Ch 12-18 DNA Repair Mechanisms

DNA Repair Mechanisms

Chapter Overview

• Focus on the study of DNA repair mechanisms as presented in chapters 12 and 18.

• Key topics include mutations, DNA damage responses, types of errors, and various mechanisms of DNA repair.

Outline

• Mutations and Molecular Definitions

• DNA Damage Response

• Types of Errors

• Types of Damage that Occur

• Different Mechanisms of DNA Repair

• Research Applications

Mutations and Molecular Definitions

Mutation

• Definition: An alteration in DNA sequence.

• Types of alterations:

  • Single base-pair substitution.

  • Deletion or insertion of base pairs.

  • Major alteration in chromosomal structure.

• Occurrence:

  • Can happen in somatic (body) or germ cells (reproductive cells).

  • Can be found in coding (gene sequences) or noncoding regions (regions without genes).

Classification of Mutations

By Molecular Change

• Point Mutation or Base Substitution: Change from one base pair to another.

• Missense Mutation: Results in new triplet code for a different amino acid.

• Nonsense Mutation: Results in a triplet code for a stop codon, leading to premature termination of translation.

• Silent Mutation: New triplet code still encodes for the same amino acid.

By Phenotype

• Loss-of-function mutations.

• Gain-of-function mutations.

• Visible (morphological) mutations.

• Nutritional (biochemical) mutations.

• Behavioral mutations.

• Regulatory mutations.

• Lethal mutations.

• Conditional/temperature-sensitive mutations.

By Location

• Somatic Mutations: Occur in any cell except germ cells; not heritable.

• Germ-line Mutations: Occur in gametes; heritable.

  • Autosomal mutations: Within genes on autosomes.

  • X-linked and Y-linked mutations: Within genes on X or Y chromosomes, respectively.

Mutations Occur Spontaneously and Randomly

Spontaneous Mutations

• Definition: Changes in nucleotide sequence that occur naturally.

• Arise from normal biological or chemical processes that alter nitrogenous bases.

• Spontaneous mutation rates are low across organisms.

Induced Mutations

• Definition: Result from external factors, whether natural or artificial.

• Examples:

  • Radiation (e.g., UV light).

  • Natural and synthetic chemicals.

DNA Damage Response

Triggers of DNA Damage

• Cellular processes, viral infections, exposure to radiation, chemicals, replication errors causing DNA damage.

Response Mechanisms

• Activation of cell cycle checkpoints.

• Induction of transcriptional programs involved in DNA repair or apoptosis.

Importance of DNA Repair

Implications

• Genetic Disorders: Failure in DNA repair can lead to genetic instability, contributing to genetic disorders.

• Cancer: Defective or incomplete DNA repair mechanisms can lead to cancer development and hereditary diseases.

Error Rates of DNA Polymerases

• DNA polymerase is generally accurate, with an initial mistake frequency of ~$10^{-5}$.

• Proofreading function improves accuracy with a second-pass mistake frequency of ~$10^{-2}$, leading to a net error rate of approximately $10^{-7}$.

Additional Error-Searching Processes

Mismatch Repair (MMR)

• Mechanism activated when distortions in DNA double helix due to mismatched base-pairs are detected.

• MMR adds an extra $10^{-2}$ to error probability, resulting in an overall frequency of errors getting through the process of approximately $10^{-9}$.

Replication Slippage

• Definition: Occurs when loops form in the template strand during replication, leading to missed nucleotides and small insertions or deletions.

• More frequent in repeat sequences (mutation hot spots).

• Associated with hereditary diseases like Fragile-X syndrome and Huntington's disease.

Tautomers

• Concept: Purines and pyrimidines can exist in different tautomeric forms, which increases the likelihood of mispairing during DNA replication.

Types of DNA Damage

Common Types

• Deamination (conversion of C → U or A → Hypoxanthine).

• Depurination (loss of A or G).

• T-T and T-C dimers (cross-linking of bases, prominent due to UV light exposure).

• Alkylation (addition of alkyl groups to bases).

• Oxidative damage (e.g., guanine oxidizing to 8-oxo-guanine).

• Double Strand Breaks (DSBs), which can be caused by factors like ionizing radiation and transposons.

Mechanisms of DNA Repair

Overview of Repair Process

• Steps:

  1. Damage occurs either spontaneously or through external agents.

  2. Damage is identified.

  3. A repair mechanism is mobilized.

  4. The damaged strand is excised.

  5. The undamaged strand serves as a template for correct base insertion.

  6. The repair is sealed, restoring DNA integrity.

Types of DNA Repair Mechanisms

• Nucleotide Excision Repair (NER).

• Base Excision Repair (BER).

• Double Strand Break Repair (DSBR) including Homologous Recombination (HR) and Non-Homologous End Joining (NHEJ).

• Mismatch Repair (MMR).

Nucleotide Excision Repair (NER)

• Responsible for repairing bulky, helix-distorting lesions such as thymine dimers.

• Genetic mutations in NER proteins lead to conditions like Xeroderma Pigmentosa and Cockayne’s syndrome.

Base Excision Repair (BER)

• Focuses on removing small, non-helix-distorting base lesions.

• Targets oxidized, alkylated, and deaminated bases.

Double Strand Break Repair (DSBR)

• Two main pathways:

  • Homologous Recombination (HR).

  • Non-Homologous End Joining (NHEJ).

• HR is accurate and uses a sister chromatid as a template while NHEJ is more error-prone and rapid.

Non-Homologous End Joining (NHEJ)

• Involves using short homologous sequences called microhomologies to guide repair and is active throughout the cell cycle, particularly in G1 phase when no homologous template is present.

Experimental Applications of DNA Repair Knowledge

Ames Test

• Procedure:

  • Two cultures of histidine-dependent Salmonella are treated with a suspected mutagen (in conjunction with rat liver extract).

  • Cultures are plated onto media lacking histidine, incubated, and compared for the number of revertant colonies.

• Interpretation: Increased numbers of revertants in experimental samples suggest potential mutagenic properties of the tested compound.

Conclusion

• Understanding DNA repair mechanisms is critical for insights into genetic disorders, cancer biology, and therapeutic applications such as gene editing.