Synthesis and physical characterization of DNA fragments containing N4-methylcytosine and 5-methylcytosine.

The synthesis of N4-methyl-2'-deoxycytidine and its fully protected mononucleotide, suitable for the oligonucleotide synthesis by phosphotriester method is described. A set of octanucleotides - d(CGCGCGCG), d(CG5mCGCGCG), d(CG4mCGCGCG) and dodecanucleotides - d(GGACCCGGGTCC), d(GGA5mCCCGGGTCC), d(GGA4mCCCGGGTCC) has been synthesized in a solution. Physical characterization of the oligonucleotide duplexes by means of UV and CD spectrometry provides the evidence that 4mC similarly to 5mC favours the B--greater than Z transition, although both of these methylated cytosines inhibit the B--greater than A conformational change. N4-Methylcytosine in contrast to 5-methylcytosine reduces the DNA double helix thermal stability.     

Effect of N4-methylcytosine and 5-methylcytosine on the stability of the DNA helix

The thermodynamic parameters (delta H, delta S) of the helix-coil transition of self-complementary oligonucleotides d(CGCGCGCG), d(CG5mCGCGCG), d(CG4mCGCGCG), d(GGACCCGGGTCC), d(GGA5mCCCGGGTCC), and d(GGA4mCCCGGGTCC) were determined. The substitution of 4mC for C was found to decrease the melting temperature of the oligonucleotides. The destabilization effect of the two substitutions is equivalent to the change of A.T for G.C pair. The free energy decrease of the helix-coil transition due to the introduction of two 4mC into an octanucleotide was estimated to be 1,24 kcal/mol.     

Phosphopeptides designed for 5-methylcytosine recognition

An artificial phosphopeptide has been developed through rational design of the interaction with 5-methylcytosine in duplex DNA. The peptide consists of two tandem zinc finger motifs, in one of which the glutamate was replaced with a phosphotyrosine, the phosphotyrosine in the peptide being effective for methylcytosine selectivity of DNA binding. The flexible modulation of the target methylated sequence by rearrangement of zinc finger peptides is possible, and the phosphopeptide provided us an important hint for expansion of the codes for the interactions of zinc fingers with DNA to methylated DNA sequences. The fluorescence-labeled phosphopeptide provided information on the methylation status of genomic DNA through fluorescence anisotropy after a 10 min incubation.     

DNA of Drosophila melanogaster contains 5-methylcytosine

It is commonly accepted that the DNA of Drosophila melanogaster does not contain 5-methylcytosine, which is essential in the development of most eukaryotes. We have developed a new, highly specific and sensitive assay to detect the presence of 5-methylcytosine in genomic DNA. The DNA is degraded to nucleosides, 5-methylcytosine purified by HPLC and, for detection by 1D- and 2D-TLC, radiolabeled using deoxynucleoside kinase and [gamma-(32)P]ATP. Using this assay, we show here that 5-methylcytosine occurs in the DNA of D. melanogaster at a level of approximately 1 in 1000-2000 cytosine residues in adult flies. DNA methylation is detectable in all stages of D.melanogaster development.     

DNA methylation in thermophilic bacteria: N4-methylcytosine, 5-methylcytosine, and N6-methyladenine.

While determining the minor and major base composition of the DNA from 17 types of thermophilic bacteria by high performance liquid chromatography (HPLC) of enzymatic digests, we have discovered a novel base, N4-methylcytosine (m4C). Its structure was proven by comparison of the DNA-derived nucleoside to the analogous authentic compound by HPLC, UV spectroscopy, and mass spectroscopy. Eight of the bacterial DNAs contained m4C. Only two contained the common minor base, 5-methylcytosine (m5C), and neither of these was from an extreme thermophile. The other prevalent modified base of bacterial DNA, N6-methyladenine (m6A), was found in nine of the DNAs. Restriction analysis revealed that four of the DNAs had dam-type (Gm6ATC) methylation patterns. Due to the propensity of m5C residues to be deaminated by heat to thymine residues and to inefficient repair of the resulting mismatched base pairs, thermophiles with optimal growth temperatures of greater than or equal to 60 degrees C generally may avoid having m5C in their genomes. Instead, some of them have deamination-resistant m4C residues.     

