Flashcards in Lecture 4 - DNA Repair Deck (58):
What is the Ames test used for? Explain how it works.
To test the mutagenic potential of a chemical aka whether its a potential carcinogen
1. Start with a strain of bacteria that has a mutation in the gene required for His synthesis (aka requires external His to survive) and plate them with minimal His media
2. Add mutagen to one and the other is the control
3. Few colonies grow on the control: natural revertants (spontaneously mutate to produce His) but many grow on the other because were able to mutate to synthesis His
What is another way of saying carcinogen?
What happens when too much mutagen is added to the plate in the Ames test?
Too high concentration can be lethal
What is the purpose of DNA repair?
To repair post-replication mutations
How often do accidental base changes in DNA result in a permanent mutation?
Less than 1 in 1000
Why does DNA methylation occur?
Many reasons but mainly gene expression regulation = epigenetics
Where does methylation and mismatch repair primarily happen?
Where is DNA methylated in both prokaryotes and eukaryotes?
At the N6 of adenines in (5′)GATC sequences, which are palindromes present in opposite orientations on the two strands
How does methylation help mismatch repair post-replication? How is the DNA referred to at this time?
Following replication, the new daughter strand for a short period of time, is not methylated = hemimethylated DNA
The repair mechanisms need to know which is the parent and which is the daughter because if there is a mismatch they want to repair the daughter strand as the parent one is a high fidelity template
What enzyme methylates the daughter strand post-replication? Can the daughter and parent strand be distinguished at this point?
Describe post-replication mismatch repair. 6 steps
1. Proteins MutS and MutL recognize a mismatch and form a complex on the DNA using ATP
2. DNA is threaded through the complex until it reaches MutH bound at a DNA methyl group to identify the parent strand (usually about 12 base pairs before reaching one)
3. MutH cleaves the unmethylated strand on the 5′ side of the G in this sequence
4. A complex consisting of DNA helicase II and one of several exonucleases then degrades the unmethylated DNA strand from that point to just beyond the mismatch
5. DNA Pol III adds the correct bases
6. Nick is sealed by DNA ligase
What determines what kinds of exonucleases are used during post-replication mismatch repair?
Whether the daughter strand was cleaved on the 3' or 5' side of the mismatch
Why is our DNA a target for spontaneous modifications? How frequent are these?
Because it resides in an aqueous environment where other chemicals reside and can make spontaneous modifications to it
What are 4 example of spontaneous modifications of DNA?
1. Oxidative damage
2. Hydrolytic attacks: depurination and deamination
3. Uncontrolled methylation by S-adenosylmethionine
4. UV damage causing dimerization of adjacent Ts
What is depurination of DNA?
An entire base is removed from a nucleotide
How often does DNA depurination happen?
5000 x / cell / day
What happens during deamination?
NH3 is removed and replaced with C=O
What happens if you deaminate a C base?
You get a U
What happens if you deaminate a 5'-methylated C base?
You get a T
What happens if you deaminate an A base?
You get hypoxanthine
What happens if you deaminate a G base?
You get xanthine
Which deamination is an epigenetic mutation?
Deamination of 5-methylcytosine to thymine
Which deamination is the most common? How is it repaired?
Deamination of cytosine to uracil
Base excision repair
What are the 2 ways in which UV light dimerizes T residues?
1. Results in formation of a cyclobutyl ring involving C-5 and C-6 of adjacent T residues = cyclobutane pyrimidine dimer => introduces a kink in DNA
2. Results in a 6-4 photoproduct, with a linkage between C-6 of one T and C-4 of its neighbor T
What is most easily recognized by DNA repair mechanisms?
Deformations of the DNA helix
What can also cause T dimerization other than UV?
How are T dimers repaired? Describe the mechanism.
Nucleotide excision repair
1. Excision nuclease nicks the strand with the dimer several nucleotides away on both sides of the dimerization
2. Helicase removes that chunk of the strands
3. DNA polymerase I/epsilon replaces the nucleotide gap (and some) using the 3' free (-OH) on the nick of the strand as a primer
4. DNA ligase seals nick
How are spontaneous DNA alterations repaired?
What are the 2 types of excision repairs?
1. Base excision repair
2. Nucleotide excision repair
How can spontaneous DNA alterations result in a sustained mutation?
If deamination or depurination has occured, DNA replication will create one unchanged daughter strand and one mutated strand with the correct base pair added (for deamination) or a point deletion (for depurination)
Are the consequences of a sustained mutation more severe in a germ cell or in a somatic cell. Why?
Germ cell because it'll be reproduced indefinitely
Describe the mechanism of base excision repair.
