Lecture 18: DNA Repair

Aims

To provide an overview of DNA damage and the molecular mechanisms that underpin DNA repair.

Learning Objectives

●      Describe the different types of DNA damage and the causes.

●      Understand that specific types of damage have specific mechanisms of repair

●      Describe the single strand repair pathways

●      Describe the double-strand break repair pathways

●      Understand how defects in DNA repair can lead to cancer

Importance of DNA Stability and Genetic Maintenance

• DNA encodes the essential genetic instructions required for the development and functioning of every known living organism.

• The stability of the DNA molecule is critical for cell survival and is the most robust biological defense against cancer.

• DNA is unique among biological macromolecules; while proteins and lipids are replaced when damaged, DNA is the only biological macromolecule that is actively repaired to maintain its integrity.

Sources and Categories of DNA Damage

• Cells are under constant attack from both internal (endogenous) and external (exogenous) sources.

• Endogenous Sources: These arise from spontaneous reactions occurring within the cell.

  • Hydrolysis reactions.

  • Reactive oxygen species (ROS).

  • By-products of normal cellular metabolism.

• Exogenous Sources: These originate from molecules or energy sources outside the cell.

  • Ultraviolet (UV) radiation.

  • X-rays and ionizing radiation.

  • Environmental carcinogens.

  • Chemotherapeutic agents.

Specific Types of DNA Lesions

• DNA damage varies in its physical effect on the helix and chemical effect on the bases:

  • Nucleotide Base Effects: These involve chemical changes to the bases themselves (e.g., deamination, methylation).

  • Single-Strand Effects: These involve breaks or modifications to only one strand of the DNA helix.

  • Double-Strand Effects: These affect both strands of the DNA helix and are generally more toxic to the cell.

Endogenous Lesions and Spontaneous Events

• Deamination: This is the most common spontaneous event, involving the removal of an amino group by hydrolysis. For example, the deamination of cytosine results in uracil, leading to a base transition ($CG \rightarrow TA$).

• Depurination (Abasic/AP Sites): The N-glycosidic bond is susceptible to hydrolysis, leading to the loss of a purine base. This occurs at a rate of approximately $18,000$ per genome per day. Failure to repair these results in frameshift mutations.

• Methylation: Often induced by S-adenosylmethionine (SAM), creating lesions such as $7\text{-methylguanine (7meG)}$ or O^6\text{-methylguanine (O^6meG)}.

• Oxidation: Reactive oxygen species can generate lesions such as $8\text{-oxoguanine (8oxoG)}$.

Exogenous Lesions and Physical Damage

• Pyrimidine Dimers: Induced by UV light, these lesions involve the covalent joining of adjacent bases (typically thymine). This distorts the DNA helix and reduces flexibility.

• Crosslinks: UV light and other agents can cause interstrand DNA crosslinks or DNA-protein crosslinks, both of which are highly toxic because they block the essential processes of replication and transcription.

• Phosphate Backbone Damage:

  • Single-strand breaks (SSBs): Caused by reactive oxygen species, Hydroxyurea, or Camptothecin.

  • Double-strand breaks (DSBs): Induced by X-rays, ionizing radiation, and Topoisomerase II inhibitors.

Quantitative Data on DNA Lesions

• Endogenous Frequency (Lesions per cell per day):

  • Depurination: $10,000$

  • Cytosine deamination: $100-500$

  • SAM-induced methylation: $600$

  • Oxidation ($8oxoG$): $4,000$

• Exogenous Frequency (Estimated per cell):

  • Peak hour sunlight (Pyrimidine dimers): $100,000/day$

  • Cigarette smoke (Aromatic adducts): $45-1029$

  • Dental X-rays ($0.005†mSv$): $0.0002$ DSBs

  • Chest X-rays ($0.02†mSv$): $0.0008$ DSBs

  • Body CT ($7†mSv$): $0.28$ DSBs

  • External beam therapy ($1800-2000†mSv$): $72-80$ DSBs

  • Space mission ($60†days$): $2$ DSBs

Mutational Consequences of Unrepaired Damage

• Transitions vs. Transversions:

  • Transitions (substituting a double-ring purine for another purine, or a single-ring pyrimidine for another pyrimidine) are more likely than transversions (substituting a purine for a pyrimidine or vice versa).

  • Examples of transitions: $C \rightarrow T$ or $A \rightarrow G$.

