5: DNA cell damage and Repair Flashcards
Why can DNA easily be damaged?
because the DNA bases are planar carbon rings –> easily chemically activated and then
- be reactive, react with other molecules
- are chemically similar –> can be transfered into one another
What is Deamination in the process of modification of an AA?
What does it lead to?
The loss of one amine group of a DNA base (often converted to keto group)
- can lead to mutation via conversion of
- cytosin to uracil (essentially thymine)
- adenine to hypoxanthine
- guanine to xanthine
- and 5-methyl cytosine to thymine.
What are “other chemical modificatios” of DNA?
What do they lead to?
Many reacgtions, often induced by free radicals or hyper-reactive oxygen molecules (byproducts of normal motabolism or can be produced by ionising radioation)
Leading to
- formation of double-bonds
- methylation, alkylation of DNA bases
- adduct formation
What is photodamage?
How does it change the DNA?
intra-strandal change by UV light being absorbed by the DNA bases
- UV light activates and causes
- pyrimindien dimer (often thyamine dimers)
- driver in skin cancer
What are the different modifications/damages that can occur in DNA change?
- Deamination
- other chemical modification
- methylation
- alkylation
- adduct formation
- photodamage
What is the consequence a Base-mismatch in the DNA?
There will be a bulge in DNA –> will be used to find the site of damage and repair of the DNA
How does Radiodamage modifies the DNA
It can sometimes break the phospho-diester bonds–> leading to gap in DNA
- also used to find the site of damage and repair
Overall spoken: What causes DNA damage?
- Carcinogens
- dietary
- lifestyle
- environmental
- occupational
- medical
- endogenous
- Radiation
- ionizing
- solar
- cosmic
Why is DNA damage problematic?
Damage can lead to mutation –> mutations can lead to cancer
- damaging DNA is an important strategy in cancer therapy (might lead to apoptosis)
Explain how polycyclic aromatic hydrocarbons might lead to DNA damage?
Where are they found?
- Common environmental pollutants
- Formed from combustion of fossil fuels
- Formed from combustion of tobacco
E.g. Benzo(a) pyrene (B(a)P)
- itself it nor carcinogenic but gets activated by metabolism (highly carciongenic)
- Forms DNA adduct when activated
Name an example of a food-born carcinogen and how it could cause cancer
Aflatoxin B1– >formed by fungi on grains and peanuts
- metabolism converts it into carcinogen
- activation–> highly reactive
- adduct formation
How does UV radioation cause cancer?
UV radiation might induce
- Pyrimidine (thymine) dimers
- mutation
- melanoma
Explain how ionizin radioation can cause DNA damage
- Generates free radicals in cells
- Includes oxygen free radicals
- super oxide radical: O2•
- hydroxyl radical: HO•
- Includes oxygen free radicals
- Possess unpaired electrons
- electrophilic and therefore seek out electron-rich DNA
Explain the effects of free radicals on the cell
They attack the DNA and cause:
- Double and single strand breaks
- Apurinic & apyrimidinic sites
- Base modifications
- ring-opened guanine & adenine
- thymine & cytosine glycols
- 8-hydroxyadenine & 8-hydroxyguanine (mutagenic)
Why is DNA repair so important?
What are its physiological capacities?
There is a lot of DNA damage going on all the time needs to be repaired to not cause cancer
–> normally: cell has a lot higher capcity to repair than damage happens
Explain adduct formation in DNA
Reactive proteins can bind to DNA (or DNA gets activated and forms Adduct itself) and alter its chemical structure by binding to it
When would Base-excision repair take place?
How does it waork?
BER repairs damages base with intace phosphate diester bonds
- DNA glycosylase removed wrong DNA
- AP endonuclease cuts phospho-diester bond
- Polymerase fills the gap with corect DNA
- Ligase binds the DNA pieces
When is Nucleotide excision repair used?
Explain its principle
Used when there is damages Base with damages phosphate-diester bond (e..g in removal of large adducts)
- Xeroderma pigmentosum proteins (XP proteins) assemble at the damage
- Endonuclease cuts edges of diester bonds that need to be removed (?) –> larger area around damage
- Helicase cuts off large DNA piece
- DNA polymerase synthesises new DNA
- DNA ligase binds them
Which factors activate p53?
What are the effects of p53 activation?
Upregulation of cellular pathways leading to
inhibits progression of cells with damaged DNA from S-phase, later produced CKI-21
- In mild and moderate stress
- antioxidant defence
- DNA repair
- growth arrest
- senescense
- In severe cellular stress
- apoptosis
What are the consequences carcinogen-DNA damge?
- Normal repair–> normal gene and function is restored
- Apoptosis –> damage too severe
- Incorrect repair+ altered primary structure
- Fixed mutations leading to
- Aberrant protiens (abweichend)
- If that is in TSG/oncogenes: carcinogenesis
- Fixed mutations leading to
How do you determine, wether a substance is a carcinogen or not?
- just looking at the chemical strucutre: is it likely to be a carcinogen?
- AMES test (in vitro) –> bacteria
- Chromosomal damge (in vitro), mammalian cells
- Micro-nuclei around DNA formed (in vitro), mammalian cells
- Murine Bone Marrow Micronucleus assay (in vivo)
Explain the AMES test in testing for Carcinogens
- A bacterium that cannot produce histidin is cultured in a histidine free environment
- Chemical to be tested + metabolite enzymes are added –> converstion to active metabolite?
- Given to bacteria –> see if they can cultivate
- If yes: mutation has occured and bacteria make histidin –> carciongenic
Why do you look at micronuclei in mamalian cultivated cells when testing for a carcinogen?
- Binucleate cells assessed for presence of micronuclei
- Can stain the kinetochore proteins to determine if chemical treatment caused clastgenicity(chromosomal breakage) or aneuploidy (chromosomal loss)
–> It shows severe chormosomal damage
Explain the Murine Bone Marrow Micronucleus Assay in testing for carciongens
Treat animals with chemical and examine bone marrow cells or peripheral blood erythrocytes for micronuclei
What is an apurinic/ Apyrimidinic site?
It is a site in DNA that has no base (mosltly due to DNA damage)
