DNA Mutation and Repair
This comprehensive summary draws on the current lecture material regarding DNA Mutation and Repair (Lecture 8 of SCIE1106), building upon the foundational concepts of DNA structure (Lecture 6) and replication (Lecture 7) discussed previously.
SCIE1106 Lecture 8: DNA Mutation and Repair
This lecture focuses on defining DNA mutation, describing the processes that cause DNA damage, detailing the consequences of this damage, and explaining the two primary cellular mechanisms for DNA repair: Mismatch Repair and Homologous Recombination. The content is largely supported by figures and concepts from Molecular Biology of the Cell (MBC) and Essential Cell Biology.
I. Definition, Consequences, and Fidelity of DNA Replication
Definition and Consequences of Mutation
A mutation is defined as any permanent and heritable change in the DNA sequence of an organism. The change must be permanent and heritable for the daughter cells to receive the mutated sequence.
Damaged DNA causes problems with DNA replication because the protein machinery required for replication recognises the DNA strand; if there are any distortions, protein binding and function can be offset. Repair mechanisms restore DNA replication and the correct nucleotide sequence.
High Fidelity and Proofreading
DNA replication is known for its "High Fidelity". Incorrect copying by DNA Polymerase is extremely rare, resulting in only one error in a billion bases (1:10⁹).
This high accuracy is achieved due to three factors:
Base-paired structure of DNA.
The primer requirements of all DNA polymerases. The primer is synthesised by DNA primase (an RNA polymerase).
The "proofreading" ability of DNA polymerases.
DNA Polymerase Proofreading: DNA polymerase possesses 3' to 5' exonuclease activity. If DNA polymerase attempts to attach an incorrect base (one that does not base pair correctly, such as Cytosine trying to bind with Adenine), the resulting distortion blocks any further elongation of the primer strand. The 3' to 5' exonuclease activity then chews back the strand, removing the incorrect base until a correctly base-paired 3' hydroxyl end is restored, allowing DNA polymerase to resume synthesis.
II. Causes of DNA Mutation
DNA is constantly susceptible to attack from environmental factors, including methylation, oxidative damage, and hydrolytic attack, which can target both the sugar phosphate backbone and the bases. These causes can be categorized into three main groups: Chemical, Radiation, and Mobile DNA.
1. Chemical Factors
Chemical factors lead to nucleotide instability or involve mutagenic chemicals.
Damage Type | Description & Consequences | Examples / Details |
|---|---|---|
Nucleotide Instability | The base is lost from the DNA backbone. DNA polymerase randomly assigns nucleotides to match the damaged site, altering the sequence. | |
Depurination | Loss of a purine (Guanine or Adenine) from the sugar phosphate backbone. | Purines are the two-ring bases (Guanine and Adenine). |
Deamination | Loss of an amine group. This changes the genetic code. | Cytosine Uracil (U); Adenine Hypoxanthine; Guanine Xanthine. |
Mutagenic Chemicals | ||
Alkylation | Electrophiles add alkyl groups (like methyl groups) to nitrogenous bases (e.g., Guanine N7 methylated guanine). This creates a distortion that stalls replication because DNA polymerase cannot recognise it. | Examples include carcinogens and methylmethane sulphonate (MEMS). |
Intercalation | A compound inserts or intercalates into the double-stranded helix. This causes distortion and stops DNA replication without changing the chemical structure of the bases. | Ethidium bromide (a carcinogen) is a classic example, formerly used routinely in labs to visualise DNA. |
2. Radiation Factors
Radiation causes physical damage to the DNA structure.
Radiation Type | Description & Consequences |
|---|---|
UV Light | Causes the formation of Thymine dimers. Adjacent Thymine bases become covalently attached to each other. This creates a bulge and distortion in the helix. |
Gamma and X-Rays | These high-energy rays attack DNA bonds by directly producing free electrons which attack the DNA backbone, OR indirectly generating hydroxide free radicals. Both mechanisms result in damage leading to single and double-stranded breaks (DSBs). |
3. Mobile DNA and Infectious Agents
Mobile DNA has the ability to insert or recombine into a target DNA molecule. Recombination is the breaking and rejoining of DNA molecules to form new combinations.