Organization of 5-methylcytosine in chromosomal DNA

The 5-methylcytosine residues of L-cells have been labeled with [methyl-3H]-L-methionine and their chromatin localization studied using deoxyribonucleases. The kinetics of micrococcal nuclease digestion showed that the methylated cytosine residues are concentrated within regions resistant to nuclease digestion and preferentially missing from those regions between nucleosomes which are nuclease sensitive. Using DNA hybridization kinetic analysis, it is shown that 5-methylcytosine is abundant in highly repeated sequences but is also present in middle repetitive and unique sequence DNA.     

DNA from Aspergillus flavus contains 5-methylcytosine

DNA from Aspergillus sp. has been reported not to contain 5-methylcytosine. However, it has been found that Aspergillus nidulans responds to 5-azacytidine, a drug that is a strong inhibitor of DNA methyltransferases. Therefore, we have re-examined the occurrence of 5-methylcytosine in DNA from Aspergillus flavus by using a highly sensitive and specific method for detection of modified bases in genomic DNA comprising high-performance liquid chromatography separation of nucleosides, labeling of the nucleoside with deoxynucleoside kinase and two-dimensional thin-layer chromatography. Our results show that 5-methylcytosine is present in DNA from A. flavus. We estimate the relative amounts of 5-methylcytosine to cytosine to be approximately 1/400.     

Oxidative damage to 5-methylcytosine in DNA.

Exposure of pyrimidines of DNA to ionizing radiation under aerobic conditions or oxidizing agents results in attack on the 5,6 double bond of the pyrimidine ring or on the exocyclic 5-methyl group. The primary product of oxidation of the 5,6 double bond of thymine is thymine glycol, while oxidation of the 5-methyl group yields 5-hydroxymethyluracil. Oxidation of the 5,6 double bond of cytosine yields cytosine glycol, which decomposes to 5-hydroxycytosine, 5-hydroxyuracil and uracil glycol, all of which are repaired in DNA by Escherichia coli endonuclease III. We now describe the products of oxidation of 5-methylcytosine in DNA. Poly(dG-[3H]dmC) was gamma-irradiated or oxidized with hydrogen peroxide in the presence of Fe3+ and ascorbic acid. The oxidized co-polymer was incubated with endonuclease III or 5-hydroxymethyluracil-DNA glycosylase, to determine whether repairable products were formed, or digested to 2'-deoxyribonucleosides, to determine the total complement of oxidative products. Oxidative attack on 5-methylcytosine resulted primarily in formation of thymine glycol. The radiogenic yield of thymine glycol in poly(dG-dmC) was the same as that in poly(dA-dT), demonstrating that 5-methylcytosine residues in DNA were equally susceptible to radiation-induced oxidation as were thymine residues.     

Detection of 5-methylcytosine in DNA sequences.

Col E1 DNA has methylated cytosine in the sequence 5'-CC*(A/T)GG-3' and methylated adenine in the sequence 5'-GA*TC-3' at the positions indicated by asterisks(*). When the Maxam-Gilbert DNA sequencing method is applied to this DNA, the methylated cytosine (5-methylcytosine) is found to be less reactive to hydrazine than are cytosine and thymine, so that a band corresponding to that base does not appear in the pyrimidine cleavage patterns. The existence of the methylated cytosine can be confirmed by analyzing the complementary strand or unmethylated DNA. In contrast, the methylated adenine (probably N6-methyladenine) cannot be distinguished from adenine with standard conditions for cleavage at adenine.     

Involvement of 5-methylcytosine in sunlight-induced mutagenesis.

In human skin cancers, more than 30 % of all mutations in the p53 gene are transitions at dipyrimidines within the sequence context CpG, i.e. 5'-TCG and 5'-CCG, found at several mutational hotspots. Since CpGs are methylated along the p53 gene, these mutations may be derived from solar UV-induced pyrimidine dimers forming at sequences that contain 5-methylcytosine. In Xorder to define the contribution of 5-methylcytosine to sunlight-induced mutations, we have used mouse fibroblasts containing the CpG-methylated lacI transgene as a mutational target. We sequenced 182 UVC (254 nm UV)-induced mutations and 170 mutations induced by a solar UV simulator, along with 75 mutations in untreated cells. Only a few of the mutations in untreated cells were transitions at dipyrimidines, but more than 95% of the UVC and solar irradiation-induced mutations were targeted to dipyrimidine sites, the majority being transitions. After UVC irradiation, 6% of the base substitutions were at dipyrimidines containing 5-methylcytosine and only 2.2% of all mutations were transitions within this sequence context. However, 24% of the solar light-induced mutations were at dipyrimidines that contain 5-methylcytosine and most of them were transitions. Two sunlight-induced mutational hotspots at methylated CpGs correlated with sequences that form the highest levels of cyclobutane pyrimidine dimers after irradiation with sunlight but not with UVC. The data indicate that dipyrimidines that contain 5-methylcytosine are preferential targets for sunlight-induced mutagenesis in cultured mammalian cells, thus explaining the large proportion of p53 mutations at such sites in skin tumors in vivo.     

