DNA single-stranded break repair; How does it take place at the molecular level

Question 1

How could you ascertain that APTX process DNA-AMP adducts in vivo (in cells), and therefore apply this concept for other processes.

Answer 1

You have two cells, patients and control.

Expect you have more AMP on the DNA in the patient cells.

Extract genomic DNA, incubate with recombinant aprataxin to release the 5’-AMP.

Therefore, release more 5’-AMP in supernatant.

Measure by mass spec you’ll see higher amount in patient’s supernatant.

Apply concept to anything.

Question 2

SCAN1

Answer 2

A mutation in an enzyme that causes a very similar phenotype to AOA1.

Pathology largely restricted to nervous system (no predisposition to cancer)

Variable onset 15 years

Cerebellar degeneration

Spinocerebellar ataxia

Question 3

What’s mutated in SCAN1?

Answer 3

TDP1 - Tyrosyl-DNA phosphodiesterase 1
Phosphodiester domain.

Question 4

Abortive Topoisomerase 1 events (listen to this slide and look a ppt slides)

Answer 4

Any event that causes misalignment of the 3’-OH
Presence of nearby breaks, collision with transcription or replication machines.

Question 5

What are topisomerases?

What does Topoisomerase cleave?

Answer 5

DNA topoisomerases (or topoisomerases) are enzymes that catalyze changes in the topological state of DNA, interconverting relaxed and supercoiled forms, linked (catenated) and unlinked species, and knotted and unknotted DNA.

Double strand break at collapsed DNA replication fork.
Topoisomerase cleaves the DNA and this intermediate is a covalent link between enzyme, DNA and 3’.
Same enzyme seals the break and then released.
Cleaves, swivels, seals, release.
Can end up with abortive events.

Question 6

Which bond does TDP1enzyme cleave?

Answer 6

Cleaves bond between tyrosine, topoisomerase and 3’ terminus of DNA.

Tyrosine is a phosphotyrosine bond.

Question 7

What’s been known for a long time about TDP1 in yeast? What is the problem?

Answer 7

In yeast is TDP1 removes topoisomerase during DNA replication. Known for long time.

Problem – mutation of this enzyme causes degeneration in cerebellum – cells in cerebellum don’t divide.

Question 8

What does human TDP1 have that yeast TDP1 doesn’t?

Answer 8

Human TDP1 have an N terminus domain whereas yeast does not.

The N-terminus domain allows this protein to jump into single strand breaks and perform this function.

Question 9

What is TDP1 physically coupled with?

Answer 9

Physically coupled to SSBR machine.
Interacts with Lig2Alpha

Point- TDP1 in physically linked to single strand break repair machinery (Lig3,XRCC1).

Question 10

How would you test if TDP1 is required for SSRB in cells? Explain the assay.

Answer 10

How can you measure SSB in cells?

Comet assay – single cell gel electrophoresis

Allows measurement of SS breaks.

Introduce drug with induces SSB’s.

Electrophoretic current will carry fragments that are broken much faster for longer than the DNA bits that are not broken. End up with tail, the longer the tail the more DNA breaks. The more DNA intensity in tail, the more DNA breaks.

Question 11

Difference between Alkaline comet and Neutral comet?

Answer 11

Alkaline comet: SSB and DSB

Neutral comet: DSB

Question 12

What is CPT

Answer 12

Cancer therapy drug

Question 13

Comet tail moment

Answer 13

Measures number of breaks in the cell.

Question 14

Describe the figure given (look at figure)

Answer 14

A, C and D are patients due to the long tails.

Make sure you look at different time points.

For control the breaks are able to be repaired, by timepoint 60min the tail gets smaller. Even if it was long at 20. The patients never decline.

Losing one copy/retaining one copy is sufficient to form function. Therefore, a recessive disorder.

Question 15

Give two examples of endogenous activities that can lead to cerebellar degeneration (SSB repair)

Answer 15

Aprotaxin removes AMP from DNA from 5’ terminus of DNA. That can happen during halted ligation. If you don’t have aprotaxin, you have AOA1 – cerebellar degeneration.

Another example TDP1
TDP1 removes topoisomerase from aborted topoisomerase events. If you don’t have it you also have cerebellar degeneration.
You need these two enzymes to prevent the degeneration of the brain of the cerebellar.

Question 16

Why and how… un-repaired SSB (DNA-TOP1 or DNA-AMP) cause disease?

Answer 16

Stalled transcription:
PARP is important for detection of breaks. Uses NAD to make the polymers (PARP polymers). NAD is important substrate for energy.
If you have excessive PARP activation by SSB you have excessive depletion of NAD. Neurons/cells can die this way.
i.e depleting NAD pool due to hyperactivation of PARP

Which can explain neurodegeneration – explain neurodegenerative defects.
Developmental defects -
Is you have lots of SSBs during development you can have collapsed DNA replication forks. This can also lead to development defects.

Question 17

What can SSB interfere with?

Answer 17

SSBs can infere with replication as well. During brain development you need lots of replication during neurogenesis and that cause problems is not resolving SSBs.

Question 18

Explain 3 ways SSBs mainfest in neuronal disease

Answer 18

1- if they persist long enough and try to replicate this piece of DNA - replication fork runs into SSB - you can see one sitochromatid is completely broken, resulting in a DSB which is lethal, event in replicating cells that is why it hits primarily progenitors. neurodevelopmental defects

2- there are consequences of SSBs in non proliferating cells: deplete NAD component and imbalance of energy metabolism (this one going into further)

3- SSB in active gene, the same way DNA pol will struggle, RNA pol may also struggle, or active signalling mechanisms leading to cell death.

Question 19

How can you examine the hyperactivation of PARP in the lab?

Answer 19

How do you test for high number of SSBs.

Hyperactivate the enzyme- measure the product.

If you have more product, you have hyperactivation.

Or depletion of NAD.

Measure PARP up or NAD down. Prove PARP is activated.

Question 20

How can you make sure the signal is specific (to PARP)?

Answer 20

Delete/remove the enzyme – you should not see the polymer?

Question 21

Identifying if its physiologically significant?

Answer 21

When delete XRCC1 in the mouse, they found that the cells in this molecular layer were gone.

Good model to confirm SSB affected the cerebellum.

Question 22

How can you make sure that the cells which are dying, is because of PARP hyperactivation due to loss of XRCC1 (leading to depletion of NAD, no energy and result in neuronal death)?

Answer 22

Remove important repair protein – you lose cells in cerebellum. Think cell death is because of hyperactivation of enzyme PARP1. How do you confirm this?

Remove the enzyme – PARP1. – cells have appeared again.
Good evidence cells death was due to PARP1 hyperactivation.

Question 23

Will you predict that XRCC1 mutations cause human disease?

Answer 23

How come a deficiency in an essential protein result in a viable human?

Still low levels of XRCC1 being produced in patients.

XRCC1 is so essential, but a mutation that allows the suppression of a very low amount of XRCC1 that it is sufficient for embryonic development, but insufficient for cerebellar function.

Question 24

Figure on hyperactivation of PARP (listen to him explain figure)

Question 25

Listen to summary questions