DNA modifications - lecture 16 Flashcards
What is the most common DNA mutation in mammals?
5 - methylcytosine (5mC)
What is the name of the enzyme that transfers the methyl group onto the cytosine?
DNMT - (DNA methyl transferase)
Where does the methyl group come from which is on the 5mC?
SAM (S – Adenyl Methionine)
What common DNA mutation is present in bacterial cells?
N6 - methyladenine
What happens when the DNA is methylated?
- Reduced binding of transcription factors
- Inactive gene , reduced gene expression
Which DNA sequence is normally methylated?
CG dinucleotide. It is palindromic and methylated in both strands.
What happens to the DNA methylation when the cell replicates?
DNA becomes a semi - methylated state, only one strand methylated
How do you methylate both strands after cell synthesis?
Via DNMT1 enzyme
How does DNMT1 methylate the un- methylated DNA strand?
NP65 protein binds to any methylated DNA. This protein recruits DMT1 , which methylates the opposite strand
What enzymes add the methyl group to the cytosine?
DNMT3A and 3B.
What enzyme maintains the methyl group on the cytosine?
DNMT1
What enzyme de-methylates the cytosine?
TET1-3
What happens to methylation after fertilisation
Methyl groups wiped out and then begin again through growth
Why do cells methylate the DNA?
As the cells migrate and pick up different methyl signals , they specify into different cell types
How did scientists discover 5 -Hydroxymethylcytosine
- Extracted DNA and digested it into single nucleotides
- Then did thin layer chromatography
- All nucleotides separate , found all normal bases and then an extra
How did scientists confirm it was 5 -Hydroxymethylcytosine?
- high performance liquid chromatography
- Observed a small peak in the same place of hmdc
What happens when there is mutations in the MeCP2 gene?
Causes Rett syndrome , which is a neurodevelopment disorder
What happens when MeCP2 binds to 5hmC?
The chromosome Is less condensed and the DNA can be accessed easily
What happens when MeCP2 binds to 5Mc?
Causes DNA to be more condensed
What happens when the enzyme MeCp2 is mutated?
Can’t bind to 5hmC, but can still bind to 5mc.
What is the overall effect of a mutated MeCp2 enzyme?
less gene expression
How does bisulphate - sequencing highlight all the methylated cytosines?
- Cytosines turn to Uracil
- 5-methylcytosines stay as C
- When converted into DNA via PCR implication, Us go to Ts , but Cs stay as Cs.
What is a major drawback between bisulphate sequencing?
Cannot differentiate between 5-mC and 5 - hmC
What does TAB - sequencing do?
Tet - assisted Bisulfite Sequencing
What is the first enzyme in TAB - seq and what does it do?
Glucosyl - transferase . Adds. glucose group only to hmC
What is the second enzyme in the TAB-seq and what does it do?
TET enzyme. Converts 5mC into carboxyl cytosine. Not to 5hmC.
After bisulphie treatment?
Only the 5mC converted into U then T. 5mhC stays as c
