Transcription associated DNA damage Flashcards
IS DSB or SSB more common?
SSB
Principle of repairing SSBs
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.
4 steps in SSB repair
- DNA damage detection - Cell to identify break
- End processing - Knife with different blades
- Gap filling
- Ligation.
How is DNA damage detected?
First step is damage detection by enzyme called PARP, they mark the site of a DNA break. To flag the problem to the cell.
Consequence of end processing?
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.
How do you search for unknown interactions?
Yeast-2-hybrid.
Immunoprecipitation.
Positive and negative controls for Y2H
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.
Once interaction found, what is next step?
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
What gene/prteoin is muated in Ataxia Oculomotor Apraxia-1 (AOA1)
XIP1 and Aprataxin are the same gene/protein
What is AOA1?
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.
How can you find out what the protein does?
IPTG induction
Cell lysis
Purification by affinity column.
Common way to tag protein?
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.
How do you decide what in vitro substrate to use?
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.
How can you determine the function of an unknown protein?
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.
By analysis what were researchers able to show about aprataxin
Aprataxin is able to cleave covalent bonds between DNA and AMP. Does this in a concentration dependent manner.
What is ligation fundamental for?
All of the repair events in SS breaks.
How does ligation happen?
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.
Generally - how does ligase work?
Use ATP, bring AMP of the ligase,
Transferring AMP to 5’ terminus of DNA
Quickly transfers the last step, seals it.
Why is the removal of AMP important
If you don’t have this removal - cerebellar degeneration occurs.
What happens if there is not a hydroxyl present when repair is happening?
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.
What intermeidate can be generated if cycle doesnt complete
it stalls and generates and intermediate, AMP-DNA intermediate, that needs to be fixed before the repair is completed.
Therefore, why do we have aprataxin?
it is there to reset the ligation cycle, buy for time for ligase to try again – hope the 3’ terminus is now fixed.
What intermediate can accumulate and lead to abortive ligation (endogenous threat)
(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.
Therefore what is the mean concept for when activity is not complete.
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.
Look at the summary questions and listen to them
