L3 DNA Replication & Repair III

DNA Replication & Repair III

DNA Damage and Its Implications

  • Types of DNA Damage:

    • DNA replication errors can lead to mutations.

    • Example of a mutated sequence:

      • Original: GTC TAA GTT A

      • Mutated: UAA GTT ACA TGU ATG TAC A

    • Failure to repair damage leads to:

    • Compromised cell survival and function.

    • Accumulation of mutations leading to cancer and genetic disorders.

Sources of DNA Damage

  • Approximately 10410^4 to 10610^6 DNA damage events occur per cell per day.

  • Causes of DNA damage include:

    • Spontaneous Hydrolysis:

    • Depurination: Loss of purines (adenine or guanine).

      • On average, ext 5000purineslost/cell/dayext{~5000 purines lost / cell / day}, resulting in abasic sites.

    • Deamination:

    • Loss of amine groups from bases, predominantly converting cytosine to uracil or methyl-cytosine to thymine.

      • Approximately 100100 bases deaminate per cell per day; adenine and guanine at roughly rac110rac{1}{10} that rate.

    • Chemical Mutagens:

    • Addition of chemical adducts to DNA bases.

      • May occur as by-products of normal oxidative metabolism or exposure to external sources (e.g., UV and ionizing radiation).

    • Radiation Damage:

    • UV radiation can cause thymine dimers, leading to helical distortions.

    • Ionizing radiation can cause double-strand breaks.

Specific Types of DNA Damage

  • Depurination:

    • N-glycosyl bond hydrolysis leads to loss of purines, resulting in abasic sites (

    • Example sequences depict the loss.

    • Affects purines G and A in DNA.

  • Base Deamination:

    • Conversion of amine group (−NH2)(-NH_2) to keto group (=O)(=O).

    • Most prevalent in cytosine where it converts to uracil, creating mismatches (e.g., C to U; Me-C to T).

  • Chemical Modifications:

    • Alkylation:

    • Transfer of methyl or ethyl groups to bases, such as O6-methylguanine, which can mispair with thymine.

    • Oxidation:

    • Interaction with reactive oxygen species leading to mutations (e.g., 8-oxoguanine can pair with adenine).

  • Base Analogs and Intercalating Agents:

    • Base Analog:

    • E.g., 5-bromouracil replaces thymine, mispairing with guanine.

    • Intercalating Agents:

    • Flat molecules that can insert between bases causing small insertions or deletions (e.g., ethidium).

DNA Damage Repair Mechanisms

  • General Mechanism:

    • Recognition and Excision:

    • Detection of damage and removal of the affected region.

    • Repair approaches vary and include:

    • Direct Reversal:

      • Photoreactivation and demethylation strategies.

    • Base Excision Repair:

      • Correcting single base mispairs or damaged bases (issues like deamination).

    • Nucleotide Excision Repair:

      • For bulky DNA adducts or distortions (like those from UV).

    • Double Stranded Break Repair:

      • Utilizes homologous recombination or nonhomologous end joining.

    • Translesion Synthesis:

      • Error-prone repair pathway for DNA lesions.

Specific Repair Processes

  • Direct Reversal Repair Mechanisms:

    • Photoreactivation:

    • Process where DNA photolyase uses light energy to reverse pyrimidine dimers, restoring the original bases.

    • Demethylation:

    • Methyltransferase removes the methyl group from O6-methylguanine; the methyl group is transferred to a cysteine residue in the protein, making it non-reversible.

  • Base Excision Repair (BER):

    • Targets damaged bases (e.g., deamination and oxidation).

    • Mechanism:

    • Recognition by specific DNA glycosylases, cleavage by AP endonuclease, followed by gap filling by DNA polymerase and ligation by DNA ligase.

    • Role of DNA Glycosylases:

    • 11 glycosylases identified; each specializing in removing specific damaged bases.

  • Nucleotide Excision Repair (NER):

    • Handles helix-distorting damage.

    • Proteins involved include UvrABCD in E. coli and related mechanisms in eukaryotes.

    • Mechanism:

    • UvrAB complex recognizes damage, UvrB unwinds the DNA, while UvrC cleaves the damaged site.

  • Transcription Coupled Repair:

    • Actively transcribed regions repair more readily when RNA polymerase stalls at a lesion. Recursion of repair proteins to maintain functionality.

  • Double-Stranded Break (DSB) Repair:

    • Caused by free radicals, ionizing radiation, or chemical agents. Repair strategies include homologous recombination and nonhomologous end joining.

    • Homologous Recombination:

    • Requires a homologous template to accurately repair breaks by strand invasion and DNA synthesis, resulting in correction through sister chromatids.

    • Nonhomologous End Joining:

    • A more error-prone method resulting in the potential loss of nucleotides when joining ends of DNA strands.

  • Translesion Synthesis:

    • Allows polymerases to bypass lesions but is error-prone; activated under SOS responses to continue DNA synthesis.

Hereditary Diseases Linked with DNA Repair Defects

  • Hereditary Non-Polyposis Colorectal Cancer:

    • Mismatch Repair defect leading to colon and ovarian cancer susceptibility.

  • Xeroderma Pigmentosa:

    • Nucleotide Excision Repair defect leading to increased risk of skin cancers.

  • Bloom’s Syndrome:

    • Homologous Recombination defect exposing individuals to various malignancies.

  • Hereditary Breast Cancer:

    • Defect in homologous recombination causing increased breast and ovarian cancer risk.

Summary

  • DNA Damage Sources:

    • Can be spontaneous or due to internal/external agents.

  • Repair Mechanisms Include:

    • Direct reversal, Base excision, Nucleotide excision, Double-strand break repair, and Translesion synthesis.