• Frameshift Mutations:

  • Caused by the deletion or insertion of bases (often at abasic sites).

  • DNA is read in three possible reading frames. A frameshift shifts the codon alignment, changing every subsequent amino acid in the protein sequence (generating missense proteins) or creating premature stop codons.

  • Frameshift mutations are generally much more damaging to the cell than simple base substitutions.

DNA Repair Pathways

Base Excision Repair (BER)

• Specializes in repairing damaged single bases (e.g., uracil from deamination or abasic sites).

• Mechanism:

  1. Uracil DNA Glycosylase: Recognizes the error and uses a "base-flipping" strategy to rotate the base $180^{\circ}$ out of the helix to identify it.

  2. The glycosylase removes the damaged base, leaving an abasic (AP) site.

  3. AP Endonuclease and Phosphodiesterase: Remove the sugar-phosphate backbone at the damaged site.

  4. DNA Polymerase: Adds the correct new nucleotide using the available $3'$ hydroxyl end.

  5. DNA Ligase: Seals the remaining nick in the backbone.

Nucleotide Excision Repair (NER)

• Repairs damage involving more than one base or large helix distortions, such as pyrimidine dimers caused by UV light.

• Involves the excision of a short patch of single-stranded DNA containing the affected bases, followed by resynthesis.

Translesion Synthesis

• Employed when DNA is highly damaged. Specialized translesional DNA polymerases can replicate across damaged templates.

• These polymerases lack precision in substrate choice and do not possess exonucleolytic proofreading activity, making them responsible for most base substitutions and single-nucleotide deletions.

Double-Strand Break (DSB) Repair

• Non-Homologous End Joining (NHEJ):

  • A simple mechanism that merely joins the broken ends of the DNA together.

  • Highly error-prone; often results in the deletion of nucleotides at the break site.

  • Restricted primarily to the G1 phase of the cell cycle.

• Homologous Recombination (HR):

  • An error-free repair mechanism.

  • Uses the intact sister chromatid as a precise template for repair.

  • Occurs only during the S-phase or G2 phase when sister chromatids are available.

  • Involves the formation of a structure called a Holliday junction.

Cell Cycle Control and Damage Detection

• The cell cycle is arrested at three main checkpoints to allow for repair: G1, entry to S-phase, and G2 (entry into mitosis).

• Detection Mechanism:

  1. Kinases ATM and ATR are activated and associate with the site of damage.

  2. These kinases activate downstream signaling pathways.

  3. The protein p53 is stabilized and activates p21.

  4. p21 acts as an inhibitor, rendering G1/S-CDK and S-CDK complexes inactive, thereby stopping the cell cycle.

• If repair is impossible, the cell undergoes apoptosis (programmed cell death).

Biomedical Consequences and Clinical Applications

Inherited Syndromes and Repair Defects

• Lynch Syndrome (MSH2, MLH1, etc.): Defect in Mismatch repair, leading to colon cancer.

• Xeroderma Pigmentosum (XP):

  • Autosomal recessive disease (1 in 250,000 in Europe; 1 in 40,000 in Japan).

  • Defect in Nucleotide Excision Repair (NER).

  • Results in a 2000-fold increased risk of skin cancer; mean onset age is 12 years (50 years earlier than average).

• Ataxia Telangiectasia (AT): Defect in ATM protein kinase; leads to leukemia, lymphoma, and gamma-ray sensitivity.

• BRCA1 and BRCA2 Defects:

  • Associate with breast, ovarian, and prostate cancer.

  • Defect in Homologous Recombination (HR).

  • BRCA1/2 carriers have an $80\%$ lifetime risk of breast cancer (10x higher than normal).

Synthetic Lethality in Cancer Treatment

• Cancer cells often have existing defects in DNA repair and replicate very fast even in the presence of damage.

• Targeting the Achilles Heel: We can use the interactions between repair pathways to selectively kill cancer cells.

• Synthetic Lethality Concept: If a cell has a defective gene in one repair pathway (e.g., BRCA2-deficient), it relies on a second pathway (e.g., PARP-mediated repair) for survival. By inhibiting that second pathway with a drug (e.g., PARP inhibitor), the cell is unable to repair any DNA damage and dies.

• Example: Specific killing of BRCA2-deficient tumors using inhibitors of poly(ADP-ribose) polymerase (PARP), such as Lynparza (Olaparib).