DNA damage and Repair

Question 1

What are the consequences of damaged DNA?

Answer 1

If not repaired, damaged DNA can either lead to a mutation or can lead to transcription and replication blockages.
Transcription and replication blockages may lead to ageing.

Question 2

What is the result of a mutation in a germ line cell?

Answer 2

Can either give rise to a:

• Selective advantage
• genetic disorder

Question 3

What can be the result of a mutation in a somatic cell?

Answer 3

A mutation in a somatic cell can lead to cancer or even cell death

Question 4

What are the causes of spontaneous damage and mutation?

Answer 4

Errors in DNA replication - incorporating the wrong nucleotide

Spontaneous damage arising from chemical nature of DNA

• Deamination of cytosine
• Depurination - loss of purine base

Damage due to reactive oxygen species (ROS)

Question 5

When cytosine is deaminated, what does it form and what is the result of this?

Answer 5

When cytosine is deaminated it forms uracil. If not repaired before replication, deamination may lead to replacement of CG base pairs with TA base pairs.

Question 6

What are the consequences of depurination?

Answer 6

As a result of lack of base because of depurination;
- when the DNA molecule is replicated, any random base can be added opposite the AP site

• a deletion opposite the AP site may occur

Question 7

What types of DNA damage can ROS induce?

Answer 7

Chemical modifications of bases e.g. Guanine –> 8 oxo-guanine

Strand breaks –> single stranded and double stranded breaks

Inter strand cross-links (covalent bonds between complementary strand)

Formation of cyclopurines causing structural distortion

Question 8

What is the result of DNA exposed to UV?

Answer 8

Cyclobutane pyrimidine dimer - covalent bonds forming between two adjacent pyrimidines (Cs and Ts)

Formation of 6-4 photoproducts between pyrimidines

These both cause structural distortions in DNA. These distortions prevent the passage of normal DNA polymerase and RNA polymerase enzymes.

Question 9

In what order of frequency do cyclobutane pyrimidine dimers form between bases?
E.g. CC, CT, TC and TT

Answer 9

T=T occurs most frequently
T=C occurs is the next most frequent
C=T is next
C=C occurs the least frequent

Question 10

What mechanisms are there to repair DMA damage?

Answer 10

• Base excision repair
• Nucleotide excision repair
• Mismatch repair
• Homology directed repair
• Translesion synthesis
• Non-homologous end joining

Question 11

What can the base excision pair repair mechanism be used for?

Answer 11

Damaged or abnormal bases e.g. U arising from deamination of C or 8 oxo-guanine arising from action of ROS

Missing bases from depurination

Mechanism: damage specific (e.g. For uracil) glycosylase enzymes slide along DNA and recognise and excise (get rid of) damaged bases. This leaves an AP site.
AP endonuclease makes a nick and leaves a phosphorites term bond 5' to the AP site
DNA polymerase (lyase activity) removes deoxyribose and phosphate previously linked to damaged base 
DNA polymerase adds in missing nucleotide and DNA ligase seals the gap

Question 12

What can the nucleotide excision pair repair mechanism be used for?

Answer 12

This is a a mechanism found in all organisms that accurately repairs damage to DNA that generates structural disorders e.g. CPDs and 6-4 photoproducts

Mechanism: In Global Genome Nucleotide Excision Repair structural distortion at the site of damage is recognised by XPC (all DNA is subject to this) - UV-DDB protein binds if CPD

In Transcription coupled NER structural distortion at the site of damage is recognised by stalled RNA polymerase (subject to actively transcribed template strand). CSA and CSB recruited. RNA polymerase backtracks so repair machinery can get to damaged site

• Unwinding of DNA by helicase
• Damaged strand cleaved either side by endonuclease XPF and XPG
• Release of 22-30 nucleotide DNA fragment that includes damaged site
• Synthesis of new DNA
• DNA ligase adds missing phosphodiester bond

Question 13

What mechanisms are less accurate for DNA repair?

Answer 13

Non-homologous end joining - which is used in repair of double strand breaks
(Ku protein binds to either end of double strand break, ku recruits nucleases, DNA polymerase and DNA ligase).

Translesion repair - Translesion DNA polymerase so can read through damaged DNA, it inserts random nucleotide and so could not be correct nucleotide

Question 14

What is an example of a disease associated with DNA repair mutations?

Answer 14

Xeroderma pigmentosum
Disease arises due to recessive mutations in genes involved in GG-NER such as XPC.
Symptoms are:
Heightened sensitivity of skin and eyes to sunlight
>1000x greater risk of developing skin cancers
Neurological degeneration in 20-30% of patients