DNA Methlases and methylation

The nucleotides in DNA can be covalently modified, and in vertebrate cells the methylation of cytosine seems to provide an important mechanism for distinguishing genes that are active from those that are not. The covalently modified 5-methylcytosine (5-methyl C) has the same relation to cytosine that thymine has to uracil and likewise has no effect on base-pairing. The methylation in vertebrate DNA is restricted to cytosine (C) nucleotides in the sequence CG, which is base-paired to exactly the same sequence (in opposite orientation) on the other strand of the DNA helix. Consequently, a simple mechanism permits the existing pattern of DNA methylation to be inherited directly by the daughter DNA strands. An enzyme called maintenance methylase acts preferentially on those CG sequences that are base-paired with a CG sequence that is already methylated. As a result, the pattern of DNA methylation on the parental DNA strand will act as a template for the methylation of the daughter DNA strand, causing this pattern to be inherited directly following DNA replication.

DNA Methylation

DNA methylation is an epigenetic modification that occurs as a result of a biological mechanism that leads to the attachment of a methyl group onto the 5thcarbon of cytosine (5mC).

DNA methylation processes are controlled by the DNA methyltransferase enzyme (DNMTs) family, which create 5 methylcytosine (5mC) via transferring a methyl group from a donor S- adenosyl methionine (SAM) to the 5th carbon of cytosine.

DNA according to methylation status is either Hypomethylated or hypermethylated.

A: CH₃ is added to cytosine in the presence of DNMT enzymes and SAM (donor). B: Symmetrical CpG methylation. C: Diagram shows relation between DNA methylation and gene expression.

What is DNA Methylation

DNA methylation is a biochemical process involving the addition of a methyl group (−CH₃) to the DNA molecule, typically at the cytosine base in a cytosine-guanine (CpG) dinucleotide context. This modification plays a critical role in gene regulation and other cellular processes.

Requirements for DNA Methylation

• S-Adenosine Methionine
• DNA fragment with Cysteine rich area.
• DNA Methylase (Also known as DNA methyl Transferase).

Process

Bacteria produce enzymes that are useful for studying methylation in vertebrate cells. They use the methylation of either an A or a C at a specific site to protect themselves from the action of their own restriction nucleases. The restriction nuclease , cuts the sequence CCGG but fails to cleave it if the central C is methylated. Thus the susceptibility of a DNA molecule to cleavage by HpaII can be used to detect whether CG sequences at specific DNA sites are methylated. The inheritance of methylation patterns can be studied in vertebrate cells in culture by first using bacterial methylating enzymes to introduce methyl groups on cytosines and Genetic Mechanisms then using bacterial restriction nucleases to follow the inheritance of these groups. The enzyme used to introduce 5-methyl C bases into specific CG sequences is the HpaII-methylase that normally protects the bacterium against its own HpaII restriction nuclease. If this enzyme is used to methylate the central C in the sequence CCGG on a cloned DNA molecule that is introduced into cultured vertebrate cells, the maintenance methylase can be shown to work as expected: each individual methylated CG is generally retained through many cell divisions, whereas unmethylated CG sequences remain unmethylated.

Types of DNA Methylases:

1.De novo Methylases: These enzymes establish new methylation patterns on previously unmethylated DNA. Examples include DNMT3A and DNMT3B.

2.Maintenance Methylases: These enzymes maintain existing methylation patterns during DNA replication. DNMT1 is the primary maintenance methylase, ensuring that newly synthesized DNA strands retain the methylation status of the parent strands.

The maintenance methylase explains the automatic inheritance of 5-methyl C nucleotides, but since it normally does not methylate fully unmethylated DNA, it leaves unanswered the question of how the methyl group is first added in a vertebrate organism. If a fully unmethylated DNA molecule is injected into a fertilized mouse egg, methyl groups will be added to nearly every CG site (an important exception will be described below). This is presumed to reflect the presence of a novel establishment methylase activity in the egg. As we shall see, de novo methylation can also occur during the differentiation of specialized cell types, although it is not known how it occurs.

Key Functions of DNA Methylases

1.Gene Regulation: Methylation typically represses gene expression. When methyl groups are added to the promoter regions of genes, it can inhibit the binding of transcription factors, preventing transcription.

2.Genomic Imprinting: In some cases, only one allele of a gene is expressed while the other is silenced, often through methylation. This phenomenon is important for development and can have implications for diseases.

3.X-Chromosome Inactivation: In female mammals, one of the two X chromosomes is randomly inactivated, a process that involves extensive methylation.

4.Transposon Silencing: Methylation helps suppress the activity of transposable elements, preventing potential genomic instability.

5.Developmental Processes: DNA methylation patterns are crucial during development, influencing cell fate and identity.

Transcription can also be silenced by methylation of DNA by enzymes called DNA methylases. This kind of silencing is not found in yeast but is common in mammalian cells. Methylation of DNA sequences can inhibit binding of proteins, including the transcriptional machinery, and thereby block gene expression. But methylation can also inhibit expression in another way: some DNA sequences are recognized only when methylated by specific repressors that then switch off nearby genes, often by recruiting histone modifying enzymes.

Switching a gene off through DNA methylation and histone modification

In its unmodified state, the mammalian gene shown can readily switch between being expressed or not expressed in the presence of activators and the transcription machinery, as shown in the top line. In this situation, expression is never firmly shut off—it is leaky. Often that is not good enough; sometimes, a gene must be completely shut off, on occasion permanently. This is achieved through methylation of the DNA and modification of the local nucleosomes. Thus, when the gene is not being expressed, a DNA methyltransferase (a methylase) can gain access and methylate cytosines within the promoter sequence, the gene itself, and the upstream activator binding sites. The methyl group is added to the 5’ position in the cytosine ring, generating 5-methylcytosine. This modification alone can disrupt binding of the transcription machinery and activators in some cases. But it can also increase binding of other proteins (e.g., MeCP2) that recognize DNA sequences containing methylcytosine. These proteins, in turn, recruit complexes that remodel and modify local nucleosomes, switching off expression of the gene completely.