Lecture 19 - DNA damage & repair

Question 1

What does DNA do?

Answer 1

DNA encodes the genetic instructions used in the development and functioning of all known living organisms.

The stability of DNA is therefore essential for cell survival

Question 2

What is our most robust defence against cancer?

Answer 2

Genetic stability

Question 3

What is the only biological macromolecule to repair?

Answer 3

DNA - all others replaced

Question 4

What are endogenous sources that put cells under constant attack?

Answer 4

• reactions with other molecules within the cell
• hydrolysis, oxygen species, by-products of metabolism

Question 5

What are exogenous sources that put cells under constant attack?

Answer 5

• UV, X-rays, carcinogens, chemotherapeutics
  Other molecules (proteins/lipids) are also susceptible to such insults, BUT these do not harbour (much) information can be replaced.

Question 6

What are examples of endogenous DNA damage?

Answer 6

• Depurination (creating an abasic site)
• Deamination
• methylation
• replication errors

Question 7

What are examples of exogenous DNA damage?

Answer 7

• Pyrimidine dimers
• Single strand breaks
• Double strand breaks
• Interstrand crosslinks

Question 8

What types of DNA damage effect the nucleotide bases of the DNA molecule?

Answer 8

• Depurination (creating an abasic site)
• Deamination
• methylation
• replication errors

Question 9

What types of DNA damage effects one strand of the DNA helix?

Answer 9

• Pyrimidine dimers
• Single strand breaks

Question 10

What types of DNA damage effects both strand of the DNA helix?

Answer 10

• double strand breaks
• interstrand crosslinks

Question 11

What is deamination?

Answer 11

Removal of the amino group by hydrolysis results in changes to the DNA bases

Question 12

What is a transition mutation?

Answer 12

• purine-purine transition/pyrimidine-pyrimidine transition

e.g. A <–> G, C <–> T

Question 13

What is a transversion mutation?

Answer 13

• purine-pyrimidine transition
• E.g. A <–> C, G <–> T

Question 14

What type of mutation is more common - transition or transversion?

Answer 14

Transition mutations more likely than transversion

Substituting a double ring structure for another double ring structure is more likely than substituting a double ring a single ring and vice versa.

Question 15

What is depurination (abasic site)?

Answer 15

• The N-glycosidic bind is a common substrate for hydrolysis = abasic site (AP site)
• More frequent at purine bases - approx 18,000 per genome per day

Question 16

What does a failure to undertake DNA repair result in?

Answer 16

Results in mutation

Question 17

What type of mutation has the most potential to be damaging to the cell?

Answer 17

Frameshift mutation - more so than substitution mutations

Question 18

What do frameshift mutations generate?

Answer 18

Missense proteins

Question 19

What does UV light induce?

Answer 19

• The formation of pyrimidine dimers - distorting DNA
• UV can also cause interstrand DNA crosslinks and DNA-protein crosslinks

Both are highly toxic to the cell as they block replication & transcription

Question 20

What types of damage affect the phosphate backbone?

Answer 20

• single strand break
• double strand break

Question 21

What are examples causes of double strand break inducers?

Answer 21

• X-rays
• Ionising radiation
• Topoisomerase II inhibitors

Question 22

What are examples causes of double strand break inducers?

Answer 22

• Reactive oxygen species
• Hydroxyurea
• Camptothecin

Question 23

How can DNA repair itself?

Answer 23

Each type of DNA damage lesion is repaired by a specific repair pathway

Question 24

What is the process of Base excision repair (BER)?

Answer 24

Repairs base damage - e.g. abasic sites, amination. Base-flipping strategy to identify errors

Question 25

What is the process of Nucleotide excision repair (NER)?

Answer 25

- Repairs damage when more than one base is involved - e.g. pyrimidine dimers (caused by UV) - Involves the excision of short patches of single stranded DNA to remove the affected bases

Question 26

What is translesion synthesis?

Answer 26

Translesional DNA polymerases can replicate highly damaged DNA They lack: - Precision in template recognition and substrate base choice - Exonucleolytic proof-reading activity They cause: - Most base substitution and single nucleotide deletion mutations

Question 27

What 2 mechanisms exist to repair double strand breaks?

Answer 27

- Non-homologous end joining (NHEJ) - Homologous recombination (HR)

Question 28

Describe features of non homologous end joining (NHEJ)

Answer 28

- Error-prone - Restricted to G1 phase - Usually results in the loss of nucleotides surrounding the break site - Important genetic information may be lost

Question 29

Describe features of homologous recombination (HR)

Answer 29

- error-free repair - occurs only in S-phase - uses intact sister chromatid as a template - double strand break is accurately repaired

Question 30

What are the 3 places where DNA damage is detected and acted upon to STOP the cell cycle?

Answer 30

- G1 - Entry to S-phase - Entry into mitosis (also check for chromosome non-disjunction)

Question 31

What type of repair occurs in G1 repair?

Answer 31

Non-homologous end joining

Question 32

What type of repair occurs in G2 repair?

Answer 32

Homologous recombination

Question 33

How is damage detected?

Answer 33

- ATM/ATR get activated and associate with the site of DNA damage - This activates other kinases to block the cell cycle - P53 is stabilized (phosphorylated) and activates P21 - P21 renders the G1/S-CDK and S-CDK complexes INACTIVATED, this preventing cycle progression - DNA is then repaired

Question 34

What happens if repair is no possible?

Answer 34

- Apoptosis

Question 35

What is an example of predisposition disease associated with defect in NER (nucleotide excision repair)?

Answer 35

- xeroderma pigmentosum - an autosomal recessive disease - 1 in 250,000 in Europe - 1 in 40,000 in Japan - 2000-fold increased risk of skin cancer - Skin cancer occurs 50 years earlier - mean onset - 12 years old

Question 36

What is an example of cancer and defects in double strand break repair?

Answer 36

- 10% breast cancer is inherited - 80-90% of all inherited breast cancers BRCA1/2 associated (also associated with ovarian and prostate cancer) - BRCA1/2 carriers have a 80% lifetime risk (10x higher than normal)

Question 37

Why are mutations associated with cancer?

Answer 37

BRCA2 deficient cells - exhibit genomic instability, are sensitive DNA damaging agents and are defective in homologous recombination --> predisposition to cancer

Question 38

Describe features of cancer cells?

Answer 38

- Very fast growing - replicate in the presence of damage - Often have defects in repair - unable to repair everyday DNA damage leading to mutations

Question 39

What is the Achilles heel of cancer?

Answer 39

DNA damaging agents are key to cancer treatments - target defects in repair to improve the effectiveness of existing therapies Use interactions between repair pathways to increase death specifically in cancer cells - synthetic lethality

Question 40

How does synthetic lethality work?

Answer 40

- Synthetic lethality occurs when two genetic mutations that are not lethal on their own cause cell death when combined. - It is used in cancer treatment, where drugs are designed to target weaknesses in cancer cells (e.g., BRCA mutations) by exploiting synthetic lethality to kill the cancer cells while sparing normal cells.