13. DNA repair.

Question 1

DNA Damage

Answer 1

DNA Lesions:
Definition: Alterations to normal chemical or physical structure of DNA.

Consequences:
- Can block replication or transcription (lethal lesions).
- May generate mutations through direct or indirect mutagenesis.

Cellular Responses to DNA Damage
1. DNA Repair (Reparation):
- Mechanisms are activated to overcome and repair the damage.

2. Programmed Cell Death (Apoptosis):
   - Mechanisms are activated to make cell death occur if the damage cannot be repaired.

Question 2

DNA Proofreading

Answer 2

During DNA replication, most DNA polymerases can “check their work” with each base they add.

Process: Proofreading.
- If polymerase detects an incorrectly paired nucleotide, it will remove and replace it immediately.
- This ensures the accuracy of DNA synthesis before continuing further.

Question 3

DNA Repair

Answer 3

Mismatch Repair
- Occurs immediately after replication
- A mismatch is detected in newly synthesized DNA
- New DNA strand is cut, and mismatched nucleotide and its neighbours are removed
- Missing patch is replaced with correct nucleotides by a DNA polymerase
- DNA ligase seals gap
- In eukaryotes, original strand is identified by recognizing nicks (single-stranded breaks) present only in newly synthesized DNA.

Direct Reparation
- Cause of Damage: Mainly induced by UV light.
- Covalent bonding of pyrimidine bases from the same DNA strand, such as T-T or C-C dimers are most common for damage
- Repair mechanism utilizes enzymes called photolyases - they catalyze the reverse reaction using UV light to break the covalent bonds.
- Process: Known as photoreactivation - is absent in mammals

Excision Repair
- Universal for all organisms.
- Based on removal of damaged base (BER – Base excision repair) or removal of damaged nucleotides (NER – Nucleotide excision repair).

-> Principle of BER
1. DNA Glycosylase: Recognizes and removes defective base from DNA strand, creating an apurinic or apyrimidinic (AP) site.

2. AP Endonuclease: Cleaves DNA strand at AP site, generating a gap in the DNA.
3. DNA Polymerase: Gap is filled by DNA Polymerase I (for prokaryotes) and β, δ and ε (for eukaryotes)
4. DNA Ligase: Stitches remaining nick

->Principle of NER
1. NER Recognition: Proteins involved in NER feel DNA distortions rather than detecting specific base damages (unlike BER).

2. Helicase Action: Helicases join at identified site and unwind a small section of the DNA double helix surrounding the damage.
3. Endonuclease: make cuts on both the 5’ and 3’ sides of damaged nucleotide, removing the distorted segment of DNA strand by doing nick.
4. Oligonucleotide Removal: The resulting single-stranded oligonucleotide containing the damage leaves from the double helix. Nick is filled with DNA
5. Nick Sealing: DNA ligase stitches the remaining nick