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BIOL 3080 Lecutre 3 - DNA Repair and Homologous Recombination

DNA Repair and Homologous Recombination

High Fidelity DNA Replication

  • Table 5-1: The Three Steps That Give Rise to High-Fidelity DNA Synthesis

    • Each step reduces the probability of final error:

    • 5' → 3' polymerization:

      • Errors per nucleotide added: 1 ext{ in } 10^5

    • 3' → 5' exonucleolytic proofreading:

      • Errors per nucleotide added: 1 ext{ in } 10^2

    • Strand-directed mismatch repair:

      • Errors per nucleotide added: 1 ext{ in } 10^3

    • Combined Error Rate: 1 ext{ in } 10^{10}

  • The probability that an incorrect nucleotide is added during polymerization is noted as an error per nucleotide, while for proofreading and mismatch repair, it denotes the chance that an error remains uncorrected.

Exonucleolytic Proofreading by DNA Polymerase

  • If the wrong nucleotide is added, DNA polymerase cannot maintain a tight grip on the template strand.

Strand-Directed Mismatch Repair - Prokaryotes

  • Strand Distinction:

    • Depends on methylation;

    • GATC sequences are methylated, but this methylation is not immediate.

  • MutS recognizes mismatched bases in DNA and initiates the repair process:

    • Parental Strand vs Newly Synthesized Strand

    • Mismatch is corrected:

    • MutL and MutH proteins are recruited.

    • MutH breaks the DNA backbone some distance away.

    • An exonuclease removes successive nucleotides encompassing the mismatched base.

    • A DNA polymerase fills in the gaps left behind, and DNA ligase seals the backbone.

Strand-Directed Mismatch Repair - Eukaryotes

  • Distortion in the DNA helix results from mismatched base pairs which are not complementary.

  • MutS binds to the mismatched base pair:

    • MutL scans surrounding DNA for a nick.

    • Exonuclease action removes successive nucleotides, including the mismatch.

    • Missing nucleotides are filled by DNA polymerase, and the backbones are repaired by DNA ligase.

DNA Damage

  • Definition: A DNA lesion results from inherent chemical reactions within cells.

  • Implications: Without DNA repair, spontaneous DNA damage would lead to rapid changes in DNA sequences.

Table 5-2: Inherited Human Syndromes with Defects in DNA Repair

  • Names and Phenotypes

    • Mutations in MSH2, 3, 6, MLH1, PMS2: leads to colon cancer.

    • Xeroderma pigmentosum (XP) groups A-G: leads to skin cancer, UV sensitivity, and neurological abnormalities.

    • Cockayne Syndrome: caused by UV sensitivity and developmental issues.

    • Ataxia telangiectasia (AT): results in leukemia, lymphoma, and genome instability.

    • BRCA1 / BRCA2: linked to breast, ovarian, and prostate cancer predispositions.

    • Werner Syndrome: characterized by premature aging and cancer.

    • Bloom Syndrome: involves cancer at multiple sites and stunted growth.

    • Fanconi Anemia groups A-G: recognized for congenital abnormalities and hypersensitivity to DNA-damaging agents.

  • Processes Affected:

    • Mismatch repair, nucleotide excision repair, translesion synthesis, homologous recombination, and more implicated in various syndromes.

Spontaneous Nucleotide Modifications

  • Types of Damage:

    • Oxidative Damage: indicated by red arrows.

    • Hydrolytic Attack: indicated by blue arrows.

    • Methylation: indicated by green arrows.

    • The width of each arrow indicates the relative frequency of the respective event.

The Impact of DNA Repair

  • Without DNA Repair: Spontaneous DNA damage would lead to significant alterations in DNA sequences.

    • Depurination Effects:

      • Removal of guanine or adenine from DNA, leading to mutations.

    • Deamination Effects:

      • Converts cytosine into uracil, creating unnatural bases in DNA, and can occur on other bases too.

Thymine Dimer Formation

  • Caused by UV irradiation, introducing covalent linkages between neighboring pyrimidine bases (C or T).

Base Excision Repair (BER)

  • Uracil DNA Glycosylase removes deaminated cytosine.

  • AP Endonuclease recognizes and repairs a deoxyribose sugar with a missing base:

    • Removal of the sugar phosphate with a missing base occurs through sequential enzymatic activity.

    • A gap of a single nucleotide is filled and sealed by DNA polymerase and DNA ligase.

Nucleotide Excision Repair (NER)

  • A multi-enzyme complex identifies lesions such as pyrimidine dimers:

    • Cuts are made on either side of the lesion.

    • An associated DNA helicase eliminates the damaged strand portion.

    • DNA polymerase and DNA ligase repair fill in the missing DNA, coupled to transcription.

Emergency Contingencies: Translesion DNA Polymerases

  • Description: Polymerase can stall at sites of DNA damage.

  • Process: Specialized translesion polymerases address the damage, allowing less accurate but versatile replication until normal polymerases take over again.

Double-Strand Break Repair (DSB)

  • Mechanisms:

    • (A) Non-homologous End Joining:

    • Broken ends are ligated without the presence of a homologous template and typically utilize short homologous DNA sequences for guidance.

    • Ku Protein plays a role by grasping broken ends and employing additional proteins for repair.

    • (B) Homologous Recombination:

      • Used when homologous DNA is available in the nucleus; this mechanism is more complex but maintains the original DNA sequence post-repair.

Replication Fork Rescue

  • When a replication fork encounters a single-strand break, it may collapse:

    • It can subsequently be repaired via homologous recombination.

    • The repair follows similar steps as homologous recombination with new DNA synthesis occurring post-collapse.