DNA Mutations, Repair Mechanisms, and Chromosomal Change

Fundamental Principles of DNA Structure and Replication Errors

• DNA in living organisms is strictly double-stranded, except for brief instances during replication and transcription when strands must unwind to serve as templates.

• While single-stranded DNA viruses exist, viruses are not considered living cells (they are neither prokaryotes nor eukaryotes) and thus do not violate the general rule for living cellular organisms.

• DNA mutations can occur spontaneously during replication. Although DNA polymerase has proofreading capabilities, errors persist due to specific mechanical and chemical reasons.

Mechanisms of Replication Errors

Tautomerization

• Nitrogenous bases in every nucleotide can undergo tautomerization, existing in different versions based on the organization of their atoms.

• This allows bases to form hydrogen bonds with non-standard partners that the predominant form would not normally permit.

Strand Slippage

• This occurs frequently in regions of repeat sequences.

• If DNA polymerase slips during synthesis, the repeat sequences allow the DNA to kink or loop, causing polymerase to reattach in the wrong position.

• Insertions: If a loop forms on the newly synthesized (daughter) strand, an extra base or repeat is added.

• Deletions: If a loop forms on the template strand, DNA polymerase skips those bases, resulting in a deletion.

Trinucleotide Repeats and Genetic Diseases

• Repeated triplets, particularly those ending in Cytosine ($C$) and Guanine ($G$), are highly prone to strand slippage.

• Triple hydrogen bonds between $C$ and $G$ stabilize the loop structure, overcoming the lack of hydrogen bonding in other parts of the loop (e.g., Adenine ($A$) mismatches).

• This results in the expansion or reduction of repeat numbers over generations.

• Fragile X Syndrome: Associated with expansions in the promoter region. Characterized by three levels:

  • Normal: $6$ to $59$ repeats.

  • Premutation: $60$ to $200$ repeats (individuals are at risk for further expansion in offspring).

  • Full Mutation: Over $200$ repeats (causes clinical symptoms).

• Huntington’s Disease: Associated with expansions in the coding region. Levels include:

  • Normal: $6$ to $28$ repeats.

  • Premutation: $29$ to $35$ repeats.

  • Full Mutation: $36$ to $120$ repeats.

• The risk of expansion increases as the number of repeats increases, a phenomenon observed in pedigrees where premutations lead to affected subsequent generations.

Chemical and Environmental Mutagens

Spontaneous DNA Damage

• Depurination: Breaking of the bond between the sugar and the nitrogenous base.

• Deamination: Removal of an amino group from a base (e.g., Cytosine becomes Uracil). Uracil in DNA pairs with Adenine, causing a transition in the next round of replication.

• Metabolic Byproducts: Oxygen radicals, hydrogen peroxide ($H_2O_2$), and superoxides can damage bases (e.g., converting Guanine to a damaged form that pairs with Adenine).

Induced Mutations

• Base Analogs: Bromouracil mimics Thymine but can tautomerize to pair with Guanine, leading to $TA \rightarrow CG$ substitutions.

• Alkylating Agents: EMS (Ethyl Methane Sulfonate) adds ethyl groups to Guanine, making it pair with Thymine. This leads to $GC \rightarrow AT$ transitions.

• Modifying Agents: Nitrous acid modifies Cytosine into Uracil and Adenine into bases that pair with Cytosine.

• Intercalating Agents: Ethidium bromide ($EtBr$) inserts itself into the double helix, causing structural distortions that stall replication/transcription or trigger repair machinery.

• Ultraviolet (UV) Light: Causes adjacent Thymines to form thymine dimers ($T=T$), creating structural barriers.

• Ionizing Radiation (X-rays): Causes high-energy double-stranded breaks, the most damaging type of environmental mutation.

DNA Repair Mechanisms

• DNA Polymerase Proofreading: The $3' \rightarrow 5'$ exonuclease activity allows for immediate correction, reducing the error rate from one in $10^5$ to one in $10^7$ bases.

• Direct Repair: specific light-regulated enzymes can reverse thymine dimers.

• Excision Repair:

  • Base Excision Repair (BER): Removes single altered bases (non-bulky damage).

  • Nucleotide Excision Repair (NER): Removes entire nucleotides or multiple bases (bulky damage/kinks).

  • Process: Damage recognition (detecting kinks/distortions), clearing out the surrounding area to provide a $3'$ hydroxyl ($OH$) overhang, DNA polymerase filling the gap, and DNA ligase sealing the phosphodiester backbone.

• Double-Stranded Break (DSB) Repair:

  • Nonhomologous End Joining (NHEJ): Enzymes trim the ends (chewing back overhangs) and ligase joins them together. This can involve the loss of genetic material since there is no template.

  • Synthesis Dependent Strand Annealing (SDSA): Occurs after replication when sister chromatids are available. The broken strand invades the sister chromatid to use it as a template.

• Apoptosis: If damage is beyond repair, the cell undergoes programmed cell death to prevent the propagation of mutations in a multicellular organism.

