Maintenace of Genomic Integrity

Question 1

what are the 5 types of DNA repair?

Answer 1

1. direct reversal (enzymatic)
2. base excision
3. nucleotide excision repair
4. homologous recombination repair + non homologous end joining
5. DNA mismatch repair

Question 2

How may DNA damage occur?

Answer 2

Copying errors during DNA replication (majority)
Spontaneous depuration
Exposure to different agents e.g. ionising radiation, UV, tobacco

Question 3

Types of DNA damage

Answer 3

1. Ethyl methane sulphonate –> ethylation of guanine at position 6
2. UV induced DNA lesions
3. Strand breaks in sugar phosphate backbone

Question 4

How does ethyl methane sulphonate cause DNA damage?

Answer 4

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

Question 5

How does UV cause DNA damage?

Answer 5

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

Question 6

What 2 categories can DNA repair be categorised into?

Answer 6

Enzymatic repair

Removal + replacement methods (4 types within)

Question 7

Types of direct enzymatic reversal

Answer 7

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

Question 8

What is base excision repair used for?

Answer 8

• 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

Question 9

How does base excision repair occur?

Answer 9

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)
3. DNA ligase (fixes backbone)

Question 10

What is a DNA glycoslyase?

Answer 10

Cut glycosidic bonds between sugar + base by recognising DNA adducts
Different glycoslyase recognise diff adducts
CAN’T ACT ON DOUBLE STRANDED DNA!

Question 11

WHat is nucleotide excision repair used for?

Answer 11

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

Question 12

How does NER occur?

Answer 12

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

Question 13

Why can’t NER occur with ssDNA?

Answer 13

• during replication, gaps are left opposite dimers as they’re not recognised
• Over time, gaps filled by daughter strand gap repair mechanism

Question 14

What is homologous recombination repair + non homologous end joining used for?

Answer 14

Repairing DNA double strand breaks, done by BRCA 1 + 2

- caused by replication errors, ROS from metabolism, ionising radiation!

Question 15

What does sensitivity to ionising radiation suggest?

Answer 15

Inability to repair DNA double strand breaks, hence sensitivity to radiation used as diagnostic tool

Question 16

How does homologous recombination repair worK?

Answer 16

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

Question 17

How does non homologous end joining occur?

Answer 17

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)

Question 18

What is DNA mismatch repair used for?

Answer 18

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

Question 19

How is homologous recombination repair affected in BRCA2 mutation?

Answer 19

Rad51 is immobile + cannot aid recombination

Question 20

What can defects in DNA mismatch repair lead to?

Answer 20

mutator phenotype - role in tumour progression, not initiation