Gene Mutation, DNA Repair, and Homologous Recombination

Gene Mutation, DNA Repair, and Homologous Recombination

Genetic Variation and Its Importance

  • Genetic variation among individuals provides the raw material for evolution.
  • Genetic analysis relies on variants, which are individuals showing phenotypic differences in one or more characters.
  • Variants arise through:
    • Mutation
    • Recombination Processes Leading to Genetic Variation

Empirical Evidence for Random Mutation

  • Results from studies demonstrated considerable variation in the numbers of bacteriophage-resistant bacteria in cultures, which supports the random mutation hypothesis. This was investigated through Luria and Delbrük’s Fluctuation Test.

Types and Mechanisms of Mutation

  • Spontaneous mutations occur naturally and randomly, typically linked to normal biological or chemical processes within the organism.
  • Induced mutations result from the influence of external (extraneous) factors, which can be either natural or artificial.
  • Mutations can also be categorized as follows:
    • Somatic mutations: Occur in any cell except germ cells and are not heritable.
    • Germline mutations: Occur in gametes and are inherited.

Types of Mutations

  • Regulatory Mutations:

    • Affect regulatory regions such as promoters and 5' & 3' UTR segments.
    • Promoter mutations: Specifically alter consensus sequence nucleotides of promoters affecting transcription initiation (impact ranging from mild to complete failure).

  • Splicing Mutations:

    • Alter intron/exon boundaries and target splicing processes.
    • Can lead to splicing errors and generation of mutant proteins due to intron retention in the mRNA.
    • A cryptic splice site is a new splice site that competes with the original for splicing.

Point Mutations

  • Types of point mutations include:
    • Transition: A pyrimidine replaces a pyrimidine, or a purine replaces a purine.
    • Transversion: A purine is interchanged with a pyrimidine.
    • The transition/transversion ratio is generally around 2 but is typically higher in coding regions, as transversions are more likely to affect the amino acid and may result in fatal mutations.
    • Silent substitutions: Point mutations which do not change the encoded amino acid.
    • Stop-loss mutations: Change a stop codon to an amino acid, lengthening the polypeptide during translation.

Consequences of Point Mutations

  • Various mutations in a DNA sequence can lead to distinct outcomes in the translated polypeptide, such as:
    1. Wild-type Sequence:
    • DNA Strand: 5' ||TTA TTT AGA TGG TGT || 3'
      Template Strand: 3' ||AAT AAA TCT ACC ACA || 5'
      mRNA: 5' UUA UUU AGA UGG UGU || 3'
      Polypeptide: Leu Tyr Arg Trp Cys C
    1. Missense Mutation: Changes one amino acid (example given).
    • DNA Strand: 5' TTA TTT AGA AGG TGT 3'
      mRNA: 5' UUA UUU AGA AGG UGU || 3'
      Polypeptide: Leu Tyr Arg Arg Cys C
    1. Synonymous Mutation: Does not change the amino acid.
    2. Nonsense Mutation: Results in a stop codon, leading to termination of translation.

Frameshift Mutations

  • Frameshift mutations may arise due to insertion or deletion of nucleotides, altering the reading frame of the genetic code. There are two varieties:
    1. Insertion of a single base pair: Alters the polypeptide sequence.
    2. Deletion of a single base pair: Also leads to an altered amino acid sequence.

Reversion Mutations

  • Forward mutation: Converts a wild-type allele to a mutant allele.
  • Reverse mutation (reversion): Converts mutant alleles back to wild-type or near wild-type alleles.

Inducing Mutations with Mutagens

  • Mutagens are agents that induce mutations, either natural or artificial. Different mutagens induce mutations with varying specificities.
    • Examples of mechanisms include:
    • Nucleotide Analogs: Mispairing during replication leading to mutation.
      • Example: 5-Bromouracil leads to base mispairing.
    • Chemical Mutagens: Alteration of base structure which can lead to mispairing.
    • Nitrous Acid: Induces transitions by converting adenine to hypoxanthine.
    • Alkylating Agents: Modify bases in a way that leads to mispairing during replication.
    • Hydroxylamine: Converts cytosine to a compound that mispairs with adenine.

Spontaneous Mutations

  • Spontaneous mutations arise without external inducing agents, primarily due to errors during DNA replication or spontaneous base changes.
    • High fidelity of DNA replication is attributed to:
    • Accuracy of DNA polymerases
    • Proofreading abilities of DNA polymerases
    • Mismatch repair mechanisms
    • Mismatches occur at a rate of about 1 imes 10^{-9} in wild-type E. coli as well as eukaryotes.
    • Strand slippage can lead to changes in the number of DNA repeats.

Insertion or Deletion from Replication Error

  • Slippage during replication results in either insertions or deletions of nucleotides:
    • Example: Repeated sequences can stabilize loops, leading to increased or decreased repeats in the daughter strand.

Trinucleotide Repeat Mutations

  • Table 11.2 Human Trinucleotide Repeat Disorders lists various diseases linked to repeat expansions and their implications.
    • Fragile X syndrome: Affects mental development, linked to the repeat of the CGG sequence.
    • Huntington's Disease: Linked to the CAG repeat, which increases with generations leading to severe movement disorders.
    • Highly expanded sequences can lead to disease phenotypes, e.g., from normal to diseased states in Huntington's Disease > 35 repeats.

DNA Repair Mechanisms

  • Incorrectly inserted nucleotides during DNA replication are corrected by:
    • DNA polymerase III: Engaged in proofreading.
  • Photoreactivation Repair: Specific to repairing UV-induced thymine dimers via photolyase enzyme.
  • Nucleotide Excision Repair (NER): Involves several key proteins (e.g., UVR genes) to remove bulky lesions, ensuring DNA integrity.
    • Base Excision Repair: Recognizes erroneous bases and cuts the DNA backbone at specific sites to replace them.
  • Nonhomologous End Joining (NHEJ): Repairing double-stranded breaks (DSB) in a way that may lead to translocations.
  • Synthesis Dependent Strand Annealing (SDSA): Utilizes sister chromatids for repair, providing an accurate template for replication, reliant on proteins such as Rad51.

Homologous Recombination (HR)

  • Meiotic recombination occurs between nonsister chromatids, significantly impacting genetic diversity.
  • Key processes include:
    • Formation of double-strand breaks (DSB) by Spo11.
    • Rad51 recognizes DSBs and facilitates strand invasion and exchange processes.
  • Formation of Holliday junctions and resolution of these junctions determines the final outcome of the recombination, which can be opposite sense or same sense, influencing genetic lineage and diversity.

Conclusion

  • Understanding gene mutation, DNA repair mechanisms, and homologous recombination is critical for fields such as genetics, molecular biology, and biomedicine.