DNA Repair

Question 1

What are the main components of UV light and what are their wavelengths?

Answer 1

UVC - 100-290nm
UVB - 290-320nm
UVA - 320 -400nm

Question 2

What is the most damaging UV forms?

Answer 2

UVC but none of this reaches the earths surface so UVB

Question 3

Which is the most penetrating form of UV radiation?

Answer 3

UVA - dermins
UVB - epidermis
UVC - blocked by atmosphere

Question 4

During sun exposure, how many how many CPDs or 6-4 photoproducts per second form in each epidermal cell?

Answer 4

50-100 CPDs or 64PPs form per second in each exposed epidermal cell

Question 5

What form of damage does base excision repair repair?

Answer 5

Damaged or abnormal bases e.g. Us, 8-oxoguanine arising from ROS

Question 6

Describe the process of base excision repair

Answer 6

• Glycosylase enzyme recognises damage
• This removes the damaged base leaving AP site
• AP endonuclease cleaves the phosphodiester bond 5’ to the site
• DNA polymerase beta (lyase activity then removes the P and dR)
• DNA polymerase beta fills the gap
• DNA ligase seals the gap

Question 7

What are the two subtypes of nucleotide excision repair?

Answer 7

GG-NER

TC-NER

Question 8

How is DNA damage detected in Global Genome Nucleotide Excision Repair.

Answer 8

XPC protein looks for single stranded regions of DNA (indicative of bulky lesions)
UV-DDB binding protein is needed to help recognise CPDs

Question 9

How is DNA damage detected in transcription coupled NER?

Answer 9

By the stalling of RNAPII and then recruitment of CSA and CSB

Question 10

What is the mutation signature for UV light?

Answer 10

> 60% of all UV induced mutations are C–>T transitions
95% of C–>T transitions affect Cs next to another pyrimidine
5% mutations are CC –> TT transitions

Question 11

How does UV light cause C–>T transitions?

Answer 11

Cytosines within a pyrimidine dimer undergo deamination to uracil approximately 10^6 more efficiently

Question 12

Describe the xeroderma pigmentosum

Answer 12

Caused by recessive mutations (usually nulls) in genes involved in GG-NER such as XPC. Symptoms are:

• Heightened sensitivity of skin and eyes to sunlight
• 1000x increase risk of developing cancer
• neurological impairment in 20-30% of patients
• Good life expectancy

Question 13

Cockayne’s syndrome is due to mutations in what genes?

Answer 13

Genes involved in TC-NER (CSA AND CSB)

Question 14

Describe the symptoms of Cockaynes syndrome?

Answer 14

• Extreme sensitivity to sunlight
• Death of non-proliferating cells
• Neurological degeneraton
• Premature ageing due to death of non-proliferating cells
• Life expectancy 12 years

Question 15

Why is cockaynes syndrome only seen in non-dividing cells?

Answer 15

In dividing cells then helicases will strip away the RNAP and this will not happen in non divding cell, so a dividing cell gets another chance at repairing the damage.

Question 16

Hazards of Human Spaceflight

Answer 16

Markus Lobrich and Penny A Jeggo - showed upregulation of DNA repair genes in astronauts

Question 17

Describe the process of Non Homologous End Joining (NHEJ)

Answer 17

Double stranded breaks in DNA are repaired by NHEJ

KU binds to broken ends of DNA
KU recruits DNA protein kinase. These two things keep the ends of the DNA molecule together

KU recruits nucleases, polymerases and ligases to seal the gaps

Question 18

What is one limitation of non homologous end joining?

Answer 18

NHEJ introduces deletions into the DNA

Question 19

Describe the process of Base Excision Repair

Answer 19

i) Damage specific (e.g. for uracil) glycosylase enzymes slide along the DNA and recognise and excise damaged bases, generating apurinic or apyrimidinic sites
ii) AP (apurinic or apyrimidinic) endonuclease cleaves phosphodiester bond 5’ to the AP site making a nick in the DNA molecule
iii) In humans, lyase (breaks a bond) activity of DNA polymerase β may remove the deoxyribose and phosphate previously linked to the damaged base
iv) DNA polymerase β replaces missing nucleotide, DNA ligase seals the gap

Question 20

Describe what happens following either recognition of damage using either the GG-NER pathway or the TC-NER pathway

Answer 20

Following recognition of damage, either by XPC and UV-DDB or stalled RNAP and CSA and CSB:

TFIIH unwinds DNA
XPF makes a cut 5’ of the site of damage (quite a way upstream)

XPG makes a cut 3’ of the damaged site

DNA polymerase and DNA ligase replace the missing nucleotides