African trypanosomes contain 5-methylcytosine in nuclear DNA

It is currently unclear if there are modified DNA bases in Trypanosoma brucei other than J-base. We identify herein a cytosine-5 DNA methyltransferase gene and report the presence and location of 5-methylcytosine in genomic DNA. Our data demonstrate that African trypanosomes contain a functional cytosine DNA methylation pathway.     

Genomic 5-methylcytosine determination by 32P-postlabeling analysis

A simple and sensitive method for the quantitation of 5-methyldeoxycytidine in DNA has been developed by the adaptation of the Randerath 32P-postlabeling technique. Nucleic acids were digested to 3'-monophosphate nucleotides, which were converted to 32P-labeled 3',5'-bisphosphate nucleotides, the 3'-phosphate was cleaved by the action of nuclease P1, and the resultant 5'-[32P]-monophosphate nucleotides were separated by two-dimensional thin-layer chromatography. Less than 1 microgram of DNA was required for the precise quantitation of 5-methyldeoxycytidine content to a detectable limit of 0.01% of the total cytidine residues methylated. The genomic 5-methyldeoxycytidine content may thus be quantitated in tissue samples, small or selective cell populations, senescing or terminally differentiating cells, or DNA from any source. We report here, for the first time, the genomic 5-methyldeoxycytidine content of normal human bronchial epithelial and normal human pulmonary mesothelial cells. The chromatographic separation of all of the normal and some of the rare monophosphate deoxyribonucleotides and ribonucleotides has been characterized. Thus, 5-bromodeoxyuridine and the RNA contamination of DNA or the DNA contamination of RNA can also be quantitated during the same analysis.     

Interaction of AluI, Cfr6I and PvuII restriction-modification enzymes with substrates containing either N4-methylcytosine or 5-methylcytosine

The cleavage specificity of R.Cfr6I, an isoschizomer of PvuII restriction endonuclease was determined to be 5'CAG decreases CTG and the methylation specificity of Cfr6I and PvuII methylases, 5'CAG4mCTG. Thus, M.Cfr6I and M.PvuII are new additions to the list of methylases with N4-methylcytosine specificity. Neither of the above RM enzymes acts on the substrates containing either N4-methylcytosine or 5-methylcytosine in a cognate methylation position.     

The 5-methylcytosine content of DNA: Tissue specificity

Using a new technique for the determination of the extent of DNA methylation by comparing the incorporation of radioactivity from deoxycytidine-2-C¹⁴ into DNA cytosine and 5-methylcytosine (5MC), the 5MC content of DNA was found to be a tissue specific property in two systems: (1) mouse tissue culture cell lines, in which the level of 5MC varied from 2.97 ± 0.03% conversion of cytosine to 5MC in the DNA of a mouse melanoma line to 4.58 ± 0.18% in a primary teratoma line, and (2) the organs of the developing chick embryo, in which the level of 5MC varied from 3.60 ± 0.04% conversion of cytosine to 5MC in the DNA of the chorioallantoic membrane to 4.19 ± 0.02% in the brain.     

Oxidatively generated damage to DNA at 5-methylcytosine mispairs

Oxidatively generated damage to DNA has been implicated as causing mutations that lead to aging and disease. The one-electron oxidation of normal DNA leads to formation of a nucleobase radical cation that hops through the DNA until it is trapped irreversibly, primarily by reaction at guanine. It has been observed that 5-methylcytosine (C(m)) is a mutational "hot-spot". However, C(m) in a Watson-Crick base pair with G is not especially susceptible to oxidatively induced damage. Radical cation hopping is inhibited in duplexes that contain C-A or C-T mispairs, but no reaction is detected at cytosine. In contrast, we find that the one-electron oxidation of DNA that contains C(m)-A or C(m)-T mispairs results primarily in reaction at C(m) even in the presence of GG steps. The reaction at C(m) is attributed to proton coupled electron transfer, which provides a relatively low activation barrier path for reaction at 5-methylcytosine. This enhanced reactivity of C(m) in mispairs may contribute to the formation of mutational hot spots at C(m).