DNA repair Knowledge-Based Study

Study notes

1. Sources and Consequences of DNA Damage

   The integrity of the genetic code is vital for cellular survival and accurate transmission of hereditary information. However, DNA is chemically reactive and constantly subject to structural modifications from both internal and external agents.

   Endogenous and Exogenous Sources

   • Endogenous sources: Metabolic by-products such as reactive oxygen species (ROS), spontaneous chemical events like depurination and deamination of cytosine to uracil, and errors introduced during replication by DNA polymerases.
   • Exogenous sources: Ultraviolet (UV) radiation (which creates pyrimidine dimers), ionizing radiation (causing double-strand breaks), and environmental chemical mutagens such as alkylating agents and polycyclic aromatic hydrocarbons.

   Uncorrected damage can alter base pairing, stall transcription or replication, lead to permanent somatic mutations, trigger apoptosis, or cause unchecked proliferation characteristic of carcinogenesis.

   Immediate Defense Mechanisms

   Cells possess mechanisms that either prevent mutations immediately during synthesis or directly reverse chemical alterations without cutting the phosphodiester backbone.

   Proofreading by DNA Polymerase

   During DNA replication, replicative polymerases (such as DNA Polymerase III in prokaryotes, or Polymerases $\delta$ and $\epsilon$ in eukaryotes) display high fidelity. When an incorrect nucleotide is incorporated, the enzyme detects the mismatched geometry:

   • The enzyme halts synthesis and moves the newly synthesized 3' end to its 3' $ightarrow$ 5' exonuclease active site.
   • The incorrect base is excised, and synthesis resumes from the corrected 3'-OH terminus.

   Direct Reversal

   Certain lesions can be directly restored to their original form without removing nucleotides:

   • Photoreactivation: The enzyme DNA photolyase absorbs visible light to monomerize cyclobutane pyrimidine dimers directly (widely found in bacteria, plants, and many animals, though absent in placental mammals).
   • Alkyltransferase action: $O^6$-methylguanine-DNA methyltransferase (MGMT) transfers an abnormal methyl group from modified guanine onto its own cysteine residue in a stoichiometric, irreversible reaction.

   Excision Repair Pathways

   When chemical modifications distort individual bases or the broader DNA helix, excision systems remove the damaged segment and use the undamaged complementary strand as a template for synthesis.

   Base Excision Repair (BER)

   BER targets non-bulky, small chemical modifications, such as oxidized bases, alkylated bases, or uracil resulting from cytosine deamination:

   1. Lesion recognition: A specific DNA glycosylase cleaves the N-glycosidic bond linking the altered base to the deoxyribose sugar, creating an apurinic or apyrimidinic (AP) site.
   2. Backbone incision: An AP endonuclease cleaves the phosphodiester backbone immediately 5' to the AP site.
   3. Resynthesis and sealing: A DNA polymerase inserts the correct nucleotide and removes the sugar-phosphate residue, after which DNA ligase seals the nick.

   Nucleotide Excision Repair (NER)

   NER resolves bulky, helix-distorting lesions, predominantly UV-induced cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts:

   1. Recognition: Multiprotein complexes scan the genome for helical distortion or transcription stalling.
   2. Dual incision: An excinuclease cleaves the phosphodiester backbone on both sides of the lesion, releasing an oligonucleotide typically 12–13 nucleotides long in prokaryotes and 24–32 nucleotides long in eukaryotes.
   3. Gap filling: DNA polymerase synthesizes replacement DNA using the intact strand, and DNA ligase restores continuity. Deficiencies in human NER result in xeroderma pigmentosum, characterized by extreme UV sensitivity and elevated skin cancer risk.

   Post-Replication Surveillance

   Mismatch Repair (MMR) operates immediately following DNA replication to capture mispaired bases and small insertion/deletion loops that escaped the proofreading activity of DNA polymerase.

   Strand Discrimination

   A critical requirement of MMR is distinguishing between the parental (correct) strand and the newly synthesized (error-containing) strand:

   • Prokaryotes (e.g., Escherichia coli): The enzyme Dam methylase adds methyl groups to adenine bases within GATC palindromic sequences. Newly synthesized strands remain transiently unmethylated (hemimethylated state), directing repair enzymes exclusively to the unmethylated daughter strand.
   • Eukaryotes: Strand discrimination is independent of DNA methylation; instead, it relies on preexisting breaks (nicks) such as those found between unligated Okazaki fragments on the lagging strand, or interactions with the replication clamp (PCNA).

   Repair Mechanism and Clinical Significance

   The repair process involves recognition by MutS homologs (MSH proteins), recruitment of MutL homologs (MLH proteins), endonucleolytic cleavage, exonuclease-mediated degradation past the mismatch, and resynthesis by DNA polymerase $\delta$ followed by ligase action. Inherited mutations in human MMR genes (e.g., MSH2, MLH1) cause Lynch syndrome (hereditary non-polyposis colorectal cancer).

   Repairing Severe Structural Breaks

   Double-strand breaks (DSBs) represent the most hazardous type of DNA damage because both strands of the duplex are broken, eliminating an intact complementary template for direct copying.

   Non-Homologous End Joining (NHEJ)

   • Mechanism: The Ku70/Ku80 heterodimer binds to the exposed DNA ends, recruits nucleases to process ragged termini, and employs DNA Ligase IV to directly ligate the ends back together.
   • Fidelity: Highly error-prone because processing often removes or adds nucleotides, causing loss of genetic sequence.
   • Timing: Operates throughout all phases of the cell cycle, serving as the dominant DSB repair pathway in non-dividing (G0/G1) mammalian cells.

   Homologous Recombination (HR)

   • Mechanism: Following nucleolytic resection that generates 3' single-stranded tails, recombinases (such as Rad51, supported by BRCA1 and BRCA2) promote strand invasion into an identical or near-identical homologous duplex.
   • Fidelity: High-fidelity (error-free) because it copies information directly from the undamaged sister chromatid.
   • Timing: Restricted primarily to the S and G2 phases of the cell cycle, when duplicated sister chromatids are readily accessible as repair templates.