5-Methyl-cytosine, deamination

Mismatch Repair. Mispairs that break the normal base-pairing rules can arise spontaneously due to DNA biosynthetic errors, events associated with genetic recombination and the deamination of methylated cytosine (Modrich, 1987). With the latter, when cytosine deaminates to uracil, an endonuclease enzyme, /V-uracil-DNA glycosylase (Lindahl, 1979), excises the uracil residue before it can pair with adenine at the next replication. However, 5-methyl cytosine deaminates to form thymine and will not be excised by a glycosylase. As a result, thymine exits on one strand paired with guanine on the sister strand, that is, a mismatch. This will result in a spontaneous point mutation if left unrepaired. For this reason, methylated cytosines form spontaneous mutation hot-spots (Miller, 1985). The cell is able to repair mismatches by being able to distinguish between the DNA strand that exists before replication and a newly synthesized strand. 

This massive amount of information should not be considered as insurmountable or only material to be marveled at but not understood. Much of the chemistry is already available to mine this information successfully. Much of it is understandable in somewhat simple terms, generally only after we have discovered the key to this simplicity. For instance, there is a marked decrease in the frequency of the dinucleotide CpG in some areas of the genome. The deficiency is believed to be due to the fact that most CpG nucleotides are methylated on the cytosine base, and spontaneous deamination of the methyl-cytosine residue creates T residues. Thus, CpG dinucleotide sequences mutate to TpG dinucleotides. But there still remain some questions. There are certain regions or islands where the CpG sequences exist in a nonmethylated form and where the frequency of CpG occurs within the expected or normal rate. Why These CpG islands are of particular interest because they are associated with the 5 ends of genes. 

CG doublets. The only modified base commonly found in eukaryotes is 5-methylcytosine,222/223 which upon deamination becomes thymine (Eq. 27-1). Most methylation occurs when C is followed by G. Usually 60-90% of all 5 -CG sequences (CpG sequences) in eukaryotic DNA are methylated. However, the fraction of methylated cytosine varies from almost zero for Drosophilia, Caenorhabditis, and Saccharomyces to as much as 30% in higher plants.224... 

DMA damage results when the amino group of cytosine residues spontaneously hydrolyzes in the cell, to produce a residue of uracil plus free ammonia. The amino group of 5-methyl-cytosine spontaneously hydrolyzes at a somewhat greater rate than cytosine. For any particular residue of 5-methyJ-cytosine, its half-life for deamination is about 40,000 years (Shen et a ., 1994). The half-life for deamination of cytosine is about twice as long. These rates are very low, and researchers who study its rate have to perform their reachons for hundreds of days to be able to detect any deamination. However, when deamination does occur in our DNA, it can result in a mutation. Research has shown that the "hotspots" for mutation in p53 all actually occur at residues of 5-methyl-cytosine, and not at cytosine (Schmutle et oi, 1996),... 

Please view the following deamination example, Deaminated 5-methyl-cytosine is identical to thymine, Hence, the overall scenario of deamination of 5-methyl-cyto-sine followed by replication involves the conversion of 5-methyl-C G to T A,... 

Deamination of 5-methyl-cytosine results in the formation of a T C mispair. This mispair is sloivly repaired by a special enzyme, which slowly hydrolyzes the resiciue of T. The enzyme is called thymine DNA glycosytase (Neddennann and jirieny, 1993)-... 

N2O3 formed by a third order reaction, can deaminate DNA bases yielding uracil from cytosine, xanthine from guanine, methyl cytosine from thymine and hypoxanthine from adenine [ 56 ]. Furthermore, it can react with secondary amines to yield carcinogenic N-nitrosoamines, which can damage DNA by alkylation, [57]. 

In human tumors, the pattern of p53 mutations is dominated by base transitions (C to T or G to A) at CpG dinucleotides (23% of all mutations). Although this type of mutation has been observed at 35 different codons, 90% of human CpG transitions are at one of six hotspot codons 175,213,245, 248, 273 and 282 (Fig. 3A) known to be important in maintaining p53 biological activity. CpG dinucleotides are sites of cytosine methylation, and deamination of 5-methylcytosine producing thymine is the most characteristic event generating spontaneous mutations in mammalian cells (Barker et... 

FIGURE 23-25 Formation of a spontaneous point mutation by deamination of 5-methyl cytosine (C) to form thymine (T). If the resulting T-G base pair is not restored to the normai C-G base pair by base excision-repair mechanisms (D), it will lead to a permanent change in sequence following DNA replication (i.e., a mutation) (B). After one round of replication, one daughter DNA molecule will have the mutant T-A base pair and the other will have the wild-type C-G base pair. 

Many spontaneous mutations are point mutations, which involve a change in a single base pair in the DNA sequence. One of the most frequent point mutations comes from deamination of a cytosine (C) base, which converts it into a uracil (U) base. In addition, the common modified base 5-methyl cytosine forms thymine when it is deaminated. If these alterations are not corrected before the DNA is replicated, the cell will use the strand containing U or T as template to form a U-A or T-A base pair, thus creating a permanent change to the DNA sequence (Figure 23-25). 

