Lecture 19 - DNA damage & repair Flashcards
What does DNA do?
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
What is our most robust defence against cancer?
Genetic stability
What is the only biological macromolecule to repair?
DNA - all others replaced
What are endogenous sources that put cells under constant attack?
- reactions with other molecules within the cell
- hydrolysis, oxygen species, by-products of metabolism
What are exogenous sources that put cells under constant attack?
- 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.
What are examples of endogenous DNA damage?
- Depurination (creating an abasic site)
- Deamination
- methylation
- replication errors
What are examples of exogenous DNA damage?
- Pyrimidine dimers
- Single strand breaks
- Double strand breaks
- Interstrand crosslinks
What types of DNA damage effect the nucleotide bases of the DNA molecule?
- Depurination (creating an abasic site)
- Deamination
- methylation
- replication errors
What types of DNA damage effects one strand of the DNA helix?
- Pyrimidine dimers
- Single strand breaks
What types of DNA damage effects both strand of the DNA helix?
- double strand breaks
- interstrand crosslinks
What is deamination?
Removal of the amino group by hydrolysis results in changes to the DNA bases
What is a transition mutation?
- purine-purine transition/pyrimidine-pyrimidine transition
e.g. A <–> G, C <–> T
What is a transversion mutation?
- purine-pyrimidine transition
- E.g. A <–> C, G <–> T
What type of mutation is more common - transition or transversion?
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.
What is depurination (abasic site)?
- 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
What does a failure to undertake DNA repair result in?
Results in mutation
What type of mutation has the most potential to be damaging to the cell?
Frameshift mutation - more so than substitution mutations
What do frameshift mutations generate?
Missense proteins
What does UV light induce?
- 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
What types of damage affect the phosphate backbone?
- single strand break
- double strand break
What are examples causes of double strand break inducers?
- X-rays
- Ionising radiation
- Topoisomerase II inhibitors
What are examples causes of double strand break inducers?
- Reactive oxygen species
- Hydroxyurea
- Camptothecin
How can DNA repair itself?
Each type of DNA damage lesion is repaired by a specific repair pathway
What is the process of Base excision repair (BER)?
Repairs base damage - e.g. abasic sites, amination. Base-flipping strategy to identify errors
What is the process of Nucleotide excision repair (NER)?
- 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
What is translesion synthesis?
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
What 2 mechanisms exist to repair double strand breaks?
- Non-homologous end joining (NHEJ)
- Homologous recombination (HR)
Describe features of non homologous end joining (NHEJ)
- Error-prone
- Restricted to G1 phase
- Usually results in the loss of nucleotides surrounding the break site
- Important genetic information may be lost
Describe features of homologous recombination (HR)
- error-free repair
- occurs only in S-phase
- uses intact sister chromatid as a template
- double strand break is accurately repaired
What are the 3 places where DNA damage is detected and acted upon to STOP the cell cycle?
- G1
- Entry to S-phase
- Entry into mitosis
(also check for chromosome non-disjunction)
What type of repair occurs in G1 repair?
Non-homologous end joining
What type of repair occurs in G2 repair?
Homologous recombination
How is damage detected?
- 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
What happens if repair is no possible?
- Apoptosis
What is an example of predisposition disease associated with defect in NER (nucleotide excision repair)?
- 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
What is an example of cancer and defects in double strand break repair?
- 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)
Why are mutations associated with cancer?
BRCA2 deficient cells - exhibit genomic instability, are sensitive DNA damaging agents and are defective in homologous recombination –> predisposition to cancer
Describe features of cancer cells?
- Very fast growing - replicate in the presence of damage
- Often have defects in repair - unable to repair everyday DNA damage leading to mutations
What is the Achilles heel of cancer?
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
How does synthetic lethality work?
- 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.
