Lecture 5-DNA repair Flashcards
What are the basic steps of a genetic screen?
Plate bacteria onto a plate, induce them to mutagens, streak out single colonies, replica plate and observe for phenotypes on interest in permissive and restrictive conditions.
When using a genetic screen to identify genes required for DNA repair, what are these called?
Rad mutants. These are the cells that cannot grow in the presence of UV radiation after replica plating.
Most yeast rad genes encode proteins required for DNA repair. The same genes are often __________ in humans.
Tumor suppresors
Without DNA repair, spontaneous DNA damage occurs which can lead to what?
mutations in tumour suppresor and oncogenes leading to cancer.
What is the lethality of rad mutants attributed to?
stalled replication and in some cases transcription failure.
What is depurination?
Purine nucleotide that loses its nitrogenous base through hydrolysis.
How are thymine dimers formed?
Spontaneously due to UV radiation.
What do thymine dimers cause?
Formed dimers need to be repaired so DNA polymerase can pass.
What are the different means in which DNA damage can be removed?
Base excision repair, nucleotide excision repair, detection of exposed ssDNA and detection of double-stranded breaks.
What is Xeroderma Pigmentosa? (XP)
disease in which individuals are extremely sensitive to sunlight.
What are some of the mutations associated with XP?
Several different genes: XPA, XPB and others which are generally identified as rad genes in yeast.
What are some of the associated phenotypes of XP? (Not sensitivity to sunlight)
High cancer rate, some diseases associated with neural disorders, reduced growth, premature aging.
What are the basic steps of NER?
XPC and Rad23 scan DNA and recognize damage.
They then recruit other proteins to excise strand, this is done by XPG nuclease.
Repair is then accomplished by DNA polymerase and ligase.
In the absence of NER, what other pathway is used?
Trans-lesion repair pathway.
What occurs in trans-lesion repair?
During DNA synthesis, DNA polymerase encounters DNA damage and stalls at this site.
DNA polymerase is then exchanged for a special trans-lesion DNA polymerase.
This DNA polymerase is able to quickly to add any nucleotide to match the other base, often the incorrect one.
This polymerase lacks exonuclease activity.
When is the error prone pathway used?
Only if the other pathways cannot cope. In XP individuals, this pathway is active which results in high mutation rates which results in cancer.
What is transcription coupled repair?
In mammalian cells, mutations seem to arise more outside of genes rather than inside them. Therefore, the repair must be different between coding sequences and non-coding sequences.
DNA repair can be linked to transcription.
What is Cockayne Syndrome?
UV sensitivity, most UV-induced Thymine dimers can still be repaired.
What are some of the diseases associated with CS?
growth defects, neurological disorders and premature aging.
What are the CS related diseases due to?
High level of cell death.
Why do CS cells fail to resume transcription after UV radiation?
CS cells lose the ability to preferentially repair DNA damage that occur within genes.
This, leads to stalling of RNA polymerase and ultimately, cell lethality.
The gene that is altered in CS must then be used for?
TCR-transcription coupled repair. This is obvious because the CS cells cannot preferentially repair the stalling lesions within genes leading to stalling of RNA polymerase.
What is CSB?
identifies stalled RNA polymerase. Recruits XPG nuclease so the NER pathway can be undergone.
What happens if XPG is mutated?
Cannot get excision of lesion.
What happens if XPC/rad23 are mutated?
UV sensitivity phenotype (related to XP).
How are double stranded breaks repaired?
Non-homologous end joining and homologous recombination?
What is NHEJ?
Error prone pathway in which ends are simply joined. If the ends are damaged or have overhangs, a small deletion ensues.
What is HR?
Accurate ds break repair in which the sister chromatid is used as a template.
What are the basic steps of the NHEJ pathway?
Start with a double stranded break in DNA.
End recognizes by Ku heterodimers.
Ku recruits other proteins such as DNA PKcs kinase which phosphorylates the proteins for processing of ends.
What is HR dependent on?
Presence of sister chromatid and cdk kinase that are only available during S and G2 phase of cell cycle.