Scale and Impact of Mutations

Large-Scale Mutations

• Usually involve chromosomal rearrangements:

  • Deletions/Duplications: Loss or gain of large chunks of chromosomes.

  • Inversions: A segment flips orientation ($180^{\circ}$ rotation).

  • Translocations: A piece of one chromosome moves to another.

  • Example: Philadelphia Chromosome (translocation between chromosomes $9$ and $22$). Fuses the $BCR$ promoter to the $c-ABL$ tyrosine kinase gene, leading to unregulated cell growth (leukemia). The drug Gleevec targets this specific fusion protein.

Small-Scale Mutations

• Point Mutations: Substitutions of single bases.

• Indels: Insertions or deletions of bases.

  • Frameshift Mutations: Caused by indels that are not multiples of $3$. This shifts the reading frame, turning subsequent codons into "gibberish."

• Open Reading Frame (ORF) Effects:

  • Synonymous (Silent): No change in amino acid due to the third-base wobble.

  • Nonsense: Creates a premature stop codon, resulting in a truncated protein.

  • Missense: Changes one amino acid for another.

  • Conservative: The new amino acid has similar properties (e.g., Alanine to Valine) and may have minimal effect.

  • Non-conservative: The new amino acid has different properties (e.g., negative charge to hydrophobic), potentially causing major functional changes.

Functional Categories of Alleles

• Wild Type: The most common allele.

• Loss of Function:

  • Null: Complete loss of function.

  • Hypomorph: Reduced function or expression (e.g., Nodal gene mutations affecting eye field separation).

  • Haploinsufficiency: One functional copy is not enough to maintain the wild-type phenotype (dominant loss of function).

• Gain of Function:

  • Hypermorph: Hyperactive protein expression.

  • Neomorph: Gaining a new function or expression in a new location (e.g., Antennapedia mutation in fruit flies causing legs to grow from the eye region).

  • Dominant Negative: A mutant protein that antagonizes or inhibits the function of the wild-type protein (often via dimerization).

Homologous Recombination (HR)

Biological Roles

• Occurs during Prophase I of Meiosis I between homologous chromosomes.

• Occurs during DNA repair (SDSA) using sister chromatids.

• Used for horizontal gene transfer and gene editing.

Molecular Mechanism (Meiotic)

1. Intentional Break: The enzyme SPO $11$ creates a double-stranded break.

2. Resection: Exonucleases chew back the DNA to create $3'$ single-stranded overhangs.

3. Strand Invasion: The single strand invades the neighboring similar DNA sequence, displacing a strand to form a D-loop (Displacement Loop).

4. Heteroduplex Formation: A region where strands from different chromosomes are entangled.

5. Holiday Junctions: X-like structures formed where strands cross.

6. Resolution: The enzyme resolvase cuts the junctions, and ligase seals them.

• Gene Conversion: Non-reciprocal transfer of information where one allele is converted to another due to mismatch repair within the heteroduplex region.

Genomic Evolution and Transposable Elements

• Syntenic Segments: Stretches of DNA that are organized similarly across species, indicating common ancestry (e.g., mouse vs. human chromosomes).

• Gene Duplication: A primary engine for evolution. Duplicated genes can acquire new functions (Neofunctionalization).

  • Example: Trichromatic vision in primates originated from a gene duplication of a photoreceptor gene, which then mutated to detect different wavelengths.

• Transposable Elements (TEs): "Jumping genes" discovered by Barbara McClintock.

  • Cut and Paste (DNA Transposons): Use the enzyme transposase to move directly to a new site.

  • Copy and Paste (Retrotransposons): DNA is transcribed to RNA, then reverse-transcribed by reverse transcriptase back into DNA, which inserts elsewhere.

  • Categories: Autonomous (carry own enzymes) vs. Nonautonomous (rely on other TEs for enzymes).

  • Impact: TEs constitute $44\%$ of the human genome and $90\%$ of the maize genome. They create genetic diversity but can also cause disease by disrupting coding sequences.

• VDJ Recombination: A specific site-specific recombination in the immune system. The RAG protein reshuffles $V$ (variable), $D$ (diversity), and $J$ (joining) exons to generate more than $1$ billion unique antibody combinations from a limited number of genes.

Questions & Discussion

• Q: Can a mutation go straight from normal to a full mutation, skipping the premutation phase?

• A: It is technically possible but mathematically unlikely. However, because the categories are based on ranges (e.g., $28$ for normal vs. $29$ for premutation in Huntington’s), the functional difference between the upper limit of one and the lower limit of the next is minimal. Most severe expansions occur progressively over multiple generations as longer repeats become increasingly unstable.

• Q: Are there mutations that create a new start codon?

• A: Yes. If a mutation creates a new $AUG$ ($ATG$ in DNA), the ribosome (which slides along the mRNA) might recognize the first $AUG$ it encounters. This can drastically change the resulting protein. The position of the mutation is critical: if it occurs in the $5'$ UTR, it might shift the entire start site, whereas if it occurs elsewhere, it might have less impact depending on the surrounding consensus sequences.