DNA Repair Lecture Notes

Chapter 11: DNA Repair

1. Introduction to Mutagenesis

  • Mutagenesis: The creation of mutations, can occur during DNA replication.

  • DNA can be damaged by various factors:

    • Heat: Speeds up chemical reactions (e.g., deamination of bases).

    • Chemicals: Can react with DNA by adding groups, breaking bonds, or fusing parts together.

    • Irradiation: Energy absorbed by DNA causes bonds to break or fuse together parts of the DNA.

2. Importance of DNA Repair

  • DNA damage differs from mutagenesis in that not all damage results in mutation.

  • Bacterial cells must possess mechanisms to repair DNA damage, which is essential for cell survival.

  • Mutagenic Treatments: Treatments that generate mutations are called mutagenic treatments or mutagens.

3. Evidence for DNA Repair

  • Comparing the number of surviving cells post-mutagenic treatment indicates the effectiveness of DNA repair mechanisms.

    • Without DNA repair, each mutation might be lethal leading to a significant drop in the survival fraction of cells.

    • With repair mechanisms, low levels of mutagenesis can be repaired, allowing some cells to survive.

    • Longer or stronger treatments lead to exponential cell death as mutations accumulate beyond repair capabilities.

4. DNA Repair Pathways

4.1 Specific Repair Pathways

  • Types of damage include:

    • Damage from deamination of bases

    • Damage due to reactive oxygen

    • Damage from alkylating agents

    • Damage due to UV radiation

4.2 General Repair Pathways

  • Include:

    • Methyl-directed mismatch repair

    • Nucleotide excision repair

5. Damage from Deamination of Bases

  • One of the most common types of damage that can result in altered base pairing.

  • Can occur spontaneously (e.g., high heat) or through deaminating agents:

    • Hydroxylamine: Deaminates cytosine to uracil.

    • Bisulfite: Deaminates cytosine in ssDNA only.

    • Nitrous acid: Deaminates cytosine, adenine, and guanine.

6. Repairing Deaminated Bases

  • DNA N-glycosylases are enzymes that break the glycosyl bond between the base and the sugar.

  • After base removal, AP endonucleases cut at specific sites.

  • 3'-OH end is utilized as a primer for DNA synthesis:

    • Exonuclease activity degrades the strand ahead of DNA polymerase, leading to replacement around the damaged site.

7. Damage Due to Reactive Oxygen

  • While molecular oxygen (O2O_2) is not very damaging, its reactive forms (e.g., superoxide radicals, hydrogen peroxide, hydroxyl radicals) are highly harmful.

  • Reactive oxygen damage is associated with increased cancer risk with aging and age-related diseases.

  • 8-oxoG is a particularly mutagenic lesion where DNA polymerase III may mispair it, notably with adenine.

7.1 Avoiding 8-oxoG Mutations

  • E. coli has several mechanisms to combat these mutations:

    • Mut M: N-glycosylase that removes 8-oxoG from DNA.

    • Mut Y: N-glycosylase that removes adenines that pair opposite 8-oxoG.

    • Mut T: Phosphatase that converts 8-oxodGTP to 8-oxodGMP.

8. Damage from Alkylating Agents

  • These agents add alkyl groups to bases, leading to potential malfunctioning of DNA.

  • Notable examples include:

    • Ethyl methanesulfonate (EMS)

    • Methyl methanesulfonate (MMS)

    • Nitrosoguanidine (NTG)

  • Repair involves the removal by specific N-glycosylases, followed by action from DNA polymerase I for gap filling.

9. Damage from UV Radiation

  • UV light causes DNA bases to absorb energy and form unwanted bonds, leading to issues like pyrimidine dimers, where adjacent pyrimidines become fused.

  • Pyrimidine dimers are repaired by mechanisms evolved specifically for this purpose.

9.1 Photoreactivation Repair System

  • Photolyase is an enzyme that separates fused bases during exposure to visible light, specifically wavelengths between 350 and 500 nm.

  • This mechanism is absent in humans and placental mammals.

10. General Repair Mechanisms

10.1 Methyl-Directed Mismatch Repair

  • Not specific—can repair various damages causing minor helix distortions such as mismatches and frameshifts.

  • Base Analogues: Chemicals that resemble normal DNA bases, potentially causing pairing errors.

    • Examples include 2-aminopurine (2-AP), 5-bromouracil (5-BU).

11. Mutagenesis by Base Analog Incorporation

  • 2-AP may incorrectly pair with __________ during DNA synthesis but could subsequently pair correctly or vice versa with cytosine or thymine.

12. Frameshift Mutagens

  • These ingredients cause distortions, preventing proper base alignment.

  • Ethidium bromide is an example that may result in deletions or insertions during DNA replication.

13. Mechanism of MDMR (Methyl-Directed Mismatch Repair)

  • Involves several genes: mutS, mutL, mutH, and dam.

  • Dam methylase marks adenine in the sequence GATC/CTAG, allowing differentiation between strands post-replication.

  • MutS dimer binds at lesions while MutH and MutL assist in excising and repairing the damage with DNA polymerases.

14. General Repair Mechanism of MDMR

  • UvrD helicase assists in unwinding damaged DNA, and subsequent repair occurs, involving repair enzymes to finalize the process and methylation by Dam methylase.

15. Isolation of Mutants

  • Phenotype: Mutants exhibit high mutation rates known as mutators.

  • Detection often involves methods like colony papillation to identify hypermutators such as lac+ revertants in E. coli.

16. Nucleotide Excision Repair

  • An essential general repair mechanism repairing wide-ranging DNA damage, excising large mutations from DNA.

  • If defective, mutants are sensitive to lower radiation doses than wild-type strains.

16.1 Mechanism of Nucleotide Excision Repair

  • Involves products from uvrA, uvrB, and uvrC genes forming a complex to identify and repair DNA distortions.

  • The process involves creating a nicked strand, with DNA pol I resynthesizing the removed part and ligase sealing nicks.

17. Induction of Nucleotide Excision Repair

  • This occurs particularly through Transcription-Repair Coupling where repair genes are induced in response to DNA damage, crucial for maintaining transcriptional integrity.

  • Mfd protein functions to release stalled RNA polymerase, guiding repair mechanisms.

18. SOS Inducible Repair

  • The SOS pathway is activated under severe DNA damage, leading to expression of din genes involved in repair, regulated by LexA repressor protein, facilitating transcription upon extensive damage.

  • RecA assists in the process, binding to single-stranded DNA (ssDNA) and triggering LexA cleavage to allow for SOS gene transcription.

19. Mechanism of SOS Mutagenesis

  • Involves UmuC and UmuD, proteins enabling the DNA polymerase V to bypass damaged regions, albeit at the cost of introducing mutations.

  • The increase in DNA damage leads to a cascade resulting in active translesion synthesis, although it may result in an increased mutation rate during replication following damage repair processes.

20. Next Lecture Preview

  • Upcoming discussion on Plasmids:

    • Understanding plasmid properties and replication.