Mutations and DNA Repair Mechanisms Detailed Notes

Mutations and DNA Repair Mechanisms

Mutations

• Changes in the structure of DNA, occurring spontaneously or by induction.
• Mutagens: Physical or chemical agents causing mutations.
• Somatic mutations: Occur in somatic cells (body cells), not passed to future generations.
• Germline mutations: Occur in germ cells (egg, sperm, or their precursors), inherited across generations.

Factors Causing Mutations

Endogenous Factors

• Chemical changes: Deamination, methylation.
• Loss of bases: Depurination, depyrimidination.
• Oxidative damage
• Replication errors
• Mismatchings: Insertions, deletions

Exogenous Factors

• Physical agents: UV and ionizing radiation.
• Chemical agents: Alkylating agents, chemotherapy agents.

Examples of Endogenous Factors

• Loss of Bases (Depurination): Spontaneous loss of purine bases.
• Chemical Changes (Deamination): Loss of amino groups from bases. For example, Cytosine becomes Uracil.

How Chemical Modifications Produce Mutations

(A) Cytosine Deamination:

• Cytosine loses its amino group, becoming Uracil.
• During DNA replication, Uracil pairs with Adenine, leading to a CG to TA change.

(B) Adenine Depurination:

• Replication machinery skips the missing purine on the template strand.
• Results in nucleotide deletion in the newly synthesized strand.

Examples of Exogenous Factors

• UV-induced thymidine dimers interfere with DNA conformation and stop DNA replication.
• Ionizing radiation breaks covalent bonds in DNA, causing chromosome breaks, leading to deletions and translocations.
• Chemical agents cause point mutations like deamination, depurination, base changes, insertion, and deletion.
• Physical agents: UV and ionizing radiation.

Mutation Types

• Gene Mutations: Occur in the base sequence of DNA; can involve single or multiple nucleotide pairs.
• Chromosomal Mutations: Changes in the number or structure of chromosomes.

Gene Mutations - Base Changes (Point Mutations)

• Base pair is replaced by another base pair.
  • Transition: Purine replaced by purine or pyrimidine replaced by pyrimidine.
  • Transversion: Purine changed to pyrimidine or vice versa.
• Insertion
• Deletion
• Frameshift mutations: Insertion or deletion of a nucleotide may cause frameshift mutations.

Gene Mutations - Point Mutations (Detailed)

• Silent mutations: No change in the synthesized amino acid.
  • Example: TTA (leucine) → TTG (leucine)
• Missense mutations: Alters the synthesized amino acid.
  • Example: GCA (alanine) → GAA (glutamic acid)
• Nonsense mutations: Generates a stop codon, resulting in a short protein.
  • Example: TTA (alanine) → TGA (stop codon)

Gene Mutations - Insertion/Deletion & Frameshift Mutations

• Insertion or deletion of a nucleotide in the coding region causes a shift in triple codons during reading, known as frameshift mutations.
• Alters the reading frame, causing amino acid changes and synthesis of nonfunctional proteins.

Chromosomal Mutations

• Changes in chromosomes, classified into:
  • Numerical anomalies
  • Structural anomalies

Chromosomal Anomalies

• Structural anomalies: Occur in all cells, early in development due to abnormal egg/sperm formation, fertilization, or defects in early embryonic life.
• Somatic / Gain anomalies: Observed in certain cells or tissues of an individual; the person is a mosaic.

Chromosome Set (n) & Ploidy

• Chromosome Set (n): Number of chromosome types in a cell with a nucleus.
  • Human: $n = 23$
• Ploidy: Number of chromosome sets (n) cells have.
  • Haploid: Single chromosome set (n) (e.g., sperm and egg).
  • Diploid: Two copies of chromosome set (2n).

Chromosomal Mutations - Numerical Anomalies

• Euploidy: Increases or decreases the number of chromosomes in multiples of n.
  • Types:
    • Monoploidy: Having (n) number of chromosomes.
    • Polyploidy: Increase in chromosome number in multiples of n.
      • Triploid (3n), Tetraploid (4n), Polyploid (> 4n).
• Aneuploidy: Decrease or increase in chromosome number other than multiples of n.
  • Examples: Trisomy, Monosomy.

Mechanism for Formation of Numerical Chromosomal Anomalies

• Mitotic Nondisjunction: Slow chromosome motion or chromatid in anaphase results in exclusion from new nuclei; delayed chromosomes are lost (anaphase delay).

Chromosomal Mutations - Structural Anomalies

• Deletion: Loss of segments of a chromosome.
• Inversion: Part between two breaks reinserts after rotating 180°.
• Translocation: Exchange of parts between 2 chromosomes.
• Isochromosome: Error in centromere division resulting in chromosomes with two copies of one arm and no copies of the other.

