DNA Repair Mechanisms and Transcription Processes

Types of DNA Repair

• Direct Reversal
• Replacement Repair
  • Categories:
  • Base Excision Repair
  • Nucleotide Excision Repair
  • Mismatch Repair
• Recombination Repair
• Non-Homologous End Joining (NHEJ)
• Resynthesis
  • Involves stretches of replacement DNA

Types of DNA Damage

• Single Base Changes
  • Affect DNA sequence but not structure
  • Examples:
  • Mutation (e.g., cytosine to uracil, deamination of methyl-cytosine to thymine)
  • Errors during replication
• Structural Distortion Mutations
  • Affect replication and transcription
  • Examples:
  • Thymine-Thymine dimers from UV irradiation
    • Fixed by bacterial photolyase or by excision repair
  • Bulky adducts (e.g., from alkylation)
    • Distorts the DNA structure and stalls polymerases
    • Caused by:
    • Cigarette smoke (polycyclic aromatic hydrocarbons)
    • Heterocyclic aromatic amines (from BBQ meat)

Prokaryotic Repair Systems

Excision Repair

• Fixes mismatches/distorted DNA
• Repair Steps:
  1. Incision: Endonuclease cuts the strand
  2. Excision: 5’–3’ exonuclease or helicase removes damaged segment
  3. Synthesis: Single-strand region serves as a template for nucleotide replacement
  4. Ligation: DNA ligase seals the phosphodiester backbone
• Key Enzymes:
  • UvrA: Recognition
  • UvrB and UvrC: Endonuclease activity
  • UvrD: Helicase

Base Flipping

• Removes only the damaged base rather than the entire nucleotide
• Uses Glycosylases to cleave bonds and flip out bases with adducts
• Lyases: Sometimes linked to glycosylases; they open sugar rings and break the backbone

Error-Prone Repair

• Last-ditch mechanism during replication stalls to avoid cell death
• Allows incorporation of any base opposite the template, ignoring complementary base pairing.
• Umu System:
  • Induced by DNA damage; includes UmuD and UmuC genes
  • Forms a complex (umuD’2C) that aids in filling replication stalls and causes mutations

Mismatch Repair

• Precautionary mechanism to correct mismatches on the damaged strand
• General Mut System: Includes genes such as MutT, MutM, and MutY
• Detects and repairs oxidative damage (e.g., 8-oxo-Gs)
• Uses proteins such as MutS, MutL, and MutH for identification and repair

Recombination Repair

• Uses similar mechanisms found in genetic recombination
• Repairs roadblocks during replication

Eukaryotic Repair Systems

General Characteristics

• Repair mechanisms have conserved elements across species
• Mammalian examples include xeroderma pigmentosum (XP)
  • Result from mutations affecting nucleotide excision repair.
  • Sensitivity to sunlight; defects in one of eight critical genes

Double-Stranded Breaks (DSB)

• Repair through homologous recombination or NHEJ
• Involves key proteins such as Ku70, Ku80, and DNA-dependent protein kinase (DNA-PK)
• NHEJ Mechanism:
  • Recognition: Broken ends are recognized by Ku proteins
  • Trimming: Artemis helps refine ends
  • Filling: Remaining gaps filled by an unspecified DNA polymerase
  • Joining: DNA ligase and XRCC4 complete the repair

Chromatin Remodeling Following Damage

• Damaged DNA stimulates chromatin remodeling to remove nucleosomes
• Post-repair, Asf1 and Caf-1 restore chromatin structure

Mobile Genetic Elements

Transposons

• Sequences that can move within the genome and are a major source of mutations
• Prokaryotic Transposons:
  • Simple IS elements
  • Composite transposons carry additional genes; e.g., drug resistance.

Transposition Mechanisms

• Replicative: Copy-and-paste mechanism mediated by transposase
• Non-Replicative: Cut-and-paste such that original site loses the element

Consequences of Transposition

• Can cause gene disruptions and rearrangements.
• Precise excision can lead to a true reversion of sequences

Eukaryotic Transposons

Example: P-Elements in Drosophila

• P-elements are not present in lab stocks but are found in wild stocks
• Their presence can lead to hybrid dysgenesis when interbreeding with M-strains

Retroviruses

• Infectious elements spreading between cells
• Consist of ssRNA, which replicates via a dsDNA intermediate
• Integration requires a transposition-like event into host genomes

Function of Viral Genes

• Gag: Nucleoprotein core components
• Pol: Reverse transcriptase and integrase
• Env: Components of the viral envelope

Oncogenes and Cancer

• Retroviruses can acquire host genes that may lead to cancer
• V-onc: Mutant viral genes causing transformation of normal cells into cancer cells