thymine, suggesting that alkyl DNA glycosylase enzyme cannot discriminate this damaged nucleobase from a normal thymine nucleobase. This result indicates that the conjugated base anion of O⁴-methylthymine would be a worse leaving group and O⁴-methylthymine is repaired in genome by demethylation rather than enzyme-catalyzed excision at N1.     

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.     

96 Study of the stacking of DNA homoassociates by the extended cluster approach

Metropolis Monte Carlo method based on the extended cluster approach (Danilov, Dailidonis, van Mourik, & Fruchtl, 2011a, 2011b; Dailidonis, Danilov, Früchtl, & van Mourik, 2011) is used to investigate adenine–adenine (AA), guanine–guanine (GG), thymine–thymine (TT), and cytosine–cytosine (CC) homoassociates in a cluster consisting of 400 water molecules. The starting structures taken were AA N(7) amino symmetric, TT N(3) – O(4) symmetric, GG N(1) – O(6) symmetric, and CC N(3) amino symmetric base pairs. A water spherical cluster with the density of water at room temperature and a radius sphere equal 13.9 Å was used, which corresponds to the most difficult conditions for the formation of stacks (see Abraham, 1982). In spite of such initial conditions, it is shown that during the simulation, each base pair is transformed into a more favorable stacked configuration. The results obtained allow to observe the whole process of convergence for the first time (for more information, visit the Website http://biophys.in.ua/).     

The Effect of a G:T Mispair on the Dynamics of DNA

Distortions in the DNA sequence such as damages or mispairs are specifically recognized and processed by DNA repair enzymes. A particular challenge for the enzymatic specificity is the recognition of a wrongly-placed native nucleotide such as thymine in T:G mispairs. An important step of substrate binding which is observed in many repair proteins is the flipping of the target base out of the DNA helix into the enzyme’s active site. In this work we investigate how much the intrinsic dynamics of mispaired DNA is changed compared to canonical DNA. Our molecular dynamics simulations of DNA with and without T:G mispairs show significant differences in the conformation of paired and mispaired DNA. The wobble pair T:G shows local distortions such as twist, shear and stretch which deviate from canonical B form values. Moreover, the T:G mispair is found to be kinetically less stable, exhibiting two states with respect to base opening: a closed state comparable to the canonical base pairs, and a more open state, indicating a proneness for base flip. In addition, we observe that the thymine base in a T:G mispair is significantly more probable to be flipped than thymine in a T:A pair or cytosine in a C:G pair. Such local deformations and in particular the existence of a second, more-open state can be speculated to help the target-site recognition by repair enzymes.     

The stability of oligodeoxyribonucleotide duplexes containing degenerate bases.

Oligodeoxyribonucleotides containing N4-methoxycytosine (mo4C), N4-methoxy-5-methylcytosine (mo4m5C) and other base-analogues were synthesised and used to compare the stabilities of duplexes containing mo4C.A and mo4C.G base pairs with those containing normal and mismatch pairs. The Tm values and other thermodynamic parameters are recorded. The otherwise identical duplexes containing a mo4C.A and a mo4C.G base pair have closely similar stabilities to each other and to the corresponding duplexes containing normal base pairs, considerably greater than the stabilities of those containing mismatch pairs. Corresponding observations are recorded in dot-blot experiments using M13 cloned DNA carrying an insert complementary to the oligonucleotides; approximate Td values are given.     

Excision of thymine and 5-hydroxymethyluracil by the MBD4 DNA glycosylase domain: structural basis and implications for active DNA demethylation

The mammalian DNA glycosylase-methyl-CpG binding domain protein 4 (MBD4)-is involved in active DNA demethylation via the base excision repair pathway. MBD4 contains an N-terminal MBD and a C-terminal DNA glycosylase domain. MBD4 can excise the mismatched base paired with a guanine (G:X), where X is uracil, thymine or 5-hydroxymethyluracil (5hmU). These are, respectively, the deamination products of cytosine, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Here, we present three structures of the MBD4 C-terminal glycosylase domain (wild-type and its catalytic mutant D534N), in complex with DNA containing a G:T or G:5hmU mismatch. MBD4 flips the target nucleotide from the double-stranded DNA. The catalytic mutant D534N captures the intact target nucleotide in the active site binding pocket. MBD4 specifically recognizes the Watson-Crick polar edge of thymine or 5hmU via the O(2), N(3) and O(4) atoms, thus restricting its activity to thymine/uracil-based modifications while excluding cytosine and its derivatives. The wild-type enzyme cleaves the N-glycosidic bond, leaving the ribose ring in the flipped state, while the cleaved base is released. Unexpectedly, the C(1)' of the sugar has yet to be hydrolyzed and appears to form a stable intermediate with one of the side chain carboxyl oxygen atoms of D534, via either electrostatic or covalent interaction, suggesting a different catalytic mechanism from those of other DNA glycosylases.     