Infectious Agents (Viruses and Bacteriophages): Retroviruses (e.g., HIV, the cause of AIDS) and bacteriophages utilise the host cell replication machinery. HIV, an RNA virus, encodes the reverse transcriptase enzyme, converting its RNA into double-stranded DNA. This viral DNA then integrates into the host DNA, causing a permanent inheritable change. This insertion can physically disrupt a coding region (gene). HIV often integrates into transcriptionally active genes to ensure its own replication.
Transposons (Jumping Genes): These are linear DNA sequences that move within and between chromosomes. They insert into various DNA sequences, which can physically disrupt genes. Their excision can also result in small DNA duplications. Transposons are critical in bacterial DNA rearrangement and can transfer antibiotic resistance genes between species.
Mechanism: Transposons carry the enzyme transposase, which binds to short inverted repeat sequences flanking the transposon. This allows the transposon to be excised, forming a transpososome intermediate, which then integrates into the target DNA at a location signalled by short direct repeats.
Non-Homologous Transposition (Cut-and-Paste): The transposon is cut out of the donor DNA (leaving a DSB) and pasted into the target DNA. This requires no similarity between donor and target DNA molecules and is catalyzed by integrases and transposases.
Replicative Transposition: The donor DNA is used as a template, resulting in both the donor and target molecules acquiring the transposon.
III. DNA Repair Mechanisms
All living cells possess mechanisms for DNA repair to overcome the constant barrage of errors and damage.
1. Mismatch Repair (MMR) System
The MMR system repairs errors in the newly synthesised DNA strand to restore the correct sequence based on the template strand. This system is extremely important; without it, the error rate of replication is 1 mistake per $10^7$ nucleotides, but with MMR, the rate drops to 1 mistake per $10^9$ nucleotides.
Four Steps of Mismatch Repair:
Recognition: Repair proteins patrol the DNA, looking for mismatches (mis-paired nucleotides) and binding to the sequence.
Excision: Nucleases degrade the mismatched region, excising the strand containing the error and creating a single-strand DNA (ssDNA) patch.
Synthesis: Repair DNA Polymerase (e.g., Pol I and Pol II in E. coli, as discussed previously) uses the free 3' hydroxyl () group as a primer to synthesise the correct complementary strand, using the parental strand as a template.
Ligation: DNA Ligase seals the remaining nick in the DNA backbone.
2. Homologous Recombination (HR)
Homologous recombination is essential for repairing double-stranded breaks (DSBs) in the phosphodiester backbone of DNA. This mechanism requires that both the donor and acceptor DNA molecules have extensive sequence similarity (are homologous) so that one can act as a template for the other.
Consequences: HR can result in the accurate restoration of the original sequence. However, it can also lead to rearrangements or new combinations of DNA (recombinant genotypes), which is a source of natural genetic diversity (e.g., during meiosis).
Mechanism of HR Repair:
A double-stranded break is introduced into the damaged chromosome.
Specialised nucleases (exonucleases) digest the 5' ends of the broken strands, creating single-stranded 3' overhangs.
Strand Invasion: The single-stranded 3' overhang migrates into the homologous, undamaged recipient chromosome, complementary base-pairing with it.
Synthesis: Repair Polymerases synthesise new DNA using the undamaged homologous strand as a template.
Exchange: The crossed strands form Holliday junctions. Rotation of these junctions allows sections of the strands to be exchanged.
Ligation: DNA synthesis continues using complementary strands as a template, and DNA Ligase seals the nicks. The nucleotide sequence at the exchange site is unaltered (no additions or subtractions), ensuring accurate DSB repair.