Transcription associated DNA damage

Question 1

IS DSB or SSB more common?

Answer 1

SSB

Question 2

Principle of repairing SSBs

Answer 2

In a situation of SSB, DNA loses its termini, to bring two DNA ends together you need a 3’ hydroxyl and a 5’ phosphate to form the phosphodiester bond. 3’ is no longer hydroxyl and 5’ is no longer phosphate.

Question 3

4 steps in SSB repair

Answer 3

1. DNA damage detection - Cell to identify break
2. End processing - Knife with different blades
3. Gap filling
4. Ligation.

Question 4

How is DNA damage detected?

Answer 4

First step is damage detection by enzyme called PARP, they mark the site of a DNA break. To flag the problem to the cell.

Question 5

Consequence of end processing?

Answer 5

Once removed you have hydroxyl and phosphate. During any cutting/trimming, you run risk of losing DNA. Cutting may not be precise. Sometime enzymes cut a bit further, you lose nucleotides which you need to replace – done by DNA polymerase.

Question 6

How do you search for unknown interactions?

Answer 6

Yeast-2-hybrid.
Immunoprecipitation.

Question 7

Positive and negative controls for Y2H

Answer 7

Control plate – empty vector.

Beta-gal = blue means interaction.

False positive – if you put with empty vector you will see interaction.
You know you have a positive interaction as the bait on its own (or with empty vector) does not grow/show a response/ activate interaction.

Question 8

Once interaction found, what is next step?

Answer 8

Blast test.
Look at nucleotide, does it look like anything familiar.

Found XIP1 has FHA domain – which bind phosphorylated proteins.

Zinc finger -likly that protein binds to DNA.

Then looks for another type of blast

Question 9

What gene/prteoin is muated in Ataxia Oculomotor Apraxia-1 (AOA1)

Answer 9

XIP1 and Aprataxin are the same gene/protein

Question 10

What is AOA1?

Answer 10

What is AOA1?

3 million affected wide world

Early onset 1-16 years

Pathology largely restricted to nervous system

Variable mental retardation

Ocular motor apraxia – inability to coordinate eye coordination

Cerebellar degeneration

Spinocerebellar ataxia.

Get neurological symptoms but don’t get cancer.

Question 11

How can you find out what the protein does?

Answer 11

IPTG induction
Cell lysis
Purification by affinity column.

Question 12

Common way to tag protein?

Answer 12

When you clone the DNA from bacteria, you put a tag called histidine which binds to nickle columns.

Or biotin which binds to streptavin (spelling?) well. Can then purify your protein from bacteria protein.

Question 13

How do you decide what in vitro substrate to use?

Answer 13

Start thinking about what this protein does.

Use substrate to test enzymatic activity of this protein.

Normally substrate comes from looking at active site.

All mutation in this disease occur in this hit domain. – activity likely important

Look at what HIT domain does, HIT protein cleave ADP, AMP attached to DNA etc.

Question 14

How can you determine the function of an unknown protein?

Answer 14

Need to visualise on denaturing gel.

Separate strands from eachother.

Be able to see migration.

Label with radioactive phosphorus.

Look for band shift. If protein is able to cleave AMP, then you will see a band shift. If it doesn’t, you won’t see a band shift.

Question 15

By analysis what were researchers able to show about aprataxin

Answer 15

Aprataxin is able to cleave covalent bonds between DNA and AMP. Does this in a concentration dependent manner.

Question 16

What is ligation fundamental for?

Answer 16

All of the repair events in SS breaks.

Question 17

How does ligation happen?

Answer 17

DNA ligase transfers AMP to itself using ATP.
Then transfers AMP to the 5’ terminus of DNA.
The same DNA ligase then catalyses this attack of hydroxyl on the bonds - i.e seals the snip.

Question 18

Generally - how does ligase work?

Answer 18

Use ATP, bring AMP of the ligase,

Transferring AMP to 5’ terminus of DNA

Quickly transfers the last step, seals it.

Question 19

Why is the removal of AMP important

Answer 19

If you don’t have this removal - cerebellar degeneration occurs.

Question 20

What happens if there is not a hydroxyl present when repair is happening?

Answer 20

The enzyme buys more time for an hydroxyl to appear and for cells to fix 3’ terminus.

This is why you get a build up of AMP DNA adducts if you dont have aprataxin.

Question 21

What intermeidate can be generated if cycle doesnt complete

Answer 21

it stalls and generates and intermediate, AMP-DNA intermediate, that needs to be fixed before the repair is completed.

Question 22

Therefore, why do we have aprataxin?

Answer 22

it is there to reset the ligation cycle, buy for time for ligase to try again – hope the 3’ terminus is now fixed.

Question 23

What intermediate can accumulate and lead to abortive ligation (endogenous threat)

Answer 23

(listen to again to see what he means)
Ribose.
DNA-AMP adducts - once intermediate is formed it jumps creating a DNA-AMP intermediate. That intermediate can accumulate and cause AOA1 genomic instability in cerebellum. You need aprataxin to snip, buying more time for this pathway to fix the ribose.

Question 24

Therefore what is the mean concept for when activity is not complete.

Answer 24

Normal activity that is not complete, and you need the repair enzyme to reset back again. To give cell more chance to fix it. Or you end up with build up of breaks. Causing cerebellar degeneration.

Question 25

Look at the summary questions and listen to them