The "Basics"

 The "Basics"

Having gone over some backround information in the introduction, in this base of the blog we will introduce the basic mechanisms in which further discussion will be based.

• What is DNA methylation?

DNA methylation is a species specific process. In this blog we focus on eukaryotic methylation. In eukaryotic methylation, a methyl group is added to the fifth position of a pyrimidine ring of a cytosine forming 5-methylcytosine. Modification mostly occurs in GpC palindromic sequences. Such sequences in mamalian genomes are mostly found upstream of genes and are referred to as GpC islands.

There are two types of methylation De Novo and Maintenance. De Novo methylation occurs at non-methylated DNA while, Maintenance methylation occurs at the hemi-methylated daughter strands. A schematic of these two processes is shown below. DNMT1 is the main enzyme responsible for maintenance methylation in mammals.

The two types of methylation[Graph taken from http://www.atdbio.com/content/56/Epigenetics#Inheritance-of-DNA-methylation-patterns]

The fashion in which methylation inhibits transcription is not really understood. One hypothesis suggests that methylation directly interferes with the binding of transcriptional activators by making the target sequences unreachable. Another hypothesis is that methylcytosines bind a family of proteins with a specific methyl-CpG-binding Domain(MBD) which bind in the DNA's major groove and attract complexes of proteins which alter chromosome structure and inhibit transcription.

• DNMT1 

DNMT1 as mentioned before is a methyl transferase and since it performs maintenance methylation it functions best in hemi-methylated DNA. 5-methylcytosine transferaces share their catalytic mechanism and use the same cofactor as a methyl donor, S-Adenosylmethionine(SAM).

The principle of the mechanism is that SAM electrophilically attacks the C(5) donating a methyl group and is converted to S-adenosyl-1-homocysteine(AdeHcy). Methyltransferases,in our case DNMT1, assist by increasing the nucleophilicity of the cytosine and promoting the reaction.  DNMT1 contains a cytosine residue which gets deprotonated to a thiolate ion and acts acts a strong nucleophile.

The mechanism that takes place is the following

1. The reaction is initiated by nucleophilic attack of the enzymes cysteine thiolate to the C(6) of cytosine,resulting in its activation
2. Activated C(5) nucleophilically attacks SAMs methyl group
3. A covalent bond is  formed between the cysteine sulfur of SAM  and the C(6) of cytosine, forming a covalent intermidiate, converting SAM to AdeHcy
4. beta-elimination follows and the covalent intermidiate is degraded  and thus producing 5-methylcytosine, AdeHcy and releasing the enzyme
 

 It should be also noted that a base is required for the deprotonation of the cytosine in DNMT1 to form the nucleophilic thiolate ion.It has been posited that this base could be a DNA phosphate group however, this has not been confirmed. 

Methyltransferase methylation mechanism [Graph taken from http://www.atdbio.com/content/56/Epigenetics#Inheritance-of-DNA-methylation-patterns]