Examples of Chromosomal Mutations

• Deletion: $46,XY,del(7)(q31q36)$
• Translocation: $t(9;22)$
• Isochromosome: $i(17)(q10)$
• Philadelphia (Ph) chromosome in CML: Resulting from $t(9;22)(q34;q11.2)$

Effects of Mutations

• Some mutations are lethal.
• Some mutations are «Loss-of- function» mutations.
• Some mutations are «gain-of function» mutations.
• Some mutations are neutral.

DNA Repair Mechanisms

• Thousands of mutations can occur in the DNA of a human cell every day.
• Organisms need to repair these mutations to maintain genetic stability through DNA repair mechanisms.
• Very few mutations (less than 0.02%) accumulate permanently due to these repair mechanisms.

Endogenous DNA Lesions and Repair

• Examples:
  • Depurination: 18,000 repairs in 24 hours.
  • Depyrimidination: 600 repairs in 24 hours.
  • Cytosine deamination: 100 repairs in 24 hours.
  • 5-Methylcytosine deamination: 10 repairs in 24 hours.
  • 8-oxo G: 1500 repairs in 24 hours.
  • Ring-saturated pyrimidines: 2000 repairs in 24 hours.
  • Lipid peroxidation products: 1000 repairs in 24 hours.
  • 7-Methylguanine: 6000 repairs in 24 hours.
  • 3-Methyladenine: 1200 repairs in 24 hours.
  • 06-Methylguanine: 20-100 repairs in 24 hours.

DNA Repair Mechanisms Overview

• Base excision repair
• Nucleotide excision repair
• Mismatch repair
• Repair of double-strand breaks

Base Excision Repair

• Repairs oxidized, alkylated, deaminated, and open-chain bases.
• 3 Stages:
  1. Defective base recognized by DNA glycosylase and removed, creating a gap. AP endonucleases cleave the phosphodiester backbone.
  2. DNA polymerase fills the gap using the undamaged DNA as a template.
  3. Ligase seals the double-stranded DNA.

Nucleotide Excision Repair

• Repairs damage caused by large defects in the DNA double helix.
• Examples: Covalent reactions of DNA bases with large hydrocarbons (e.g., carcinogenous benzopyrene) or pyrimidine dimers caused by UV light.
• Process:
  1. A large multienzyme complex scans the DNA to find the damage.
  2. The phosphodiester backbone is cut at the site of damage.
  3. DNA Helicase removes the damaged site.
  4. The gap is filled with DNA polymerase.
  5. DNA ligase seals the strand.

Molecular Mechanism of Nucleotide Excision Repair

• Key factors and their functions:
  • E. coli: UvrA (damage recognition), UvrB (damage recognition, DNA unwinding), UvrC (5' and 3' incisions), UvrD (release of damaged oligomer), Mfd (transcription-repair coupling).
  • Humans: RPA (damage recognition), TFIIH (PIC assembly, DNA unwinding), XPC (damage recognition), XPG (3' incision), XPF (5' incision), CSB (transcription-coupled repair), DDB (chromatin repair).

Repair of Double-Strand Breaks

• Dangerous DNA damages as there is no template for repair.
• Caused by ionizing radiation, oxidizing agents, and metabolites.
• If unrepaired, lead to chromosome fragmentation and gene loss.
• Cause structural chromosomal abnormalities like translocation, inversion, and insertion.

Mechanisms for Repair of Double-Strand Breaks

• 1- Nonhomologous End Joining:
  • Broken ends are joined with DNA ligation.
  • Usually results in a loss of nucleotides in the junction region.
  • Common in mammalian somatic cells.
• 2- Homologous Recombination:
  • DNA repair using sister chromatids as a template.
  • Used during or immediately after DNA replication.

Nonhomologous End Joining (Detailed)

• Ku proteins detect broken chromosome ends and bind.
• Additional proteins keep the broken ends together.
• Broken ends are then joined together.

Homologous Recombination (Detailed)

• Specific nucleases cut off the region of the double-strand break, forming single-strand protruding ends.
• One of the single-strand 3’ ends searches for a homologous sequence on the sister chromatid to base pair with.
• DNA polymerase extends the invading strand using the undamaged DNA strand as a template.
• Damaged DNA is repaired, and the ends are sealed with DNA ligation.

Repair of a Broken Replication Fork by Homologous Recombination

• The moving replication fork stops when it encounters a single-strand break.
• Damage is repaired by homologous recombination.

Mismatch Repair

• Repairs mismatching bases during replication.
• In Prokaryotes:
  • MutS detects the mismatch.
  • MutS-MutL complex activates MutH.
  • MutH creates a nick on the reverse strand; enzymes remove the defective region.
  • DNA polymerase fills the gap, and DNA ligase performs the ligation.
• In Eukaryotes:
  • Homologues of MutS: Msh2 and Msh6.
  • Homologues of MutL: Mlh1 and Pms1.

Inherited Human Syndromes with Defects in DNA Repair