Methylation underlies several important biological processes, including restriction and modification, mismatch error correction (a DNA repair process), and the control of eukaryotic gene expression. S-Adenosylmethionine (AdoMet) is the substrate for methylation of both RNA and DNA. Methylation occurs at the polynucleotide level, with transfer of a methyl group from AdoMet to a nucleotide residue.

The sole methylated base found in eukaryotic DNA is 5-methylcytosine (mC). In animals, methylation is found primarily in C residues that are immediately 5' to G residues (that is, in a sequence CpG). When such a C is methylated, so is the corresponding C in the complementary strand. In plant DNA, the methylated sequence is CpNpGp, where N can be any base.

In prokaryotic DNA the major methylated bases are N⁶-methyladenine (mA) and to a lesser extent N⁴-methylcytosine. Methylation in bacteria occurs at specific sites. In E. coli, methylation of A residues in the sequence 5´-GATC-3´ is involved in mismatch error correction, and it plays a role in controlling initiation of DNA replication. Methylation at other sites protects DNA against cleavage by restriction endonucleases (described here). Structural studies on a bacterial DNA methylase have shown that the bases undergoing methylation rotate completely out of the DNA duplex and into a catalytic pocket within the enzyme structure. Other enzymes that work on bases, such as uracil-N-glycosylase, operate similarly.

The biological significance of DNA methylation in prokaryotes is now fairly clear, but its importance in eukaryotes has not yet been defined. What is known, however, is that methylation at a particular site is a heritable phenomenon. That is, when eukaryotic DNA replicates, a maintenance methylase ensures that all of the sites that were methylated in parental DNA are methylated in daughter DNA. The process is shown in Figure 25.3a.

Methylation occurs after replication and unmethylated sites in parental DNA remain unmethylated in daughter DNA. 5-Azacytidine, a cytidine analog that is metabolized like cytidine to the analog of dCTP (but cannot be methylated), can be incorporated into DNA. After replication replaces the modified C with a real C, it too remains unmethylated (see Figure 25.3b).

Changes in methylation can have effects on transcription. For example, azacytidine treatment can cause adult bone marrow cells to reactivate the synthesis of fetal hemoglobin, which is normally turned off during development.

Methylation may be involved in carcinogenesis. Deamination of an mC residue in DNA creates a G-T base pair, an event that could create a GC ---> AT mutation. Most sequence alterations in tumor cell DNAs involve GC --->AT transitions.