DNA Repair

Question 1

DNA is subject to attack/damage from many things, such as:

Answer 1

Replication Errors 
UV Light 
Corsslinking Agents (e.g. psoralen, cisplatin)
Carcinogenic Agents
Alyklation of DNA Bases
Oxidation
Deamination
Ionising Radiation
Oxygen Free Radicals

Question 2

Possible damage to DNA:

Answer 2

Missing Bases
Altered Bases
Incorrect Bases
Linked Pyrimidines (Due to UV Light causing pyrimidine dimers)
Crosslinked Strands
Bulge Due to Insertion or Deletion
Strand Breaks (Especially Double Strand Breaks)

Question 3

3 Strategies for DNA Repair:

Answer 3

1. Reversal
2. Base Excision
3. Nucleotide Excision

Question 4

Defects in BLANK mechanisms are associated with cancer (and other diseases)

Answer 4

Defects in DNA repair mechanisms are associated with cancer (and other diseases)

Question 5

Direct Reversal of DNA Damage (Enzymes involved)

Answer 5

DNA Photolyase: Photoreactivating enzymes repair pyrimidine dimers and use light energy to repair damage. Absorbs light from 300-500nm and transfers it to FADH2, which then transfers an electron to the pyrimidine dimer, splitting it into monomers.

DNA methyltransferase: Removes the alkyl groups from oxygens of bases.

Question 6

Base Excision Repair Pathway (BER)

Answer 6

2 Steps and 2 Enzymes:

Glycosylases: Remove the damaged nitrogenous base while leaving the sugar-phosphate backbone intact, creating an apurinic/apyrimidinic site.
Note that each type of damaged nucleobase requires its own glycosylase.

AP-Endonucleases: Cleave 5’ to abasic site. All cells possess strong AP-endonuclease activity. AP=apyrinic/apyrimidinic

Question 7

Nucleotide excision repair pathway (NER)

Answer 7

The only mechanism for removing bulky adducts and is the most important repair mechanism for cell survival.
Note: Doesnt just remove the affected nucleotide, but surrounding ones as well.

In E.coli, UvrABC endonuclease: a multienzyme complex
Dimer of UvrA binds to DNA and its ATPase recognises damage.
UvrB’s ATPase activity is activated by UvrA, damaged DNA wraps around UvrB
UvrA leaves and UvrC enters.
UvrC is responsible for cleaving the nucleotides either side of the DNA damage.
DNA helicase II (UvrD) then removes the excised segment
DNA polymerase I fills in the correct nucleotides.

Question 8

Transcription-Repair Coupling

Answer 8

Bulky lesions that block RNA polymerase are repaired more rapidly in transcribed regions than in silent ones.
There is enhanced rate of repair of transcribed strand, implying that there is transcription-repair coupling.

But RNA pol. hinders access to UvrABC, so coupling factor is required: TRCF - transcription-repair coupling factor.

TRCF recognises stalled RNA Pol.
TRCF binds to the stalled complex, displacing RNA Pol.
TRCF binds to UvrAB (UvrA dimer + UvrB)
Facilitates removal of UvrA from complex, accelerating DNA repair.

Question 9

Double Strand Breaks (DSBs) and they issue they pose for DNA repair:

Answer 9

Existing repair mechanisms (BER, NER) use the undamaged strand to restore genetic information.
So when both strands are damaged there is a problem.
Some solutions are:
- Homologous Recombination
- Single Strand Annealing
- Non-Homologous End Joining

Question 10

Mismatch Correction has an issue in DNA repair, which of the two bases/strands is the correct vs incorrect one?

Answer 10

The daughter strand remains unmethylated at the DAM site for a short time after DNA replication. This gives polarity to the DNA strands,distinguishing the correct parental strand from the “incorrect” daughter strand.