It is used to maximize the maintenance of the integrity of the information encoded by DNA. Cytosine can spontaneously deaminate to form uracil this damage is repaired by base excision repair. If uracil rather than thymine were used in DNA, then correctly positioned uracil would be indistinguishable from that arising from cytosine deamination. Use of thymine (methylated uracil) in DNA avoids this problem. 

Pol L also has the unprecedented ahility to misinsert dGMP opposite a template T at a rate that exceeds that of correct dAMP incorporation. Furthermore, on templates that contain two or more consecutive Ts, preferential dGMP incorporation opposite the first T is followed by preferential incorporation of A opposite the second template T. This remarkable specificity led us to speculate that Pol l may function in a specialized BER reaction, replacing dGs that are inadvertently removed by a DNA glycosylase from G—T or G—U mismatches that arise by deamination of 5-methyl-cytosine or cytosine (Bebenek et at, 2001). 

Deamination, the hydrolytic loss of exocyclic amino groups on the DNA bases, is typically a very slow reaction. For example, deamination of cytosine residues in dnplex DNA occnrs with a half-life of about 30,000 years under physiological conditions, and the deamination of adenine residues is still more sluggish. " Alkylation at the N3-position of cytosine (Scheme 8.5) greatly increases the rate of deamination (ty2 = 406 h). Deamination of 3-methyl-2 -deoxycytidine proceeds 4000 times faster than the same reaction in the unalkylated nucleoside. Alkylation of the N3-position in cytosine residues also facilitates deglycosylation (Jy2 = 7700 h, lower pathway in Scheme 8.5), but the deamination reaction is 20 times faster and, therefore, predominates. ... 

The analogons deamination reaction is not observed in l-methyl-2 -deoxy-adenosine nncleosides. ° Rather, in the adenine series, the Dimroth rearrangement occnrs (Scheme 8.4). On the contrary, in styrene adducts of 2 -deoxyadenosine, the hydroxyl residue of the adduct undergoes intramolecular reaction with the base to initiate deamination (Scheme 8.6). ° ° Similarly, cytosine residues bearing styrene adducts at the N3-position undergo rapid deamination (nearly complete deamination is seen within 75h). °°... 

In mammalian genomes, some cytosine residues of the CpG (cytosines adjacent to guanines) sequences in DNA are methylated, forming 5-methylcytosine. Deamination of 5-methylcytosine, however, yields thymine, a normal base component of DNA. In single-stranded DNA, this is a challenging problem as cells are not able to determine that this thymine is abnormal. In double-stranded DNA, however, deamination of the 5-methylcytosine in a methylated C G base pair yields a T G mismatch. Cells are therefore able to distinguish the thymine in a T G mismatch as... 

The similarly linked dimer (155 R = Me, R = H) has been isolated213 after irradiation of DNA in vitro and in vivo possibly by combination of cytosine and thymine (accompanied by deamination). The macrocyclic tetramer (157) has also been isolated on irradiation of DNA 214 it is thought to be the dimer of the above product (155 R = Me, R = H). The trans-syn structure of 157 has been confirmed by X-ray diffraction analysis of the hexa-A -methyl derivative.215 These are all biologically significant photoreactions as described in Section III, B, 2. Further irradiation of the trans-syn and cis-syn cyclobutane-type dimers (see Section III,E, 2) of 1,3-dimethyluracil with a sensitizer has been found to produce the 5,5 -linked dimer (158).216... 

The presence in DNA of thymine rather than uracil was an enigma for many years. Both bases pair with adenine. The only difference between them is a methyl group in thymine in place of the C-5 hydrogen atom in uracil. Why is a methylated base employed in DNA and not in RNA The existence of an active repair system to correct the deamination of cytosine provides a convincing solution to this puzzle. 

The treatment consists of a chemical reaction where sodium bisulfite involves the deamination of unmethylated cytosines to uracil (Figure 3A). In contrast, the modified cytosines (5mC, 5hmC) remain unmodified. After bisulfite conversion, the uracils are amplified as thymines in a PCR, whereas 5mC or 5hmC are amplified as cytosines. Next, the comparison of sequence information between the reference genome and bisulfite-treated DNA can provide information about patterns of methylation. The major drawback to bisulfite conversion is that it does not distinguish between 5mC and 5hmC DNA (Figure 2D), and some biases may occur in the efficiency of bisulfite conversion. 

In DNA, cytosine spontaneously deaminates to uracil. The presence of the extra methyl group is a clear indication that a thymine really belongs in that position, not a cytosine that has been deaminated. 

The dinucleotide CpG is notable because it is greatly under-represented in human DNA, occurring at only about one fifth of the roughly 4% frequency that would be expected by simply multiplying the typical fraction of Cs and Gs (0.21 x 0.21). The deficit occurs because most CpG dinucleotides are methylated on the cytosine base, and spontaneous deamination of methyl-C residues gives rise to T residues. 

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