Propeller-twisted adenine.thymine and guanine.cytosine base pairs tend to buckle and stagger in opposite directions.

Base pairs are propeller-twisted, buckled and staggered in DNA fragment crystals. These deformations were analyzed with isolated Watson-Crick base pairs using empirical potentials and buckle was found to almost linearly correlate with propeller. Interestingly, the thymine.adenine pair favours negative buckling for propellers mostly observed in DNA crystals while positive buckling is preferred by the cytosine.guanine pair. The propeller also induces opposite staggers in the adenine.thymine and guanine.cytosine base pairs.     

Single base-pair mutations in centromere element III cause aberrant chromosome segregation in Saccharomyces cerevisiae.

In this paper we show that a 211-base pair segment of CEN3 DNA is sufficient to confer wild-type centromere function in the yeast Saccharomyces cerevisiae. We used site-directed mutagenesis of the 211-base pair fragment to examine the sequence-specific functional requirements of a conserved 11-base pair segment of centromere DNA, element III (5'-TGATTTATCCGAA-3'). Element III is the most highly conserved of the centromeric DNA sequences, differing by only a single adenine X thymine base pair among the four centromere DNAs sequenced thus far. All of the element III sequences contain specific cytosine X guanine base pairs, including a 5'-CCG-3' arrangement, which we targeted for single cytosine-to-thymine mutations by using sodium bisulfite. The effects of element III mutations on plasmid and chromosome segregation were determined by mitotic stability assays. Conversion of CCG to CTG completely abolished centromere function both in plasmids and in chromosome III, whereas conversion of CCG to TCG decreased plasmid and chromosome stability moderately. The other two guanine X cytosine base pairs in element III could be independently converted to adenine X thymine base pairs without affecting plasmid or chromosome stability. We concluded that while some specific nucleotides within the conserved element III sequence are essential for proper centromere function, other conserved nucleotides can be changed.     

Perturbation of Hydrogen Bonds in the Adenine…Thymine Base Pair by Na+, Mg2+, Ca2+ and NH4 + Cations

Abstract

Perturbation of the hydrogen bonds in the adenine…thymine base pair by Na⁺, Mg²⁺, Ca²⁺ and NH₄ ⁺ cations has been investigated by means of ab initio SCF calculations with the STO-3G basis set. The geometry of adenine…thymine, as well as those of the perturbed pairs were optimized. Approach of any cation to thymine at 06 leads to destabilization of the adenine…thy mine pair; divalent cations (Mg²⁺, Ca²⁺) have a profound effect on the structure of the base pair. The approach of a cation to other available sites (thymine: O2, adenine N1 and N3) leads, on the other hand, to stabilization of the base pair. If a water molecule is placed between the cation and the base pair, the structure and stability of the base pair are changed only negligibly.     

M.HhaI binds tightly to substrates containing mismatches at the target base.

The (cytosine-5) DNA methyltransferase M.HhaI causes its target cytosine base to be flipped completely out of the DNA helix upon binding. We have investigated the effects of replacing the target cytosine by other, mismatched bases, including adenine, guanine, thymine and uracil. We find that M.HhaI binds more tightly to such mismatched substrates and can even transfer a methyl group to uracil if a G:U mismatch is present. Other mismatched substrates in which the orphan guanine is changed exhibit similar behavior. Overall, the affinity of DNA binding correlates inversely with the stability of the target base pair, while the nature of the target base appears irrelevant for complex formation. The presence of a cofactor analog. S-adenosyl-L-homocysteine, greatly enhances the selectivity of the methyltransferase for cytosine at the target site. We propose that the DNA methyltransferases have evolved from mismatch binding proteins and that base flipping was, and still is, a key element in many DNA-enzyme interactions.