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