DNA Damage and Repair

DNA Structure and Supercoiling

  • Overview of DNA Structure
    • DNA is a double helix composed of deoxyribonucleic acid, with two strands running in opposite directions.
    • Supercoiling: DNA can be supercoiled to fit within cellular structures and during processes like replication.

Types of DNA Damage

  • DNA damage can arise from:
    • Endogenous by-products of metabolism
    • Exogenous environmental factors (e.g., UV radiation, chemicals)
  • Types of damage include:
    • Single and double strand breaks
    • Base oxidation and adduction
    • Crosslinking (inter- and intra-strand)
    • Potential impacts of damage include mutations and DNA insertions/deletions.

DNA Damage Mechanisms

DNA Polymerase Proofreading

  • Proofreading function: DNA polymerases possess 3' to 5' exonuclease activity that removes mis-paired bases.
    • Historical significance of maintaining genomic integrity through accurate replication.

Polymerase Errors

  • Errors arise from:
    • Altered dNTP pools or low-fidelity polymerases leading to incorrect base insertion.
    • Importance of high fidelity polymerases for genomic stability.
    • Once an error occurs, it remains in subsequent divisions unless corrected before replication.

Causes of Insertions and Deletions (Indels)

  • Insertion or Deletion (InDel): Results from polymerase slippage during replication, especially in repeat-rich sequences.

Topoisomerase Trapping

  • Endogenous and exogenous factors may cause topoisomerases to become trapped at sites of DNA damage, leading to replication obstacles.
    • Examples include drugs like doxorubicin and camptothecin.

Base Modifications

  • More frequent in single-stranded DNA during replication/transcription.
  • Caused by agents such as:
    • UV radiation
    • Nitrous acid
    • Reactive Oxygen Species (ROS)
  • Repair processes developed, focusing heavily on deamination consequences (1/3 of point mutations).

Oxidative Damage and Reactive Agents

  • Ionizing radiation and oxidative molecules significantly damage DNA, resulting in:
    • Single and double strand breaks
    • Base modifications
    • Cross-links

Summary of DNA Damage Types

  • Varied origins include:
    • Exogenous agents (UV, alkylating agents, X-rays)
    • Endogenous causes (spontaneous changes, oxidative damage).
  • DNA Damage increases the risk of ∗point mutations∗ and ∗frameshift mutations∗.

Major DNA Repair Mechanisms

Mismatch Repair (MMR)

  • Corrects errors post-replication. Proteins differentiate between old (methylated) and new (unmethylated) strands via methylation patterns.
  • Components: MutS, MutL, MutH, operate sequentially to address mismatches without altering DNA.

Base Excision Repair (BER)

  • Recognizes and repairs single base modifications and strand breaks:
    • Enzymes, e.g., DNA glycosylases, target specific lesions for enzymatic removal.
    • Offers "short" or "long" repair pathways.

Nucleotide Excision Repair (NER)

  • Removes bulky adducts and large distortions of the DNA helix through specific enzymes that identify structural changes rather than a specific base.

Non-Homologous End Joining (NHEJ)

  • Repairs double-strand breaks by recognizing and aligning broken strands before ligation, despite the risk of mutations due to processing.

Effect on Mutational Frequency

  • Mutations are spontaneous changes post-damage, influenced by mechanisms that vary in efficiency and specificity, emphasizing the need for effective DNA repair systems.