Protein-linked DNA breaks and neurological disease

Question 1

Why do SSBs primarily impact the nervous system? (5)

Answer 1

• Non-cycling cells so not able to use homologous recombination as a backup repair pathway
• High transcriptional demand
• High oxidative stress
• SSBs can accumulate, block transcription and cause cell death
• No regenerative capacity

Question 2

Why don’t SSBs affect cycling cells?

Answer 2

Cycling cells can use homologous recombination as a backup repair pathway which requires sister chromatids that are only available during cell division

Question 3

Why do SSBs primarily impact the the cerebellum? (2)

Answer 3

• 40% of the total neuronal population of the brain is in the cerebellum
• May also be differences in gene expression/epigenetic mechanisms/gene positioning in the cerebellum compared to other brain areas but unknown

Question 4

How can you examine redundancy?

Answer 4

Functional complementation

Question 5

What is functional complementation? (4)

Answer 5

• Transform human genomic library into TDP1 -/- yeast
• Plate onto medium containing CPT (DNA damage agent) and select resistant clones
• TDP1 -/- yeast will die on medium containing CPT as won’t be able to repair induced SSBs and only yeast that has taken up the human genomic library will survive as this encodes other genes that can complement TDP1 function
• Isolate resistant clones, extract plasmid DNA and sequence to identify complementary genes

Question 6

How can you confirm that TTRAP is performing a similar function to TDP1? (5)

Answer 6

• Cleavage assay because TDP1 cleaves the covalent bond between the 3’ phosphate and the tyrosine residue of Top1
• Engineer the mimic DNA substrate (dsDNA with nick in top strand and tyrosine covalently bound, radiolabel)
• Purify TDP1 and TTRAP and incubate with substrate
• Product runs further when adding TDP1 it can repair the damage
• Both TDP1 and TTRAP can cleave the substrate but TTRAP is less efficient than TDP1 because requires greater concentration for less cleavage

Question 7

What is the difference between Top1 and Top2? (2)

Answer 7

• Top1 generate SSBs and the tyrosine group covalently bonds to the 3’ end of the DNA break
• Top2 generates DSBs and the tyrosine group covalently bonds to the 5’ end of the DNA break

Question 8

How can you be sure the biochemical activity is specific to the enzyme you are incubating and not a contaminant from purification?

Answer 8

Mutate an amino acid in the active site and see if the activity changes

Question 9

How do you confirm 5’-tyrosyl phosphodiesterase activity (Top2 break resolution) in patient cell extracts? (3)

Answer 9

• Extract protein from patient and control cells
• Add proteins to same substrate (Top2 break mimic, dsDNA with covalently attached tyrosine to 5’ end)
• Observe differences in substrate cleavage: patient sample shows little to no cleavage over time in comparison to control sample suggesting the patient has a TTRAP mutation

Question 10

What happens when you incubate TDP1 and TTRAP with a mimic of Top2 DNA break? (2)

Answer 10

• TDP1 can’t resolve the Top2 break (dsDNA with covalently attached tyrosine to 5’ end) but TTRAP can
• TTRAP cleavage is absent if you mutate an amino acid in the active site which suggests the activity is specific to TTRAP and not a contaminant

Question 11

How do you measure DSBs in single cells? (3)

Answer 11

• Neutral comet assay
• Immunofluorescence for markers of DSBs e.g. γH2Ax
• Use drugs that specifically induce the damage you are interested in

Question 12

What is γH2Ax? (2)

Answer 12

• Histone protein encoded by H2AFX gene
• Phosphorylated form is γH2Ax (S139) which forms when DSBs appear so marker of DSBs

Question 13

What kind of DNA damage is induced by camptothecin (CPT)?

Answer 13

SSBs with Top1 covalently bound 3’

Question 14

What kind of DNA damage is induced by etoposide (ETOP)?

Answer 14

DSBs with Top2 covalently bound 5’

Question 15

What is TTRAP? (2)

Answer 15

• Now named TDP2
• 5’-tyrosyl phosphodiesterase that repairs Top2 DSBs

Question 16

What is TDP1?

Answer 16

Tyrosyl phosphodiesterase that repairs Top1 SSBs

Question 17

What disease is caused by TDP1 mutation?

Answer 17

SCAN1

Question 18

What disease is caused by TDP2 mutation?

Answer 18

Spinocerebellar ataxia, autosomal recessive type 23 (SCAR23)

Question 19

What is caused by SCAR23? (4)

Answer 19

• Defects in nuclear DNA DSB repair
• Developmental delay
• Epilepsy
• Ataxia

Question 20

What causes AOA1?

Answer 20

APTX gene mutation

Question 21

What causes AOA2?

Answer 21

SETX gene mutation

Question 22

What causes AOA4?

Answer 22

PNKP gene mutation

Question 23

What causes AOA-XRCC1?

Answer 23

XRCC1 gene mutation

Question 24

What causes SCAN1?

Answer 24

TDP1 gene mutation

Question 25

What causes SCAR23?

Answer 25

TCP2 gene mutation

Question 26

How do you treat inherited diseases affecting DNA repair genes? (8)

Answer 26

- No cure - Physical therapy - Educational support for learning difficulties - Speech therapy - Genetic testing - Prenatal testing - CRISPR gene editing - Stem cell therapy

Question 28

What are optical tweezers? (2)

Answer 28

- Infrared lasers that can trap and move small particles e.g. 4um beads - Exerts force on the particle to contain it within the laser which can be measured

Question 29

How can you use optical tweezers to look at DNA? (5)

Answer 29

- Use streptavidin coated beads that stick to biotin - Put biotin at the ends of DNA - Can apply flow and trap DNA between 2 beads - Can stretch the DNA and measure the force generated by the DNA pulling on the beads - Can use damaged DNA/add DNA damaging agents and reconstitute repair pathways

Question 30

How can you visualise ssDNA break repair steps using optical tweezers? (4)

Answer 30

- Trap a piece of DNA with a ss break and incubate with repair proteins - E.g. PARP labelled with yellow fluorescence protein binding to ss break site, timing of binding and turnover - E.g. apply dsDNA-specific dye to visualise polymerase gap filling of ss break - E.g. RPA labelled with eGFP to visualise ss regions

Question 31

How can topoisomerase 2A be studied using optical tweezers? (3)

Answer 31

- Fluorescently label Top2A to visualise binding to dsDNA - Use 2 DNA strands to create a DNA tangle and incubate with Top2A and ATP - Visualise Top2A resolving the tangle

Question 32

How does topoisomerase 2A work? (6)

Answer 32

- Top2A untangles DNA as it is copied during cell division - Dimer with 3 gate regions - N terminal gate opens and can capture 2 pieces of crossed DNA - One strand of the crossed DNA is cleaved forming a DSB with Top2A covalently attached at the break site - Other strand is passed through the broken strand to resolve the tangle and the DSB site is repaired - If Top2A doesn't work properly can generate genomic instability

Question 33

How does etoposide work? (3)

Answer 33

- Induces DSBs by inhibiting re-ligation of the break in the Top2 mechanism - Inhibits a single tangle resolution by Top2A by ~50% - Inhibits 4 tangles by 100% as more tangles gives more chance to inhibit by trapping Top2A at the break site

Question 34

What is TDP2? (3)

Answer 34

- Tyrosyl-DNA phosphodiesterase 2 - Removes covalently attached Top2A at DSBs and releases the broken and unbroken strand from the tangle - Broken piece can then be repaired correctly