DNA Errors (Mutations) - Lippincott Illustrated Reviews: Biochemistry, 8th ed.

a) Types of Errors (Mutations) in DNA

1. Point Mutations (Single Base Substitutions)

a. Transitions

• A purine is replaced by a purine (A ↔ G), or a pyrimidine by a pyrimidine (C ↔ T).
• Example: In sickle cell disease, GAG → GTG at codon 6 of the β-globin gene (glutamate → valine). This is a transversion (below), but Lippincott uses it as the classic point mutation example.
• Transitions are more common than transversions because the geometry of the base pair is less disrupted.

b. Transversions

• A purine is replaced by a pyrimidine, or vice versa (A/G ↔ C/T).
• Less common; cause a greater structural disturbance.

2. Based on Effect on Protein: Functional Classes of Point Mutations

3. Frameshift Mutations (Insertions and Deletions / Indels)

• One or more nucleotides are inserted into or deleted from the DNA sequence.
• This shifts the reading frame of the mRNA downstream of the mutation.
• Nearly all codons after the insertion/deletion are read incorrectly, producing a completely aberrant protein, often with a premature stop codon.
• Deletions of multiples of 3 nucleotides do not cause a frameshift - they just remove one or more amino acids.
• Example: Many cases of Duchenne muscular dystrophy are caused by frameshift deletions in the dystrophin gene.

4. Trinucleotide (Triplet) Repeat Expansions

• A specific 3-base sequence is abnormally repeated many more times than normal.
• Once the number of repeats exceeds a threshold, it causes disease - often with anticipation (worsening in successive generations).
• Examples: Huntington disease (CAG repeats in the huntingtin gene), Fragile X syndrome (CGG repeats), Myotonic dystrophy (CTG repeats).
• Lippincott notes that Southern blotting can detect trinucleotide repeat expansions.

5. Large-Scale DNA Rearrangements

• Large insertions or deletions - removing or adding entire exons or gene segments.
• Inversions - a segment is reversed in orientation within the chromosome.
• Translocations - a segment moves from one chromosome to another.
• These can be detected by Southern blotting (as noted in Lippincott, p. 1339).

6. Single Nucleotide Polymorphisms (SNPs - "snips")

• Lippincott states: ~90% of human genome variation comes in the form of SNPs, i.e., a variation involving just one base.
• They are the most common type of sequence variation at a given locus.
• Most occur in non-coding regions (introns/intergenic regions) and are harmless.
• Some SNPs in coding or regulatory regions can cause disease susceptibility or directly cause disease.
• The HapMap project catalogues common SNPs in the human genome.

b) The Most Common Error in DNA

Single Nucleotide Polymorphisms (SNPs) account for approximately 90% of all human genome variation - Lippincott Illustrated Reviews: Biochemistry, 8th ed., p. 1341.

• C → T transitions (particularly at CpG dinucleotide sites) are the single most frequent type of mutation in the human genome. This happens because cytosine residues at CpG sites are often methylated (forming 5-methylcytosine), and spontaneous deamination of 5-methylcytosine produces thymine - causing a C → T change. This is why CpG sites are mutation "hotspots."

• The most common replication errors are mismatched base pairs (e.g., G pairing with T instead of C), which if uncorrected produce transition mutations in the next round of replication.
• The proofreading activity of DNA polymerase III (3'→5' exonuclease) corrects most of these, and the mismatch repair (MMR) system catches the rest - making the overall fidelity of replication extremely high (~1 error per 10⁹ base pairs).

Summary Table

DNA Repair Mechanisms - Lippincott Illustrated Reviews: Biochemistry, 8th ed. (Chapter 30, pp. 1176-1187)

Overview

1. Recognition of the lesion
2. Excision (removal) of the damaged nucleotide(s)
3. Gap filling using the undamaged complementary strand as template
4. Ligation to restore strand continuity

Mechanism 1 - Mismatch Repair (MMR)

• The Mut proteins (MutS, MutL, MutH in E. coli; MSH and MLH homologs in humans) identify the mispaired nucleotide(s).
• The system must distinguish the correct (parent) strand from the incorrect (daughter) strand.
  • In prokaryotes: the parent strand is methylated at GATC sequences (by DAM methylase). The newly synthesized daughter strand is transiently unmethylated - this hemimethylation signals which strand needs correction.
  • In eukaryotes: the daughter strand is identified by nicks in the newly synthesized strand.
• An endonuclease nicks the daughter strand; an exonuclease removes the mismatched nucleotide(s) and additional flanking bases.
• DNA pol III (prokaryotes) / pol δ (eukaryotes) fills the gap; DNA ligase seals it.

