SSBR factor defects and human disease Flashcards
(25 cards)
What disease is caused by Aprataxin (APTX) mutation?
Ataxia oculomotor apraxia type 1 (AOA1)
What are the symptoms of AOA1? (5)
- Variable mental retardation
- Ocular motor apraxia
- Cerebellar degeneration
- Spinocerebellar ataxia
- Pathology largely restricted to nervous system, no predisposition to cancer
How do you determine if AOA1 patients with APTX mutations have more AMP-DNA? (3)
- Extract DNA from patient fibroblast sample
- Add APTX to the sample so it will cleave the AMP adducts
- Do mass spec to see AMP levels in the sample and compare to a healthy normal control
What is SCAN1? (2)
- Spinocerebellar ataxia with axonal neuropathy
- Caused by mutation of TDP1
What is TDP1? (2)
- Tyrosyl-DNA phosphodiesterase 1
- Enzyme involved in repairing DNA-TOP1 breaks
What are the symptoms of SCAN1? (4)
- Loss of motor coordination
- Cerebellar degeneration
- Spinocerebellar ataxia
- Like AOA1 pathology is largely restricted to the nervous system, no predisposition to cancer
What is the structure of TDP1? (2)
- Large N terminal domain
- Phosphodiesterase domain
What mutation is seen in SCAN1 patients?
H493R mutation at the active site of the phosphodiesterase domain
What is the function of Top1? (2)
- Topoisomerase 1 introduces a transient break in one of the DNA strands to release supercoil tension which is quickly resealed
- If the reaction isn’t complete due to presence of nearby breaks/collision with transcription or replication machinery, a covalent bond is formed between the tyrosine residue of Top1 and a 3’ phosphate in the DNA break site
What is the function of TDP1?
Breaks the covalent bond between the Top1 tyrosine and 3’ DNA phosphate
What is the difference between human and yeast TDP1?
Yeast TDP1 doesn’t have the N terminal domain but does have the phosphodiesterase domain
How is TDP1 physically coupled to SSBR machinery?
Binds to Lig3 which binds to XRCC1
What is a comet assay? (5)
- Single cell gel electrophoresis for measuring DNA damage
- Isolate cells and embed into agarose on a cover slip
- Add lysis buffer to release the DNA and run electrophoresis
- Broken DNA runs further than unbroken DNA
- Stain with fluorescent dye
What do healthy vs damaged cells look like in a comet assay? (3)
- Healthy cells with normal DNA remain intact
- Damaged cells with broken DNA look smeared with a ‘head’ and a ‘tail’
- Calculate tail moment length to find how much DNA damage is in the cell (bigger = more damage)
What are the types of comet assay? (2)
- Alkaline conditions for measuring SSBs and DSBs because DNA strands separate
- Neutral conditions for measuring DSBs because DNA strands remain together
What is CPT? (2)
- Camptothecin
- Top1 inhibitor
What are examples of types of unrepaired SSBs that can cause disease? (2)
- DNA-TOP1
- DNA-AMP
How can SSBs cause neurodegenerative defects? (2)
- Stalled transcription can cause not enough protein being made and lead to cell death
- Cause excessive PARP1 activation (detects SSBs) causing NAD+ depletion as this is used to make PAR chains, leading to cell death
How can SSBs cause neurodevelopmental defects?
Cause collapsed DNA replication
How can you examine excessive PARP activation in the lab? (2)
- Substrate (NAD) goes down or the product (PAR) goes up
- E.g. PAR chain antibody immunofluorescence
How can you make sure an antibody immunofluorescence signal is specific?
Delete PARP1 and observe if the signal is still there or not
What is the impact of XRCC1 KO? (2)
- Induce lots of ss breaks and PARP overactivation
- Delete PARP1 aswell and signal disappears (shows signal is specific)
What is the impact of XRCC1 KO in mouse cerebellum? (3)
- Death of interneurons due to increased ss breaks
- XRCC1 PARP1 double KO restores normal interneuron density and reduces cerebellar ataxia
- Means that the neuronal death is due to PARP1 overactivation in response to ss breaks as deleting PARP1 rescues the phenotype
What phenotype is caused by XRCC1 mutation?
Cerebellar degeneration and loss of motor coordination