1. DNA glycosylase recognizes an incorrectly pairs base and cleaves it off
2. AP endonuclease + phosphodiesterase removes the leftover phosphodiester backbone along with its ribose
3. DNA polymerase I starts synthesis from the free 3′ hydroxyl at the nick, removing (with its 5′→3′ exonuclease activity) and replacing a portion of the damaged strand using the 3' free (-OH) on the nick of the strand as a primer
4. DNA ligase seals nick
What does the length of the DNA chunk removed during nucleotide excision repair dependent on?
Whether it's in eukaryotes (longer) or prokaryotes (shorter)
Is the repair of double strand breaks typically complete?
Are double strand breaks less or more common than spontaneous DNA alterations?
What are the 2 mechanisms to repair DNA double strand breaks? When is each used? Which one predominates?
1. Nonhomologous end joining: somatic cells - PREDOMINATES
2. Homologous recombination: can only be used shortly after replication when there is a sister chromatid available to serve as the template (in S and G2 phases of the cell cycle)
Describe the mechanism of nonhomologous end joining and the resulting DNA. 3 steps
1. End is recognized by Ku heterodimers and other proteins (eg: p53 and BRCA1) that bind to prevent degradation of the DNA and arrest the cell cycle
2. Nucleotides are chewed back to remove the nucleotide overhand that results from the break until a blunt end is obtained
3. Ends are joined by DNA ligase
Resulting DNA has suffered nucleotide deletions
Why is it important for the cell cycle to be arrested during nonhomologous end joining?
So that the cells do not divide and propagate the mutation
What can cell cycle dysfunction and propagation of mutated cells that lead to cancer be due to?
p53 and BRCA1 gene mutations
Describe the mechanism of homologous recombination and the resulting DNA. 5 steps
1. Exonucleases degrade the 5' ends of each DNA strand leaving 3' overhangs
2. Strand invasion: a single broken strand will crossover to the sister chromatid at a branch point
3. Branch point migrates along the length of the sister chromatid so that the template can be read and DNA polymerase can synthesize a new strand portion
4. The newly synthesized DNA pairs with the top-strand and DNA synthesis finishes
5. DNA ligase joins the ends
Resulting DNA: break COMPLETELY repaired
What does a mutation in BRCA2 affect? What does it cause?
Affects repair by homologous recombination
Causes breast, ovarian, and prostate cancer
What does a mutation in MSH2, 3, 6, MLH2, PMS2 affect? What does it cause?
Affects mismatch repair
Causes colon cancer
What does a mutation in xeroderma pigmentosum (XP) groups A-G affect? What does it cause?
Affects nucleotide excision repair
Causes skin cancer, UV sensitivity, neurological abnormalities
What do a lot of inherited syndromes with defects in DNA repair cause?
What does a mutation in XP variant affect? What does it cause?
Translesion synthesis by DNA polymerase
Causes skin cancer, UV sensitivity
What does a mutation in ataxia telanglectasia (AT) affect? What does it cause?
ATM protein, kinase activated by double-stranded breaks
Causes leukemia, lymphoma, gamma-ray sensitivity, genome instability
What does Werner syndrome affect? What does it cause?
Accessory 3'-exonuclease and DNA helicase
Causes premature aging, cancer at several sites, genome instability
What does Bloom syndrome affect? What does it cause?
Accessory DNA helicase for replication
Causes cancer at several sites, stunted growth, and genome instability
What do Fanconi anemias groups A-G affect? What does it cause?
DNA interstrand cross-link repair
Causes congenital abnormalities, leukemia, and genome instability
What does a 46 BR patient have affected? What does this cause?
DNA ligase I
Causes hypersensitivity to DNA-damaging agents, genome instability
What happens when BRCA 1 is completely deficient?
1. Impaired DNA damage repair
2. Defective G2/M cell cycle checkpoint
3. Centrosome amplification
==> Genetic instability leading to either:
- p53 activation => cell cycle arrest OR apoptosis => development abnormalities or embryonic lethality
- p53 inactivation => activation of oncogenes => clonal expansion => tumorigenesis
What is the Peto paradox?
You would think that as animals get bigger and have more cells and as their lifespan gets longer they would have a higher cancer incidence, but this is not true.
The incidence of cancer is remarkably flat
What is the human cancer mortality?
What is the elephant cancer mortality?
What can explain the Peto paradox?
Larger animals with more cells have more genes that code for binding proteins that bind DNA for repair like p53 without which apoptosis does not occur and cancer does instead
What is a retrogene?
DNA copy of the RNA that is reinserted as a gene (aka a gene without introns)
How many alleles for TP53 in humans vs elephants? What does this mean for human vs elephant cells?
Elephants: 40 (including 38 retrogenes)
Elephant cells more readily undergo apoptosis