MMR reduces the error rate of replication from 1 in 10⁷ to 1 in 10⁹ nucleotides.

Fig. 30.28 - Methyl-directed mismatch repair in E. coli (Lippincott)

Mechanism 2 - Nucleotide Excision Repair (NER)

• UV radiation causes covalent joining of two adjacent pyrimidines (usually T-T), forming an intrastrand cross-link (dimer) that blocks DNA replication.
• A UV-specific endonuclease (UvrABC excinuclease in bacteria) recognizes the bulky dimer.
• The enzyme cleaves the damaged strand on both the 5' and 3' sides of the lesion.
• A short oligonucleotide (~12 nucleotides in prokaryotes; ~27-29 nucleotides in eukaryotes) containing the dimer is excised.
• DNA pol I (prokaryotes) / pol δ or ε (eukaryotes) fills the gap.
• DNA ligase seals the nick.
• NER occurs throughout the cell cycle (not limited to S phase).
• NER has two sub-pathways: global genomic repair (searches all chromosomes) and transcription-coupled repair (preferentially repairs lesions encountered by RNA polymerase).

Fig. 30.29 - Nucleotide excision repair of pyrimidine dimers in E. coli (Lippincott)

Mechanism 3 - Base Excision Repair (BER)

• Uracil in DNA (from deamination of cytosine, or misincorporation of dUTP during replication)
• Oxidized bases (e.g., 8-oxoguanine)
• Alkylated bases (e.g., methylated adenine from dimethyl sulfate)
• AP sites (apurinic/apyrimidinic sites) - spontaneously lost bases (~10,000/cell/day are purines)

1. A specific DNA glycosylase recognizes the abnormal base and hydrolyzes the glycosidic bond, removing the base from the deoxyribose-phosphate backbone. This creates an AP site (apyrimidinic or apurinic site).
2. A specific AP endonuclease cleaves the strand just to the 5' side of the AP site.
3. A deoxyribose phosphate lyase removes the single base-free sugar-phosphate residue.
4. DNA pol I (prokaryotes) fills the gap using the complementary strand.
5. DNA ligase seals the nick.

Key point: BER removes one nucleotide at a time (single-nucleotide BER), unlike NER which removes an oligonucleotide.

Mechanism 4 - Double-Strand Break (DSB) Repair

• Ionizing radiation (X-rays, gamma rays)
• Chemotherapeutic agents (e.g., doxorubicin, bleomycin)
• Oxidative free radicals
• Also occurs naturally during genetic recombination (meiosis)

Sub-mechanism A: Non-Homologous End Joining (NHEJ)

• Proteins recognize and bind the broken DNA ends.
• The ends are processed and ligated together directly.
• Error prone - some DNA is lost in the process (mutagenic).
• Can occur at any phase of the cell cycle.
• Defects in NHEJ are associated with cancer predisposition and immunodeficiency syndromes.

Sub-mechanism B: Homologous Recombination (HR)

• Uses the sister chromatid or homologous chromosome as a template to replace lost DNA.
• Much less error prone ("error free") because lost DNA is accurately replaced.
• Occurs in late S and G₂ phases of the cell cycle (when sister chromatid is available).

• BRCA1 / BRCA2 mutations: BRCA1 and BRCA2 proteins are involved in homologous recombination. Mutations in these genes impair HR, leading to greatly increased risk for breast cancer and ovarian cancer. This is why BRCA-mutated cancers are sensitive to PARP inhibitors (which block a backup repair pathway).
• Ataxia-Telangiectasia (AT): Caused by mutations in the ATM gene (a kinase that senses DSBs and coordinates the DSB repair response). Clinical features include cerebellar ataxia, telangiectasias, immunodeficiency, and greatly increased cancer risk (especially lymphomas).

Summary Table

Source: Biochemistry, 8th ed. Lippincott Illustrated Reviews, Chapter 30, pp. 1176-1187