In biochemistry, the DNA methyltransferase (DNA MTase) family of enzymes catalyze the transfer of a methyl group to DNA. DNA methylation serves a wide variety of biological functions.
All the known DNA methyltransferases use S-adenosyl methionine (SAM) as the methyl donor. A simple and accurate method for quantification of CpG methylation density in any DNA region can be obtained by using bisulfite PCR. Bisulfite treatment converts all unmethylated cytosine into uracil and leaves methylated cytosine intact. This modified DNA is then amplified by polymerase chain reaction (PCR). During PCR, uracil is read as thymidine? by DNA polymerase. Only methylated CpG dinucleotides in the initial sequence remain as the sequences of CpG (but are no longer methylated) whereas other unmethylated CpG sites appear as TpG sequences. Following bisulfite PCR, individual DNA strands are sequenced and all remaining cytosines are indications of a methylated cytosine in the original DNA template.
MTases can be divided into three different groups on the basis of the chemical reactions they catalyze:
- m6A - those that generate N6-methyladenine EC 220.127.116.11
- m4C - those that generate N4-methylcytosine EC 18.104.22.168
- m5C - those that generate C5-methylcytosine EC 22.214.171.124
De novo and maintenance DNA MTases
De novo methyltransferases recognize something in the DNA that allows them to methylate cytosines de novo. These are expressed mainly in early embryo development and they set up the pattern of methylation.
Maintenance methyltransferases add methylation to DNA when one strand is already methylated. These work throughout the life of the organism to maintain the methylation pattern that had been established by the de novo methyltransferases.
Mammalian DNA methyltransferase (DNMT)
Four active DNA methyltransferases have been identified in mammals. They are named DNMT1, DNMT2, DNMT3A and DNMT3B. DNMT3L is a protein that is closely related to DNMT3A and DNMT3B structurally and that is critical for DNA methylation, but appears to be inactive on its own.
DNMT1 is the most abundant DNA methyltransferase in mammalian cells, and considered to be the key maintenance methyltransferase in mammals. It predominantly methylates hemimethylated CpG di-nucleotides in the mammalian genome. This enzyme is 7-20 fold more active on hemimethylated DNA as compared with unmethylated substrate in vitro, but it is still more active at de novo methylation than other DNMTs. The enzyme is about 1620 amino acids long. The first 1100 amino acids constitute the regulatory domain of the enzyme, and the remaining residues constitute the catalytic domain. These are joined by Gly-Lys repeats. Both domains are required for the catalytic function of DNMT1.
DNMT1 has several isoforms, the somatic DNMT1, a splice variant (DNMT1b) and an oocyte specific isoform (DNMT1o). DNMT1o is synthesized and stored in the cytoplasm of the oocyte and translocated to the cell nucleus during early embryonic development, while the somatic DNMT1 is always found in the nucleus of somatic tissue.
DNMT1 null mutant embryonic stem cells were viable and contained a small percentage of methylated DNA and methyltransferase activity.
DNMT2 has been called the enigmatic methyltransferase. It has strong sequence similarities with m5C MTases of both prokaryotes and eukaryotes. Dnmt2 was recently shown to methylate position 38 in Aspartic acid transfer RNA; it does not methylate DNA (). The name has been provisionally changed to Rnmt2 to better reflect its biological function. Dnmt2/Rnmt2 is the first RNA cytosine methyltransferase to be identified in a vertebrate.
DNMT3 is a family of DNA methyltransferases that could methylate hemimethylated and unmethylated CpG at the same rate. The architecture of DNMT3 enzymes is similar to DNMT1 with regulatory region attached to a catalytic domain. There are three known members of the DNMT3 family: DNMT3a, 3b and 3L.
DNMT3a and DNMT3b can mediate methylation-independent gene repression. DNMT3a can co-localize with heterochromatin protein (HP1 ) and methyl-CpG binding protein (MeCBP). They can also interact with DNMT1, which might be a co-operative event during DNA methylation. DNMT3a prefers CpG methylation to CpA, CpT, and CpC methylation, though there appears to be some sequence preference of methylation for DNMT3a and DNMT3b . DNMT3a methylates CpG sites at a rate much slower than DNMT1, but greater than DNMT3b.
DNMT3L contains DNA methyltransferase motifs and is required for establishing maternal genomic imprints, despite being catalytically inactive. DNMT3L is expressed during gametogenesis when genomic imprinting takes place. The loss of DNMT3L lead to bi-allelic expression of genes normally not expressed by the maternal allele. DNMT3L interacts with DNMT3a and DNMT3b and co-localized in the nucleus. Though DNMT3L appears incapable of methylation, it may participate in transcriptional repression.