DNA Repair 5

Overview

Maintaining genomic integrity is vital for cell survival and the prevention of diseases like cancer. DNA is constantly subjected to damaging agents from both endogenous and exogenous sources. These notes explore the types of damage, the sources of these lesions, and the sophisticated cellular pathways designed to repair them.

DNA Damage

DNA damage refers to physical or chemical alterations to the DNA structure. If left unrepaired, these lesions can interfere with replication and transcription, leading to mutations or cell death.

Types of DNA Damage

1. Single-Base Alterations:

   • Abnormal bases: Such as uracil (from cytosine deamination) or hypoxanthine.

   • Base-pair mismatches: Occurring during replication.

   • Abasic sites: Known as AP (apurinic/apyrimidinic) sites where the nitrogenous base is missing.

2. Structural Distortions:

   • Thymine dimers: Cyclobutane pyrimidine dimers (CPDs) caused by UV radiation.

   • Chemical adducts: Large molecules attached to bases (e.g., benzo[a]pyrene).

3. Strand Breaks:

   • Single-strand breaks (SSBs): Interruptions in the phosphodiester backbone of one strand.

   • Double-strand breaks (DSBs): Breaks in both strands, often considered the most lethal form of damage.

4. Cross-links:

   • Intra-strand: Cross-links between bases on the same strand.

   • Inter-strand: Covalent links between bases on opposite strands, preventing strand separation.

Sources of DNA Damage

Spontaneous (Endogenous) Damage

• Replication Errors:

  • DNA polymerases exhibit high fidelity, but errors occur approximately once every $10^7$ base pairs.

  • Proofreading: Most polymerases have $3' \rightarrow 5'$ exonuclease activity to correct errors immediately.

  • Strand Slippage: Occurs in repetitive sequences (microsatellites), leading to insertions or deletions.

• Tautomerisation:

  • Bases exist in dynamic equilibrium between keto/enol or amino/imino forms.

  • If a base is in its rare enol form during replication, Guanine ($G$) might pair with Thymine ($T$) instead of Cytosine ($C$).

• Hydrolytic Reactions:

  • Deamination: Loss of an amino group. Cytosine deaminates to Uracil (~100 per cell per day). 5-methylcytosine deaminates to Thymine, creating a "mutation hotspot" because $T$ is a natural DNA base.

  • Depurination: Hydrolysis of the $N$-glycosidic bond. Approximately 10,000 purines ($A$ and $G$) are lost per cell per day, resulting in AP sites.

• Oxidative Damage:

  • Reactive Oxygen Species (ROS) like $OH^\bullet$ (hydroxyl radicals) and $O_2^-$ (superoxide) modify bases.

  • A common lesion is 8-oxo-guanine, which can mispair with Adenine.

Induced (Exogenous) Damage

• Chemical Mutagens:

  • Intercalating agents: Proflavine or ethidium bromide wedge between base pairs, distorting the helix and causing frameshift mutations during replication.

  • Base analogues: 5-bromouracil ($5-BU$) mimics thymine but frequently shifts to an enol form that pairs with Guanine.

  • Alkylating agents: Chemicals like ethylmethane sulfonate (EMS) add alkyl groups (e.g., methyl or ethyl) to bases, changing their pairing properties.

• Physical Mutagens:

  • UV Radiation: Specifically $UV-C$ and $UV-B$ create covalent bonds between adjacent pyrimidines (usually Thymine), creating bulky dimers that stall DNA polymerase.

  • Ionising Radiation (X-rays, Gamma rays): Generates ROS and directly breaks the DNA sugar-phosphate backbone, leading to SSBs and DSBs.

Repair Mechanisms

Cells have evolved distinct pathways to handle specific types of lesions. The repair process generally involves detection, excision, DNA synthesis, and ligation.

1. Direct Reversal of Damage

Some lesions can be repaired without removing any nucleotides.

• Photoreactivation: DNA photolyase (found in many organisms, but not placental mammals) uses visible light energy to break the bonds of thymine dimers.

• Demethylation: The protein $O^6$-methylguanine-DNA methyltransferase (MGMT) transfers a mutagenic methyl group from Guanine to its own cysteine residue. This is a "suicide enzyme" mechanism because the protein is inactivated after one use.

2. Base Excision Repair (BER)

Repairs non-bulky damage like deaminated or oxidized bases.

1. Detection: A specific DNA Glycosylase recognizes the damaged base and cleaves the $N$-glycosidic bond, leaving an AP site.

2. Incision: AP Endonuclease cuts the phosphodiester backbone at the $5'$ end of the AP site.

3. Excision: Phosphodiesterase removes the remaining sugar-phosphate residue.

4. Gap Filling: DNA Polymerase \beta (in eukaryotes) adds the correct nucleotide.

5. Sealing: DNA Ligase restores the backbone.

3. Nucleotide Excision Repair (NER)

Repairs bulky, helix-distorting lesions (e.g., thymine dimers).

• In E. coli (UvrABC pathway): The UvrAB complex scans DNA; UvrC (exinuclease) cuts the damaged strand on both sides of the lesion; UvrD (helicase) removes the segment.

• In Eukaryotes: Divided into Global Genome NER (surveys the whole genome) and Transcription-Coupled NER (repairs DNA that is being actively transcribed).

4. Mismatch Repair (MMR)

Corrects errors that escaped proofreading during replication.

• Strand Discrimination: In E. coli, the parental strand is methylated at $GATC$ sites by Dam methylase. The unmethylated (new) strand is targeted for repair. In humans, the mechanism likely relies on nicks in the lagging strand.

• Proteins: Involves MutS (recognizes mismatch), MutL (links MutS to MutH), and MutH (cleaves the unmethylated strand in bacteria).

5. Double-Strand Break (DSB) Repair

1. Non-Homologous End Joining (NHEJ): Ends are simply ligated back together. It is fast but error-prone because nucleotides are often lost or added at the junction.

2. Homologous Recombination (HR): Uses a sister chromatid as a template for error-free repair. This occurs mainly in the $S$ and $G_2$ phases of the cell cycle.

Insights from Disease

• Xeroderma Pigmentosum (XP): Caused by mutations in NER proteins. Patients are extremely sensitive to sunlight/UV, leading to severe skin lesions and a 1000-fold increase in skin cancer risk.

• Lynch Syndrome (HNPCC): Caused by mutations in human mismatch repair genes (e.g., $MSH2$, $MLH1$). It results in microsatellite instability and a high predisposition to colon and endometrial cancers.

• BRCA1/BRCA2 Mutations: Deficiencies in these genes impair Homologous Recombination, significantly increasing the risk of breast and ovarian cancers.