Lecture 7 - DNA Repair

Question 1

How would you identify the genes required for DNA repair - Rad genes - from large-scale mutagenesis of haploid yeast?

Answer 1

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

Question 2

What is an underlying cause of human familial cancers?

Answer 2

Mutation in the DNA repair pathway - mutations are acquired rapidly and develop into cancer later

Question 3

What are the two general categories of DNA damage with respect to the effect on the DNA?

Answer 3

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

Question 4

What are the results of chemical alteration of a nucleotide?

Answer 4

• 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)

Question 5

What are the results of a break in the DNA backbone?

Answer 5

Failure of DNA polymerase (and RNA polymerase) to progress past the break

Question 6

What are some specific DNA damaging agents that can greatly increase the frequency of specific types of DNA damage?

Answer 6

1) UV light - increase pyrimidine dimers
2) Nitrous acid - increases cytosine deamination
3) X-rays - increase double-stranded breaks

Question 7

What is depurination?

Answer 7

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

Question 8

What is deamination?

Answer 8

Cytosine becomes uracil and will now be a template for adenine which introduces a mutation

Question 9

What are pyrimidine dimers?

Answer 9

Any combination of two pyrimidines joining which permits DNA polymerase to pass it

Question 10

What pathways can DNA damage repair be carried out by?

Answer 10

Error-free or error-prone pathways

Question 11

What can error-prone pathways lead to?

Answer 11

Mutations and cancer

Question 12

How is base excision repair (BER) carried out?

Answer 12

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

Question 13

How is xeroderma pigmentosa caused and what does it lead to?

Answer 13

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

Question 14

How is nucleotide excision repair carried out?

Answer 14

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

Question 15

What happens if the error-free nucleotide excision repair pathway fails?

Answer 15

Error-prone translesion repair pathway used

Question 16

How is the translesion repair pathway carried out?

Answer 16

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

Question 17

What mutation causes the translesion repair pathway to be used a lot?

Answer 17

XPG mutation

Question 18

Do mammalian cells accumulate mutations more outside or within genes?

Answer 18

Outside - therefore, DNA transcribed is less likely to be mutated or DNA damage is repaired more efficiently in transcribed genes

Question 19

What is Cockayne syndrome?

Answer 19

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

Question 20

How do we know that the altered gene in Cockayne syndrome is necessary for transcription-coupled DNA repair?

Answer 20

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

Question 21

How is transcription-coupled repair used for those with Cockayne syndrome?

Answer 21

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

Question 22

Why does the error-free pathway of a pyrimidine dimer only work in genes?

Answer 22

Because it is recruited by a stalled RNA polymerase

Question 23

What branch does transcription-coupled repair come from?

Answer 23

Nucleotide excision repair

Question 24

What does coupling nucleotide excision repair to transcription do?

Answer 24

Ensures that the cell’s most important DNA is efficiently repaired and that transcription can occur through damaged DNA

Question 25

Summary of nucleotide excision repair.

Answer 25

• 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

Question 26

What are the two major pathways to repair double-stranded breaks?

Answer 26

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

Question 27

What is the NHEJ pathway?

Answer 27

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

Question 28

How are telomeres protected by DNA repair machinery?

Answer 28

By telomere binding proteins - sheltrin complex

Question 29

What is the pathway for homologous recombination?

Answer 29

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

Question 30

How is a double-stranded break in genomic DNA mediated by Cas9 repaired?

Answer 30

Non-homologous end joining pathway (NHEJ)

Question 31

How is CRISPR used to generate small deletions?

Answer 31

Introduce Cas9 and a single guide RNA corresponding to a site within the gene

Question 32

How is CRISPR used to introduce specific point mutations in a gene?

Answer 32

Add guide RNA and some DNA that serves as a template for homologous recombination to plasmids with sequences homologous to the gene

Question 33

How can CRISPR be used to introduce an epitope tag?

Answer 33

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