DNA SSBR Flashcards
(20 cards)
What is the most common type of DNA break? (2)
- Single strand breaks
- Double strand breaks are more toxic but less common
What are ‘clean’ DNA ends?
5’ phosphate and 3’ OH groups that are easily re-ligated
What are the steps of single strand break repair (SSBR)? (4)
- Damage detection
- End processing
- Gap filling
- Ligation
What is damage detection in SSBR?
Poly(ADP) ribose polymerase (PARP) detects the damage and creates poly(ADP) ribose (PAR) chains at the break site which signals to the cell that repair is needed
What is end processing in SSBR? (3)
- DNA ends in a single strand break (SSB) are ‘dirty’ and need to be cleaned up
- Each type of DNA end has a specific set of proteins for processing
- Involved XRCC1 scaffold protein for recruitment of other processing proteins
What are the 2 methods of gap filling in SSBR?
- Short-patch repair done by polymerase beta which accurately synthesises DNA across the break site
- Long-patch repair done by polymerase delta/epsilon which is less accurate and overshoots the break site creating a 5’ flap which is removed by Fen1 nuclease
What is ligation in SSBR? (2)
- Nicks in the phosphate backbone are re-joined
- Ligase 3 is in short-patch repair and ligase 1 is in long-patch repair
How does the yeast two hybrid system work? (3)
- A reporter gene has a promoter with a binding site for the Gal4 transcription factor
- Fuse the bait protein (e.g. XRCC1) to the Gal4 binding domain and fuse the prey protein to the Gal4 activating domain
- If the bait and prey proteins interact, the Gal4 domains come close together enough to cause expression of the reporter gene which allows the yeast to grow on dropout media/beta galactosidase expression causing a colour change
How can you confirm the results of a yeast two hybrid experiment?
Reverse the bait and prey proteins and see if you get the same result
What is the structure of Xip1/aprataxin? (4)
- Forkhead associated domain (FHA) for binding to other proteins i.e. XRCC1
- Nuclear localisation sequence (NLS)
- Histidine triad (HIT) domain active site
- Zinc finger binding motif for DNA binding
What is Xip1? (2)
- Involved in SSBR
- Same gene/protein as aprataxin which is mutated in ataxia oculomotor apraxia-1 (AOA1)
What is AOA1? (5)
- Ataxia oculomotor apraxia-1
- Causes variable mental retardation, ocular motor apraxia, cerebellar degeneration, spinocerebellar ataxia
- Pathology restricted to the nervous system with no predisposition to cancer
- Affects 3 million worldwide
- Early onset 1-16 years
Where are mutations in aprataxin located in AOA1?
Cluster in the HIT active site domain
How do you isolate a protein of interest in order to study its enzymatic function in the lab? (3)
- Put protein of interest sequence (i.e. aprataxin) into a plasmid vector with a His tag
- Transform bacteria with the plasmid and induce protein expression with IPTG which removes repressors from the lac operon to allow gene expression
- Collect and purify the protein of interest by doing cell lysis and putting the lysate through a nickel column which pulls out the protein via binding to the His tag
How do you know which in vitro substrate to use when studying enzymatic activity of aprataxin? (2)
- HIT proteins are known to interact with nucleotide adducts
- Systematically mix purified protein with different DNA adducts to see which interact
How can you determine the function of aprataxin? (2)
- Incubate aprataxin with DNA with an AMP adduct and run on a denaturing gel
- Increasing concentrations of aprataxin cause the DNA to run at a smaller size suggesting it repairs the AMP adduct
How do DNA-AMP adducts arise in cells? (4)
- During normal ligation of compatible DNA ends, DNA ligase uses energy from ATP to adenylate itself
- Ligase transfers the AMP to the 5’ end of the SSB (phosphate end)
- 3’ OH forms a phosphodiester bond with the phosphate group via nucleophilic attack to seal the nick
- 3’ end of SSB is often not clean so can’t form a phosphodiester bond with the 5’ AMP which results in a build up of DNA-AMP adducts
What is the role of aprataxin?
Remove DNA-AMP adducts to allow for SSBR
What endogenous event can lead to abortive ligations resulting in DNA-AMP adducts?
Ribonucleotides being incorporated into the DNA duplex instead of deoxyribonucleotides (ribose contamination)
How is ribose contamination fixed? (3)
- RNase H2 detects the ribose and tags them resulting in an AMP group replacing the 5’ phosphate group
- Aprataxin cleaves off the AMP and allows the cell time to recognise the ribose and remove it via ribonucleotide excision repair (RER)
- Leads to genomic instability and cell death if not fixed as seen in AOA1 when there is no aprataxin
