DNA Repair Mechanisms and Mutation Effects

Biological DNA Repair Mechanisms Overview

• DNA repair mechanisms exist to preserve genomic stability, correct errors introduced during replication, and reverse damage caused by environmental or metabolic agents.

• DNA repair functions through three overarching strategies:

  • Prevention of DNA damage prior to occurrence.

  • Direct reversal of damaged bases.

  • Homology-dependent repair and error-prone repair pathways.

Error Prevention Mechanisms Prior to DNA Damage

• Proofreading by DNA Polymerase:

  • Proofreading is the primary and most frequent mechanism for preventing DNA replication errors.

  • Replicative DNA polymerases detect mismatched base pairs as synthesis occurs.

  • Polymerases utilize a $3' \rightarrow 5'$ exonuclease activity to excise the incorrectly incorporated nucleotide.

  • Polymerization then resumes in the $5' \rightarrow 3'$ direction.

• Elimination of Free Radicals and Reactive Oxygen Species (ROS):

  • Superoxide dismutase (SOD) converts superoxide free radicals ($\text{O}_2^{\bullet-}$) into hydrogen peroxide ($\text{H}_2\text{O}_2$).

  • Catalase converts hydrogen peroxide ($\text{H}_2\text{O}_2$) into water ($\text{H}_2\text{O}$):          $\text{O}_2^{\bullet-} \xrightarrow{\text{SOD}} \text{H}_2\text{O}_2 \xrightarrow{\text{catalase}} \text{H}_2\text{O}$     

  • Oxidative stress caused by unmitigated ROS produces severe base modifications:

  • Thymidine glycol: Distorts DNA and blocks replication fork progression.

  • 8-oxo-7,8-dihydrodeoxyguanine (8-oxo-dG): Mispairs with adenine ($\text{A}$) during replication, producing a $\text{G}\cdot\text{C} \rightarrow \text{T}\cdot\text{A}$ transversion mutation.

Direct Reversal of DNA Damage

• Photoreactivation of UV-Induced Damage:

  • Ultraviolet (UV) radiation induces covalent cross-linking between adjacent pyrimidines, creating a cyclobutyl ring and forming cyclobutane pyrimidine dimers (CPDs or CPPs).

• CPD Photolyase Enzyme:

  • Binds to cyclobutane pyrimidine dimers.

  • Uses energy absorbed from visible light ($350\,nm - 450\,nm$, or $>300\,nm$) to cleave the cyclobutyl ring.

  • Directly restores native pyrimidine bases without breaking the phosphodiester backbone.

  • Functional CPD photolyase is absent in placental mammals.

• Hierarchy of UV Damage Processing:

  • Direct reversal via CPD photolyase (when available).

  • Nucleotide Excision Repair (NER) if photolyase is absent or fails.

  • SOS Repair / Trans-lesion Synthesis if NER is unable to clear the damage.

  • Unrepaired UV lesions lead to permanent mutations, with $\text{C} \rightarrow \text{T}$ transitions being the most common.

Homology-Dependent Repair Pathways

• Homology-dependent repair utilizes the undamaged complementary strand as a template to excise lesions and direct repair synthesis.

• Includes three main pathways:

  1. Base Excision Repair (BER)

  2. Nucleotide Excision Repair (NER)

  3. Post-Replication Mismatch Repair (MMR)

Base Excision Repair (BER)

• Target Lesions:

  • Repairs minor, non-bulky DNA damage that does not distort the double helix.

  • Examples: Depurination/depyrimidation (abasic sites), base deamination (e.g., cytosine deaminated to uracil), and oxidation products (e.g., 8-oxoguanine).

• Steps in Base Excision Repair:

  1. Base Removal: DNA glycosylase recognizes specific damaged or modified bases and cleaves the N-glycosidic bond linking the base to the deoxyribose sugar. This leaves an apurinic or apyrimidinic (AP) site. If the nucleotide is already depurinated, DNA glycosylase action is unnecessary.

