Lecture 7 - DNA Repair Flashcards
How would you identify the genes required for DNA repair - Rad genes - from large-scale mutagenesis of haploid yeast?
1) Plate out single cells
2) Grow colonies
3) Replica plate
4) Use low-level UV or other radiation on the replica plate
5) Isolate colonies that grow on untreated plates
What is an underlying cause of human familial cancers?
Mutation in the DNA repair pathway - mutations are acquired rapidly and develop into cancer later
What are the two general categories of DNA damage with respect to the effect on the DNA?
1) Chemical alteration of a nucleotide
* Not mutations but can lead to them
2) Break in the DNA backbone - either a single stranded or double-stranded break
What are the results of chemical alteration of a nucleotide?
- Change in DNA sequence at the next S-phase (e.g., deamination, depurination)
- Failure of DNA polymerase (and RNA polymerase) to progress past the altered nucleotide (e.g., pyrimidine dimers)
What are the results of a break in the DNA backbone?
Failure of DNA polymerase (and RNA polymerase) to progress past the break
What are some specific DNA damaging agents that can greatly increase the frequency of specific types of DNA damage?
1) UV light - increase pyrimidine dimers
2) Nitrous acid - increases cytosine deamination
3) X-rays - increase double-stranded breaks
What is depurination?
Loss of purine so the DNA synthesis for that nucleotide will be skipped and makes a smaller strand that can cause severe damage to the reading frame
What is deamination?
Cytosine becomes uracil and will now be a template for adenine which introduces a mutation
What are pyrimidine dimers?
Any combination of two pyrimidines joining which permits DNA polymerase to pass it
What pathways can DNA damage repair be carried out by?
Error-free or error-prone pathways
What can error-prone pathways lead to?
Mutations and cancer
How is base excision repair (BER) carried out?
1) Scans double helix to detect altered bases
2) Glycosylases specific to each type of altered base - removes the base
3) AP endonuclease and phosphodiesterase removes the sugar-phosphate
4) DNA polymerase adds a new nucleotide and DNA ligase seals the nick
*Steps 3 and 4 can be used for depurinated DNA
How is xeroderma pigmentosa caused and what does it lead to?
The nucleotide excision repair pathway has mutations in one of the genes and it makes you extremely sensitive to UV since pyrimidine dimers can’t be repaired
How is nucleotide excision repair carried out?
1) XPC-Rad23 dimer scans DNA and recognizes the damage
2) An excision nuclease, XPG, is recruited to cut on either side of the pyrimidine dimer so the strand can be removed and the 3’ end can be extended by a repair DNA polymerase and DNA ligase
What happens if the error-free nucleotide excision repair pathway fails?
Error-prone translesion repair pathway used
How is the translesion repair pathway carried out?
1) DNA synthesis can’t proceed with pyrimidine dimers so there will be covalent modifications made to the sliding clamp and the replicative DNA polymerase is released
2) Translesion DNA polymerase will be loaded by assembly factors to push through the dimer but base-pairing is not occurring properly
What mutation causes the translesion repair pathway to be used a lot?
XPG mutation
Do mammalian cells accumulate mutations more outside or within genes?
Outside - therefore, DNA transcribed is less likely to be mutated or DNA damage is repaired more efficiently in transcribed genes
What is Cockayne syndrome?
An inherited UV sensitivity syndrome like xeroderma pigmentosa, but not as severe since most UV-induced thymine dimers can still be repaired
- Causes growth defects, neurological disorders, and premature aging, all due to high levels of cell death
How do we know that the altered gene in Cockayne syndrome is necessary for transcription-coupled DNA repair?
CS cells have the same frequency of mutations in genes compared to non-coding DNA because the mutation is involved in the repair pathway - failure to repair DNA damage in coding genes results in stalling of RNA polymerase and cell lethality occurs because of stalled transcription
How is transcription-coupled repair used for those with Cockayne syndrome?
1) RNA polymerase II encounters DNA damage and stalls
2) CBS (Cockayne syndrome B) identifies stalled RNA polymerase
3) CBS recruits repair factor XPG, an excision endonuclease
4) Single-stranded DNA is removed and gets repaired by DNA polymerase and DNA ligase
Why does the error-free pathway of a pyrimidine dimer only work in genes?
Because it is recruited by a stalled RNA polymerase
What branch does transcription-coupled repair come from?
Nucleotide excision repair
What does coupling nucleotide excision repair to transcription do?
Ensures that the cell’s most important DNA is efficiently repaired and that transcription can occur through damaged DNA
Summary of nucleotide excision repair.
- Important for the repair of UV-induced thymine dimers
- Identifies and excises DNA base damage
- Two branches: General pathway identifies DNA damage anywhere in the genome and TCR pathway is triggered when transcription is stalled
What are the two major pathways to repair double-stranded breaks?
1) Non-homologous end joining (NHEJ)
- Error-prone and ends are recessed before joining so there is a resulting small deletion
2) Homologous recombination
- Accurate because it uses sister chromatids as a template for repair
What is the NHEJ pathway?
1) End recognition and binding by Ku heterodimers
2) Additional proteins used and processing of DNA ends carried out
3) Limited repair synthesis and ligation
* Loss of 1 or 2 nucleotides from either end
How are telomeres protected by DNA repair machinery?
By telomere binding proteins - sheltrin complex
What is the pathway for homologous recombination?
1) Mre11 nuclease complex recruited to DNA ends
2) Mre11 recruits CtlP and BRCA1 which digests the 5’ ends of broken strands
3) Rad51 mediates the invasion/exchange with sister chromatid
4) Repair DNA polymerase synthesizes DNA using sister chromatid template (undamaged)
5) Invading strand released and broken double helix re-formed
5) DNA synthesis continues using strand from damaged DNA as a template
6) DNA ligated
How is a double-stranded break in genomic DNA mediated by Cas9 repaired?
Non-homologous end joining pathway (NHEJ)
How is CRISPR used to generate small deletions?
Introduce Cas9 and a single guide RNA corresponding to a site within the gene
How is CRISPR used to introduce specific point mutations in a gene?
Add guide RNA and some DNA that serves as a template for homologous recombination to plasmids with sequences homologous to the gene
How can CRISPR be used to introduce an epitope tag?
Plasmid introduced with guide RNA and Cas9 has an epitope tag - double-stranded break near one end of the coding sequence can be repaired via homologous combination
