DNA Repair Flashcards
Explain how the genome can be stable, but DNA is not.
On an evolutionary scale, the genome is stable (the pool of organisms maintains the same genes). DNA, however, is a relatively transient molecule that can be degraded, damaged, mutated, etc.
What are 6 sources of DNA damaging agents? hint: 5 are natural.
SUN, oxygen, medical drugs, cellular metabolism, DNA polymerase, tobacco smoke and other pollutants.
How many mutations are sunlight responsible for, and how do these form?
Up to 100,000 UV products in DNA per cell per day. Nucleotides readily absorb UV light and are excited, causing the formation of unwanted bonds that cause kinks in DNA structure, stalling DNA and RNA pol.
How is 8-oxoguanine formed and describe its pairing.
It is formed from reactive oxygen species. Carbonyl formation on 5-member ring of guanine. Disrupts aromaticity. 8-oxoguanine pairs with adenine.
How are reactive oxygen species generated?
1% of oxygen in electron transport chain is converted to ROS. Superoxide ion is only partially reduced, and premature release can cause it to cause DNA mutations.
How might anti-cancer drugs harm normal cells?
Anti-cancer drugs act by causing DNA damage to cancer cells so they can’t divide, but sometimes noncancerous cells are damaged as well.
How can topoisomerase II inhibitors and cisplatin cause DNA damage?
Topoisomerase II inhibitors cause double strand DNA breaks. Cisplatin causes intrastrand cross links.
How does ionizing radiation damage DNA?
Ionizing radiation causes double strand breaks. This is why it is popular to use against cancer.
How does DNA polymerase damage DNA?
Some DNA polymerases have lower fidelity than others.
How does intrinsic DNA instability cause damage to DNA?
Bases can be lost by deamination (breakage of glycosidic bond) which gets in the way of polymerases. Cytosine can spontaneously be deaminated to uracil (amino group is hydrolyzed), which pairs with adenine.
What is the most frequent mutation in in human tumors?
C -> T transition is the most common mutation in human tumors. This occurs when C is deaminated to form U.
Which mutation is a signature of smoking-related lung cancer?
A G –> T transversion (purine to pyrimidine)
Which mutations are commonly found in colorectal, kidney, liver, and prostate cancers?
short deletions and insertions (1-3bp)
What type of mutation is responsible for truncation of a protein?
A chromosome deletion.
How can chromosome mutations be identified?
Cytologically using a microscope (karyotype)
How are chimeric proteins formed?
When nonhomologous chromosomes exchange material and are expressed. These proteins may have altered exprssion/activity.
Describe the Philadelphia chromosome and how it is fomred.
It is a translocation of chromosome 9 to chromosome 22, so that the transcription product is a fusion protein with the C-end of ABL (tyrosine kinase) and the N-end of BCR. The fusion protein has constitutive tyrosine kinase activity, so the cells in chronic myelogenous leukemia (CML) proliferase excessively.
Describe Down Syndrome and the type of mutation which causes it.
It is caused by genome mutation, in which there is the loss or gain of an entire chromosome. In down syndrome, there is the gain of chromosome 21. The gain or loss of larger chromosomes is embryonically lethal.
What is the difference between DNA damage and DNA mutation?
DNA damage is reversible if DNA repair machinery gets to the damage in time. DNA mutation is fixed in the genome (i.e. permanent).
When are mutations desirable?
Antibodies.
How often do DNA damage events result in a DNA mutation?
1 in 1000 DNA damage events result in DNA mutation.
What are the two cellular protection mechanisms that provide genome stability?
Damage avoidance and DNA repair (as a last resort option)
How are ROS removed from the cell?
ROS such as superoxide in the mitochondria is further reduced to hydrogen peroxide by superoxide dismutase, followed by full reduction to water by peroxidases catalase.
What is glutathione, and what role does it play in preventing DNA damage?
It is an antioxidant that prevents damage caused by ROS. It works by confering radioresistance to cells (i.e. cancer cells are often radioresistant).
How are cells shielded from UV radiation?
The melanin in skin dissipates 99.9% of UV as heat. This is why blondes and redheads are more susceptible to cancer (less melanin).
What are the 3 R’s of DNA repair?
Recognition, removal, replacement.
When does DNA mismatch repair take place?
Often right after replication, when proofreading does not catch the mismatch.
By how much does MMR increase the fidelity of DNA replication?
By 100-fold compared to DNA polymerase + exonucleolytic proofreading.
What role does MutS play in MMR in prok?
It is a homodimer that searches for mismatched DNA base pairs along the DNA (S = slide). Only one dimer subunit interact with the DNA. At the mismatch, the homodimer pauses, changes conformation, and forms a stable complex with other repair proteins.
How are MutL and MutS recruited to sites of MMR?
They are recruited when MutS finds a mismatch on the DNA.
Describe the base pairing of O-methyl guanine.
It can pair with C or T, causing mutations if it replicates after pairing with T.
Describe the mechanism of BER.
- DNA glycosylase cleaves the modified base.
- The AP site is released due to activity of AP endonuclease (nicks the 5’ end of the site) and AP exonuclease (nicks 3’ after the site).
- The single nucleotide gap is filled in by DNA pol beta, and the nick is sealed by DNA ligase I
Describe the mechanism of NER
Recognition protein binds to unaffected strand and slides along until it reaches the dimer or adduct
helicase unwinds the dna
exonuclease cuts out the affected strand
DNA pol and ligase fill in and seal the gap
Describe the steps of repair of dsDNA breaks by HR.
- dsDNA break is ressected in both directions by 5’->3’ exonuclease
- Rad51 searches for homologous sequence in sister chromatid and allows strand invasion of the 3’ ressected end with the homologous strand.
- New DNA synthesis ensues with the 3’ ressected end as a “primer” and the homologous strand as the template.
- The newly synthesized strand unwinds from the template and acts as the template for its partner DNA strand.
- DNA ligase seals.
Describe the mechanism of dsDNA repair by non-homologous end-joining.
- Ku70, Ku80, and DNA-PK join the DNA ends together.
- Ends are resected until there is microhomology by exonuclease,
- gaps are filled in with DNA pol.
- bonds are sealed using XRCC4 and DNA ligase IV.
Describe the upregulation of homologous recombination proteins during S phase.
CDK-cyclin complex is activated, E2F2 is released from Rb, and E2F2 allows the transcription of Rad51, Rad54, Bard1, MSH2, MLH1.