  2. Backbone Cleavage: AP endonuclease recognizes the abasic site and cleaves the phosphodiester bond immediately $5'$ to the AP site.

  3. Excision & Resynthesis:

  • Prokaryotes: Deoxyribose phosphodiesterase (dRpase) removes the abasic sugar-phosphate residue along with adjacent nucleotides. DNA Polymerase I synthesizes replacement DNA.

  • Eukaryotes:

    • Short-Patch BER: DNA Polymerase $\beta$ ($\text{Pol } \beta$) inserts a single replacement nucleotide and removes the $5'$\text{-deoxyribose phosphate}.

    • Long-Patch BER: DNA Polymerase $\beta$ or $\delta/\epsilon$ synthesizes a short stretch ($2-10$ nucleotides), creating a displaced single-stranded flap. Flap Endonuclease 1 (FEN1) clips off the displaced segment.

  1. Ligation: DNA Ligase (DNA Ligase I or Ligase III/XRCC1 in eukaryotes) seals the nick.

Nucleotide Excision Repair (NER)

• Target Lesions:

  • Repairs bulky, distortion-inducing DNA damage, including UV-induced pyrimidine dimers and bulky chemical adducts.

• Two Damage Recognition Pathways:

  • Global Genomic Repair (GGR / Global Genome NER):

  • Operates throughout transcriptionally silent areas of the genome.

  • Initiates relatively slowly.

  • $XPC$ and $XPE$ proteins recognize damaged bases.

  • Recruits the multiprotein $TFIIH$ complex (part of the RNA Polymerase II initiation machinery, containing $XPB$ and $XPD$ helicases).

• Transcription-Coupled NER (TC-NER):

  • Operates selectively on the template strand of transcriptionally active genes.

  • Initiates faster than GGR.

  • RNA Polymerase II stalls upon encountering bulky damage in DNA.

  • The stalled transcription complex recruits Cockayne Syndrome proteins A and B ($CSA$ and $CSB$).

  • RNA Polymerase II, $CSA$, and $CSB$ are displaced/replaced by the $TFIIH$ complex containing $XPB$ and $XPD$ helicases.

• Common Core Steps of NER:

  • $XPB$ and $XPD$ helicase subunits unwind the DNA double helix around the site of damage.

  • Replication Protein A ($RPA$) binds to and stabilizes single-stranded DNA.

  • Endonucleases execute dual incisions on the damaged strand:

  • $15-24$ nucleotides upstream ($5'$ side) from the lesion.

  • $2-8$ nucleotides downstream ($3'$ side) from the lesion.

  • The single-stranded oligonucleotide containing the lesion is excised and released.

  • PCNA and DNA Polymerase $\delta$ or $\epsilon$ ($\text{Pol } \delta/\epsilon$) synthesize replacement DNA using the intact strand as a template.

  • DNA Ligase I seals the phosphodiester backbone.

• Pathologies Associated with NER Deficiencies:

  • Xeroderma Pigmentosum (XP):

  • Caused by mutations in NER genes, including XPB ($XPB$) and XPD ($XPD$).

  • Characterized by extreme sensitivity to UV light and a high frequency of skin cancers.

  • Ocular manifestations occur in $18\%$ of patients, affecting the eyelids, conjunctiva, and cornea.

  • Neurological deficits may accompany cutaneous manifestations.

• Cockayne Syndrome (CS):

  • Caused by mutations in TC-NER genes CSA ($CSA$) or CSB ($CSB$); also known as dwarfism-retinal atrophy-deafness syndrome.

  • Stalled RNA Polymerase II fails to recruit TC-NER factors, triggering cellular apoptosis.

  • Features include dwarfism/short stature, premature aging, developmental delays, delayed neurological development, facial dysmorphism, and retinal pigmentary degeneration/retinal atrophy.

  • Impaired transcription repair permits error-prone bypass mechanisms, causing chromosomal rearrangements.

