Lecture 5-DNA repair

Question 1

What are the basic steps of a genetic screen?

Answer 1

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.

Question 2

When using a genetic screen to identify genes required for DNA repair, what are these called?

Answer 2

Rad mutants. These are the cells that cannot grow in the presence of UV radiation after replica plating.

Question 3

Most yeast rad genes encode proteins required for DNA repair. The same genes are often __________ in humans.

Answer 3

Tumor suppresors

Question 4

Without DNA repair, spontaneous DNA damage occurs which can lead to what?

Answer 4

mutations in tumour suppresor and oncogenes leading to cancer.

Question 5

What is the lethality of rad mutants attributed to?

Answer 5

stalled replication and in some cases transcription failure.

Question 6

What is depurination?

Answer 6

Purine nucleotide that loses its nitrogenous base through hydrolysis.

Question 7

How are thymine dimers formed?

Answer 7

Spontaneously due to UV radiation.

Question 8

What do thymine dimers cause?

Answer 8

Formed dimers need to be repaired so DNA polymerase can pass.

Question 9

What are the different means in which DNA damage can be removed?

Answer 9

Base excision repair, nucleotide excision repair, detection of exposed ssDNA and detection of double-stranded breaks.

Question 10

What is Xeroderma Pigmentosa? (XP)

Answer 10

disease in which individuals are extremely sensitive to sunlight.

Question 11

What are some of the mutations associated with XP?

Answer 11

Several different genes: XPA, XPB and others which are generally identified as rad genes in yeast.

Question 12

What are some of the associated phenotypes of XP? (Not sensitivity to sunlight)

Answer 12

High cancer rate, some diseases associated with neural disorders, reduced growth, premature aging.

Question 13

What are the basic steps of NER?

Answer 13

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.

Question 14

In the absence of NER, what other pathway is used?

Answer 14

Trans-lesion repair pathway.

Question 15

What occurs in trans-lesion repair?

Answer 15

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.

Question 16

When is the error prone pathway used?

Answer 16

Only if the other pathways cannot cope. In XP individuals, this pathway is active which results in high mutation rates which results in cancer.

Question 17

What is transcription coupled repair?

Answer 17

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.

Question 18

What is Cockayne Syndrome?

Answer 18

UV sensitivity, most UV-induced Thymine dimers can still be repaired.

Question 19

What are some of the diseases associated with CS?

Answer 19

growth defects, neurological disorders and premature aging.

Question 20

What are the CS related diseases due to?

Answer 20

High level of cell death.

Question 21

Why do CS cells fail to resume transcription after UV radiation?

Answer 21

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.

Question 22

The gene that is altered in CS must then be used for?

Answer 22

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.

Question 23

What is CSB?

Answer 23

identifies stalled RNA polymerase. Recruits XPG nuclease so the NER pathway can be undergone.

Question 24

What happens if XPG is mutated?

Answer 24

Cannot get excision of lesion.

Question 25

What happens if XPC/rad23 are mutated?

Answer 25

UV sensitivity phenotype (related to XP).

Question 26

How are double stranded breaks repaired?

Answer 26

Non-homologous end joining and homologous recombination?

Question 27

What is NHEJ?

Answer 27

Error prone pathway in which ends are simply joined. If the ends are damaged or have overhangs, a small deletion ensues.

Question 28

What is HR?

Answer 28

Accurate ds break repair in which the sister chromatid is used as a template.

Question 29

What are the basic steps of the NHEJ pathway?

Answer 29

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.

Question 30

What is HR dependent on?

Answer 30

Presence of sister chromatid and cdk kinase that are only available during S and G2 phase of cell cycle.