The role of protein-linked DNA breaks and neurolocial disease

Question 1

Why do SSBs primarily impact the nervous system?

Answer 1

Not cycling, have high transcriptional demand.

Limited regenerative capacity

If a neuron dies, you can’t replace it.

Question 2

Why do these breaks not affect cycling cells?

Answer 2

Cycling cells have a back-up pathway.

If single strand breaks are not repaired by this pathway, they can be repaired during replication by homologous recombination (HR).

This process requires presence of sister chromosome.

Can only happen during cell division – where neurons don’t have this pathway – derived for repairing single strand breaks by HR.

Question 3

And why do SSBs primarily impact the cerebellum?

Answer 3

Granular neurons – smallest neurons in the brain.

Cerebellum maybe more affected by those breaks because those breaks have alternative ways of clearing in other parts of the brain – due to genetic differences, epigenetic and gene positioning.

Question 4

Describe an experiment where you can look at breaks accumulating in absence/presence of TDP1

Answer 4

Kinetic experiment.
Induce breaks and then allowed to repair.
A drug that induced the breaks, mainly repaired by TDP1.
TDP1 is important for repairing break.

Question 5

What does the data (look at it) tell us of the kinetic assay

Answer 5

Tells us the TDP1 is unlikely to be the only enzyme that repairs these breaks.

Question 6

How can you examine redundancy and back-up pathways?

Answer 6

Functional complementation – complement the function which is defective in a model organism. How you discover new gene functions.

Question 7

How can we utilise yeast as a model animal?

Answer 7

Have yeast strain and defective in certain pathways – such as don’t have TDP1.

Want to discover activites that can do the function of those strains.

Human genomic library that encodes most of the human genes.

After you plate transformants into a plate.

Question 8

Resistant clones

Answer 8

Plate containing DNA damage agent (e.g CPT).

Start with yeast strain that you delete components you are interested in.

Before you plate them, you transform those yeast strains with a human genomic library.

Plate transformants.

Yeast strain that is defective and doesn’t have resue plasmid will die

Only the yeast/colonies that can take up genes from the library that allow them to repair the damage will grow.

Then select.

listen to these slides again

Question 9

ATTRAP

Answer 9

Foudn t be performing a similar function to TDP1 in these cells (Yeast?)
Cleaves same bond as TDP1 but not as efficiently.

Question 10

TOP1 and TOP2

Answer 10

Same bond but different molarity

Question 11

What happened when researchers engineered the same substrate but swapped the chemistry from 3’ to 5’ and checked the activity.

Answer 11

People have engineered the same substrate but swapped the chemistry from 3’ to 5’ and chekced the activity.

The phosphotyrosine is now on the 5’, instead of 3’ to mimic Top2.

TTRAP is able to cleave the phosphotyrosine bond between DNA and topisomerase but when it is on 5’ terminus which is present in Top2 enzymes.

How can you make sure that this activity seen here is correct and not due to contaminant during the preparation of the enzyme.

Question 12

How can you be sure the biochemical activity is specific to the enzyme you are incubating and NOT a contaminant during the preparation? Experiment you can do to make sure enzymatic activity is due to enzyme that is purified?

Answer 12

Best control is to kill that activity in the same protein and use it as a control. Any activity you have active site. If you look at active site of protein, you try to find the residues that are critical for the activity, going to mutate them. Purify in same way you purified the WT.

Have two proteins – WT and same protein with juston AA change to kill activity.

Run through same assay.

(listen again)

Question 13

How can you confirm 5’-tyrosyl phosphodiesterase activity in cell extracts?

Answer 13

In a cell depleted from TTRAP, you use this substrate with 5’ modification. You incubate with cell extract.

Question 14

How can you confirm 5’-tyrosyl phosphdiesterase activity in cells?

Answer 14

listen to

Question 15

How can you measure DSBs in single cells?

Answer 15

Neural comet assay

Immunofluorescence looking at markers of DSBs.

Use drugs that specifically induce the damage you are interested in

Question 16

What does gammaH2ax mark?

Answer 16

Mark the sites of DSBs – the more spots you have the more DSBs there are.

Question 17

How can you use drugs that specifically induce the damage you are interested in?

Answer 17

Denature the DNA, if you denature the DNA you canmeasure primarily SSBs. If you do not denature DNA you measure DSBs.

Adding Camptothecin (CPT) a toxin which induces these breaks. (induces Top1 type breaks)

Or adding Etoposide (ETOP) (induces Top2 type breaks)

Question 18

Difference between Top1 and Top2

Answer 18

Top1 induces SSBs – bind to 3’ terminus

Top2 induces DSBs – bind to 5’ terminus

Question 19

What has TTRAP been renamed to?

Answer 19

TDP2.

Question 20

Common break caused by Topisomerase 1

Answer 20

Removed by TDP1 on 3’ terminus.
i.e Top1 repaired by TDP1

Question 21

Break caused by Topoisomerase 2

Answer 21

Causes DSB and attaches itself to 5’ terminus.
i.e Top2 is repaired by TDP2.

Question 22

How was the role of TDP1 and TDP1 in Top1 and Top2 breaks discovered?

Answer 22

Functional complementation assay

Question 23

Will deficiency in TDP2 cause neurological disease?

Answer 23

Yes

Question 24

Protein-linked DNA breaks (PDBs)

Answer 24

Phosphodiesterases: TDP1, TDP2
Signalling molecules: ATM, PARP1
Nucleases: XPF, MUS81, CtIP, MRE11

Question 25

Why is TDP2 important

Answer 25

Have nucleases that can potentially remove this linkage and break repaired by homologous recombination in cycling cells or NHEJ which stiches two DNA ends together.

If you have TDP2 you don’t need to break the DNA.

Question 26

Take home message about TDP2 importance

Answer 26

Damage in cycling cells, can repair damage by HR which is error free.

Alternative to this process, is error prone.

In non cycling cells don’t have privelage by HR, only rely on nucleases for NHEJ. This is error prone, but can be error free if you use TDP2. This is because it spares DNA from cutting nucleases.

Question 27

Do PDBs arising from TDP2 defect also cause neurodegeneration?

Answer 27

After sequencing found a mutation in the TDP2 gene. Mutation is predicted to truncate the protein and lose active site of that protein.

Take home messgae – all the consequences of this mutation will give an inactive enzyme.

Question 28

How can you detect TDP2 activity in patient samples?

Answer 28

Oligonucleatide with a 3’ terminus to measure the activity of TDP1.

Can see once you incubate the suspension cells from this irish family, if you look at TDP1 activity you see a band shift. Tells us that all of these patients, controls and siblings have TDP1 activity.

Whereas on left cant see two bands, activity is probably gone.

So they do have TDP1 but not TDP2. Can confirm this by running the protein extract from those patients on a western blot.

Question 29

Three ways of blocking transcription?

Question 30

One way to look at transcriptional effects

Answer 30

is to label the newly synthesised RNA – by EU.

Question 31

Can you measure transcriptional competency?

Answer 31

listen to lecture

Question 32

You have transient Top2 throughout the cycle of the enzyme. What can happen to it? how is this counteracted?

Answer 32

Sometimes this transient event becomes permanent and you end up with a trapped Top2 DNA breaks. Causing DNA damage and that can disrupt transcription. Disturb homeostasis and cause cell death.

To counteract this cells have evovled TDP2, which was previously known as TTRAP and that TDP2 is clever as it can remove those trapped Top2 from DNA and allow normal Top2 function to promote gene expression and neuronal homeostasis.

Question 33

Summary questions