Post-Replication Mismatch Repair (MMR)

• Function:

  • Corrects replication mismatches and insertion/deletion loops that escape DNA polymerase proofreading.

  • Proofreading reduces errors to $1\text{ in } 100,000$ base pairs; MMR further increases fidelity, leaving uncorrected errors in only $1\text{ in } 10,000,000$ bases.

• Prokaryotic Mechanism:

  1. $MutS$ protein recognizes mispaired bases and small loops, binding directly to the mismatched pair near the replication fork.

  2. $MutS$ recruits $MutL$ and $MutH$ proteins.

  3. $MutH$ identifies methylated adenine bases on hemimethylated parent DNA strands at $\text{GATC}$ sites (methylated by Dam methylase).

  4. $MutL$ bridges and positions $MutH$ adjacent to $MutS$.

  5. $MutH$ nicks the unmethylated, newly synthesized daughter strand containing the error.

  6. DNA helicase unwinds the region, and an exonuclease excises the segment between the nick and the mismatch.

  7. DNA Polymerase III fills the gap using the parent strand as a template.

  8. DNA Ligase seals the nick.

• Eukaryotic Mechanism:

  • Utilizes $MutS$ homologues (e.g., MSH2, MSH6) and $MutL$ homologues (e.g., MLH1, PMS2).

  • Does not rely on DNA methylation; instead, PCNA and pre-existing strand nicks (such as Okazaki fragment junctions) direct strand discrimination.

  • Resynthesis is executed by DNA Polymerase $\delta$ or $\epsilon$ ($\text{Pol } \delta/\epsilon$).

• Clinical Relevance:

  • Mutations in human MMR genes cause Hereditary Non-Polyposis Colorectal Cancer (HNPCC), or Lynch Syndrome.

  • Causes predisposition to early-onset colon cancer, affecting up to $1\text{ in } 200$ individuals in Western populations.

Error-Prone DNA Repair Pathways

• Error-prone mechanisms serve as pathways of last resort when DNA damage blocks normal replicative machinery.

Trans-Lesion Synthesis (TLS) / SOS System

• Bypasses un-repaired DNA damage at stalled replication forks.

• Prevents replication collapse and avoids apoptosis, allowing replication to complete at the cost of fidelity.

• Mechanism:

  1. Replicative DNA Polymerase III (or Polymerase $\delta/\epsilon$ in eukaryotes) stalls upon encountering a lesion.

  2. Single-stranded DNA at the stalled fork binds $RecA$ in prokaryotes.

  3. Specialized error-prone bypass polymerases (e.g., Polymerase V in prokaryotes; at least $5$ specialized bypass polymerases in eukaryotes) replace the replicative polymerase.

  4. Bypass polymerases are recruited by sliding clamps ($\beta\text{-clamp}$ in prokaryotes; PCNA in eukaryotes).

  5. The bypass polymerase synthesizes DNA across the lesion without proofreading capability.

  6. The bypass polymerase is displaced, allowing replicative Polymerase III ($\text{Pol } \delta/\epsilon$) to resume normal synthesis.

Non-Homologous End Joining (NHEJ)

• Purpose:

  • Repairs double-strand breaks (DSBs) caused by ionizing radiation, chemicals, ROS, or replication across single-strand nicks.

  • Operates in both resting ($\text{G}_0/\text{G}_1$) and mitotically active cells to rejoin broken ends and prevent cell death.

• Mechanism:

  1. $Ku70/Ku80$ heterodimers bind directly to exposed, double-stranded DNA ends at the break site, stabilizing them.

  2. The $Ku$ complex recruits additional repair factors: DNA-dependent protein kinase ($\text{DNA-PK}$), Artemis (nuclease), $XRCC4$, $NHEJ1$ (XLF), and DNA Ligase IV.

  3. Artemis processes and trims DNA ends, removing damaged or incompatible nucleotides.

  4. $XRCC4$ and DNA Ligase IV ligate the trimmed ends back together.

• Consequences and Limitations of NHEJ:

  • Lacks proofreading capability.

