BIOS3020 Molecular and Cellular Biology

Instructor: Dr. Emma HargreavesEmail: e.j.mead@kent.ac.ukText: Essential Cell Biology, Fifth Edition, W. W. Norton & Company, 2019

Chapter Overview

Focus: DNA Replication, Repair, and RecombinationReading: Essential Cell Biology, 5th edition, Bruce ALBERTS et al. ISBN: 9780393680393 (Available in paperback, published in 2019)

Key Concepts in DNA Structure and Mutation

• Examples of DNA Mutations

  • Normal vs. Mutant Gene: Example - B-globin gene shows a mutation in a single nucleotide from A, leading to changes in protein expression, critical for oxygen transport in the body.

Types of Mutations:

1. Point Mutations:

   • Substitutions:

     • Silent: No change in protein sequence due to redundancy in genetic code (e.g., both GAA and GAG encode for glutamic acid).

     • Missense: Results in a different amino acid, potentially altering protein function (e.g., Sickle Cell Disease caused by a missense mutation in the β-globin gene where glutamic acid is replaced by valine, affecting hemoglobin's properties). Severity can depend on the position of the mutation in the protein structure.

     • Nonsense: Creates a truncated protein due to the introduction of a stop codon (e.g., β-thalassemia results from such mutations affecting hemoglobin).

2. Insertions and Deletions:

   • Cause frameshift mutations that distort the reading frame, affecting downstream amino acids (e.g., Haemophilia A linked to mutations in the F8 gene).

DNA Replication Error Rates:

• Without proofreading: 1 mistake per 10^5 nucleotides.

• With proofreading: 1 mistake per 10^7 nucleotides.

• With mismatch repair: 1 mistake per 10^9 nucleotides, making repair processes essential for fidelity.

Importance of DNA Repair:

• Errors can lead to permanent mutations if not corrected, which can manifest as various diseases, including cancers. Proofreading by DNA polymerase is crucial for minimizing replication errors.

Mechanisms of DNA Repair:

1. Recognition and Repair of Errors:

   • Distortions in the DNA backbone due to mis-pairs or chemical lesions are recognized by repair enzymes.

2. Bacterial Repair Mechanism:

   • Methylation marks the newly synthesized strand allowing for mismatch recognition and subsequent repair, utilizing proteins like MutS and MutL to identify and repair mismatches.

3. Slippage and Repeat Expansion:

   • During replication, slippage can lead to changes in repeat numbers (e.g., CAG repeat expansions), impacting genetic stability and leading to conditions like Huntington’s Disease.

Consequences of Repeat Changes:

• Generally harmless when mutations occur outside coding regions but can lead to severe conditions when within coding regions (e.g., Huntington’s Disease, Fragile X Syndrome).

Sources of Mutation:

1. Point Mutations arise from errors in replication and various forms of chemical DNA damage.

   • Transitions: Purine to purine or pyrimidine to pyrimidine (e.g., adenine to guanine).

   • Transversions: Purine to pyrimidine or vice versa (e.g., guanine to cytosine).

2. Spontaneous Mutations:

   • Depurination: Loss of purine bases occurs naturally at about 5000 bases per cell per day and can lead to substitutions if not repaired.

   • Deamination: Conversion of cytosine to uracil, which leads to transition mutations if left unrepaired at the DNA level (e.g., CG to TA transition).

3. Chemically Induced Mutations:

   • Alkylating Agents: These chemical agents modify bases (e.g., ethyl methanesulfonate alters guanine structure).

   • Base Analogues: Incorporate incorrect bases during replication (e.g., 5-bromouracil mimics thymine).

   • Intercalating Agents: These cause frameshifts by slipping between bases during the DNA replication process, leading to insertion or deletion mutations.

Specific DNA Damage Types:

• Thymine Dimers:

  • Caused by UV damage, these distort the DNA structure and facilitate nucleotide excision repair for correction, preventing replication errors.

Repair Mechanisms for Double-Strand Breaks:

1. Homologous recombination utilizes undamaged DNA as a template for accurate repair, ensuring sequence fidelity.

   • Key Steps include:

     • Strand invasion, base pairing, synthesis using complementary strands, and ligation to restore DNA integrity.

Cumulative Nature of Mutations:

• Mutations can accumulate over time, influencing long-term health and disease prevalence, such as cancer, highlighting the necessity for understanding and repairing genetic integrity.

Conclusion and Summary:

Understanding the types of mutations, their sources, and the mechanisms of repair is crucial for comprehending genetic stability and its implications in various diseases. The interplay between DNA replication fidelity and repair systems underpins the molecular basis of genetic diseases and offers insights into potential therapeutic strategies for their management.