DNA modifications - lecture 16

Question 1

What is the most common DNA mutation in mammals?

Answer 1

5 - methylcytosine (5mC)

Question 2

What is the name of the enzyme that transfers the methyl group onto the cytosine?

Answer 2

DNMT - (DNA methyl transferase)

Question 3

Where does the methyl group come from which is on the 5mC?

Answer 3

SAM (S – Adenyl Methionine)

Question 4

What common DNA mutation is present in bacterial cells?

Answer 4

N6 - methyladenine

Question 5

What happens when the DNA is methylated?

Answer 5

• Reduced binding of transcription factors

- Inactive gene , reduced gene expression

Question 6

Which DNA sequence is normally methylated?

Answer 6

CG dinucleotide. It is palindromic and methylated in both strands.

Question 7

What happens to the DNA methylation when the cell replicates?

Answer 7

DNA becomes a semi - methylated state, only one strand methylated

Question 8

How do you methylate both strands after cell synthesis?

Answer 8

Via DNMT1 enzyme

Question 9

How does DNMT1 methylate the un- methylated DNA strand?

Answer 9

NP65 protein binds to any methylated DNA. This protein recruits DMT1 , which methylates the opposite strand

Question 10

What enzymes add the methyl group to the cytosine?

Answer 10

DNMT3A and 3B.

Question 11

What enzyme maintains the methyl group on the cytosine?

Answer 11

DNMT1

Question 12

What enzyme de-methylates the cytosine?

Answer 12

TET1-3

Question 13

What happens to methylation after fertilisation

Answer 13

Methyl groups wiped out and then begin again through growth

Question 14

Why do cells methylate the DNA?

Answer 14

As the cells migrate and pick up different methyl signals , they specify into different cell types

Question 15

How did scientists discover 5 -Hydroxymethylcytosine

Answer 15

• Extracted DNA and digested it into single nucleotides
• Then did thin layer chromatography
• All nucleotides separate , found all normal bases and then an extra

Question 16

How did scientists confirm it was 5 -Hydroxymethylcytosine?

Answer 16

• high performance liquid chromatography

- Observed a small peak in the same place of hmdc

Question 17

What happens when there is mutations in the MeCP2 gene?

Answer 17

Causes Rett syndrome , which is a neurodevelopment disorder

Question 18

What happens when MeCP2 binds to 5hmC?

Answer 18

The chromosome Is less condensed and the DNA can be accessed easily

Question 19

What happens when MeCP2 binds to 5Mc?

Answer 19

Causes DNA to be more condensed

Question 20

What happens when the enzyme MeCp2 is mutated?

Answer 20

Can’t bind to 5hmC, but can still bind to 5mc.

Question 21

What is the overall effect of a mutated MeCp2 enzyme?

Answer 21

less gene expression

Question 22

How does bisulphate - sequencing highlight all the methylated cytosines?

Answer 22

• 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.

Question 23

What is a major drawback between bisulphate sequencing?

Answer 23

Cannot differentiate between 5-mC and 5 - hmC

Question 24

What does TAB - sequencing do?

Answer 24

Tet - assisted Bisulfite Sequencing

Question 25

What is the first enzyme in TAB - seq and what does it do?

Answer 25

Glucosyl - transferase . Adds. glucose group only to hmC

Question 26

What is the second enzyme in the TAB-seq and what does it do?

Answer 26

TET enzyme. Converts 5mC into carboxyl cytosine. Not to 5hmC.

Question 27

After bisulphie treatment?

Answer 27

Only the 5mC converted into U then T. 5mhC stays as c