  • Lacks a template mechanism to match correct ends when multiple double-strand breaks occur simultaneously.

  • Inherently mutagenic: always generates insertion/deletion (INDEL) mutations at the join site.

  • Can generate severe chromosomal translocations, inversions, deletions, and duplications.

  • Inhibited at telomeres to prevent inappropriate end-to-end chromosome fusion.

Homologous Recombination (HR) for Double-Strand Breaks

• An error-free double-strand break repair mechanism operating during late $\text{S}$ and $\text{G}_2$ phases, utilizing an intact sister chromatid as a template.

• Mechanism:

  1. Nucleases execute end resection, digesting $5'$ strands to generate $3'$ single-stranded DNA overhangs.

  2. The $3'$ single-stranded end invades the intact homologous duplex template, forming a displacement loop (D-loop).

  3. DNA synthesis extends the invading $3'$ end using the sister chromatid as a template.

  4. Pathways for resolution:

  • Double-Strand Break Repair (DSBR): Capture of the second $3'$ end forms double Holliday Junctions (HJs). Resolution by resolvases leads to crossover or non-crossover products.

  • Synthesis-Dependent Strand Annealing (SDSA): Helicases displace the extended invading strand from the D-loop, allowing it to re-anneal to the second resected end of the broken chromosome. Synthesis and ligation complete repair, producing exclusively non-crossover products.

Predicting Effects of Genetic Mutations

• Genetic Mapping & Nomenclature:

  • Cytogenetic Location: e.g., Chr1p31.3 ($\text{Chromosome 1, short arm [p], band 31.3}$).

  • Gene Symbol: e.g., RPE65.

  • Variant Nomenclature: e.g., c.1304A>G (p.Y435C):

  • c.1304A>G: Coding DNA nucleotide position $1304$ converted from Adenine ($\text{A}$) to Guanine ($\text{G}$).

  • p.Y435C: Amino acid position $435$ converted from Tyrosine ($\text{Y}$) to Cysteine ($\text{C}$).

  • Interpretations: Pathological, Non-pathological, or Variant of Unknown Significance (VUS).

• Classification of Mutations by Location and Type:

• Coding Region Mutations:

  • Synonymous / Silent Mutations ($\text{m5}$): Nucleotide substitution does not alter the amino acid sequence; typically retains full protein function.

  • Conservative Missense Mutations: Replaces an amino acid with one of chemically similar properties; minimal impact on protein structure.

  • Non-Conservative Missense Mutations: Replaces an amino acid with one of significantly different size, charge, or polarity; often disrupts structure or function.

  • Active Site Mutations ($\text{m2, m3}$): Occur directly within catalytic or binding pockets of active sites, typically causing complete loss of function (null mutation).

  • Non-Active Site Structural Mutations ($\text{m4}$): Occur outside catalytic domains; may retain partial activity (leaky mutation).

• Promoter & Non-Coding Regulatory Mutations ($\text{m1}$):

  • Alter transcription factor ($\text{R}$) binding sites in regulatory regions.

  • Outcomes include:

    1. Complete abolition of transcription factor binding, resulting in zero transcription (null mutation).

    2. Altered transcription factor binding affinity, producing quantitative changes in mRNA/protein levels.

    3. Ectopic expression (protein produced in incorrect cell types or at inappropriate developmental times).

• Regulatory Gene Mutations:

  • Mutations in genes encoding transcription factors or regulatory proteins disrupt expression across multiple downstream target genes, leading to multi-organ system defects.

• Splice Site Mutations ($\text{m6}$):

  • Occur at conserved splice donor ($5'$) or acceptor ($3'$) sites.

  • Creation of Cryptic Splice Sites: Promotes aberrant splicing, inserting intronic sequences or truncating exons.

  • Elimination of Canonical Splice Sites: Leads to exon skipping or intron retention.

  • Splicing errors in coding sequences typically induce reading frame shifts and premature stop codons, producing truncated, unstable, or non-functional proteins (null mutations).