Flashcards in Maintenace of Genomic Integrity Deck (20):
what are the 5 types of DNA repair?
1. direct reversal (enzymatic)
2. base excision
3. nucleotide excision repair
4. homologous recombination repair + non homologous end joining
5. DNA mismatch repair
How may DNA damage occur?
Copying errors during DNA replication (majority)
Exposure to different agents e.g. ionising radiation, UV, tobacco
Types of DNA damage
1. Ethyl methane sulphonate --> ethylation of guanine at position 6
2. UV induced DNA lesions
3. Strand breaks in sugar phosphate backbone
How does ethyl methane sulphonate cause DNA damage?
Ethyl methane sulphonate (drug) usually causes ethylation of guanine at position 6, causing it to pair w thymine instead of cytosine
Hence in replication, GC becomes AT
Drugs that cause ethylation at position 7 good as it kills cell
How does UV cause DNA damage?
Production of thymine dimers & 6-4 photoproducts
- adjacent thymines joined covalently (cyclobutane ring)
- causes DNA distortion + difficulties in replication
- can --> skin tumour (usually basal cell carcinoma)
- DNA replication machinery doesn't detect 2 thymine + inserts random base
What 2 categories can DNA repair be categorised into?
Removal + replacement methods (4 types within)
Types of direct enzymatic reversal
For UV indiced dimers - visible light + photolyase can remove cyclobutane ring --> monomerisation
O6 alkyl guanine - alkyl transferase can remove the alkyl group
For strand breaks in sugar phosphate backbone - ligase enzyme
What is base excision repair used for?
- spontaneous depurination --> apurinic site
- deamination of cytosine
- formation of DNA adducts acter exposure to reactive metabolites
Acts at single base level + only affects 1/2 bases
How does base excision repair occur?
1. Altered base is excised by DNA glycoslyase (breaks sugar-base bond + removes base)
2. Apurinic endonuclease (removes sugar + phosphate), 3. DNA polymerase (synthesises new base)
4. DNA ligase (fixes backbone)
What is a DNA glycoslyase?
Cut glycosidic bonds between sugar + base by recognising DNA adducts
Different glycoslyase recognise diff adducts
CAN'T ACT ON DOUBLE STRANDED DNA!
WHat is nucleotide excision repair used for?
similar to BER but CANNOT act on ssDNA (so used for repairing ds breaks)
non specific - doesn't recognise specific adducts but recognises distortions as a whole
can repair large adducts e.g. thymine dimers unlike BER
Efficient + error free
How does NER occur?
1. endonuclease cuts one side of dimer
2. exonuclease removes many nucleotides either side of damage
3. polymerase synthesised missing nucleotides based on other strand
4. ligase puts it together
Why can't NER occur with ssDNA?
- during replication, gaps are left opposite dimers as they're not recognised
- Over time, gaps filled by daughter strand gap repair mechanism
What is homologous recombination repair + non homologous end joining used for?
Repairing DNA double strand breaks, done by BRCA 1 + 2
- caused by replication errors, ROS from metabolism, ionising radiation!
What does sensitivity to ionising radiation suggest?
Inability to repair DNA double strand breaks, hence sensitivity to radiation used as diagnostic tool
How does homologous recombination repair worK?
1. endonuclease enzyme removes protective factors e.g. 53BPI in exposed end of DNA & forms ssDNS (aided by BRCA1)
2. Rad51 coats ssDNA causing it to pair w homologous DNA duplex --> recombination
- BRCA2 gets Rad51 to the right place for this to occur
- Release of RCA2 & Rad51 triggered by phosphorylation due to DNA damage
How does non homologous end joining occur?
INDEPENDENT OF BRCA1 + Rad51
Example = VDJ recombination
1. DS break detected by Ku70/86
2. Proteins protecting exposed ends e.g. DNA PK joined together by ligase
3. Gaps in DNA filled w random nucleotides hence is error proner (but good for VDJ)
What is DNA mismatch repair used for?
Repairing copying errors where extra base is added/replaced --> base-base mismatch
- can occur due to polymerase slippage during replication
Loss in hereditory non polyposis colorectal cancer
How is homologous recombination repair affected in BRCA2 mutation?
Rad51 is immobile + cannot aid recombination