DNA Replication & Repair

DNA Replication & Repair

DNA replication is the process of copying the double helix before cell division, ensuring each daughter cell receives an identical genome. It is semi-conservative (Meselson-Stahl experiment) — each new DNA molecule contains one original and one newly synthesized strand.

Key Properties of DNA Replication

  • Semi-conservative: Parent strand serves as template for new strand
  • Bidirectional: Proceeds in both directions from Origin of Replication (ori)
  • Semi-discontinuous: Leading strand synthesized continuously; lagging strand synthesized as Okazaki fragments (100–200 nt in eukaryotes)
  • 5'→3' direction: DNA polymerase only adds nucleotides to 3'-OH end
  • Requires RNA primer: Primase synthesizes short RNA primer (~10 nt) to provide 3'-OH for DNA pol to extend

Key Enzymes in Replication

  • Helicase: Unwinds double helix at replication fork (5'→3' direction); breaks H-bonds between strands
  • Topoisomerase I: Relieves positive supercoiling ahead of fork by nicking and re-sealing one strand
  • Topoisomerase II (Gyrase in bacteria): Relieves supercoiling by cutting both strands. Target: Fluoroquinolones (Ciprofloxacin)
  • SSB Proteins (RPA in eukaryotes): Stabilize single-stranded template
  • Primase: Synthesizes RNA primer. Cannot proofread.
  • DNA Pol III (prokaryotes) / DNA Pol δ, ε (eukaryotes): Main replicative polymerases; 5'→3' synthesis + 3'→5' proofreading (exonuclease)
  • DNA Pol I (prokaryotes): 5'→3' exonuclease removes RNA primers and fills gaps
  • DNA Ligase: Seals nicks between Okazaki fragments (uses NAD+ in bacteria, ATP in eukaryotes)
  • Telomerase: Eukaryotic RNA-dependent DNA polymerase (reverse transcriptase); extends 3' overhang of telomeres; active in germline and cancer cells; absent in somatic cells → telomere shortening → cellular senescence

DNA Repair Mechanisms

  • Direct Repair: O⁶-methylguanine methyltransferase; repairs alkylated guanine directly without strand break
  • Base Excision Repair (BER): Glycosylase removes damaged base → AP site → AP endonuclease → fills gap. Repairs: Oxidized, deaminated, or alkylated bases. AP site (abasic site) = most common DNA lesion.
  • Nucleotide Excision Repair (NER): Removes bulky lesions (UV-induced thymine dimers, crosslinks). Helicase unwinds → 12–25 nt removed → gap filled by DNA pol → ligase. Defective NER → Xeroderma Pigmentosum (UV hypersensitivity, ↑risk of skin cancer). Also Cockayne syndrome, Trichothiodystrophy.
  • Mismatch Repair (MMR): Corrects base-pair mismatches and insertion/deletion loops after replication. Proteins: MLH1, MSH2, MSH6 etc. Defective MMR → Microsatellite Instability (MSI) → Lynch Syndrome (HNPCC) → ↑colon, endometrial, ovarian cancer risk.
  • Double-Strand Break Repair: - Non-Homologous End Joining (NHEJ): Error-prone; predominant in G1 phase; joins ends directly; mediated by Ku proteins + DNA-PK. - Homologous Recombination (HR): Error-free; uses sister chromatid as template; active in S/G2 phase; requires BRCA1, BRCA2, RAD51. BRCA1/2 mutations → ↑breast and ovarian cancer risk.

DNA Damage Causes

  • UV light: Thymine dimers (CPD and 6-4 photoproducts)
  • Ionizing radiation: Double-strand breaks, oxidative damage
  • Alkylating agents (chemotherapy): Add alkyl groups to bases
  • Deamination: Cytosine → Uracil; 5-methylcytosine → Thymine
  • Depurination: Loss of purine bases spontaneously