How DNA “Proofreading” Occurs During Replication?

Sourav PanOctober 25, 2024

Answered

DNA proofreading is a critical process that ensures replication accuracy by correcting errors in nucleotide pairing. It primarily relies on the activities of DNA polymerase and exonuclease during and after replication. The following points provide a detailed explanation of how proofreading works:

• DNA Polymerase’s Role in Proofreading:
  • DNA polymerase, especially DNA Pol III in prokaryotes, carries out two main functions: polymerase activity (adding nucleotides) and exonuclease activity (removing incorrect nucleotides). During replication, the polymerase ensures that only the correct complementary nucleotide pairs are added. If an incorrect base is incorporated, DNA polymerase detects this error, reverses direction, and uses its exonuclease function to remove the mismatched nucleotide.
  • Once the incorrect base is excised, the polymerase resumes its task by inserting the correct nucleotide and continuing replication. This dual functionality—adding and removing nucleotides—enhances the fidelity of the replication process.
• Conformational Changes and Nucleotide Selection:
  • DNA polymerase undergoes conformational changes during nucleotide selection. Wrong bases have a lower binding affinity, causing the polymerase to dissociate and correct the error. Only when the correct nucleotide is added does the enzyme proceed along the DNA strand.
• Exonuclease Activity in Proofreading:
  • The exonuclease activity, especially the 3′ to 5′ exonuclease function, is key in proofreading. It breaks the phosphodiester bond between nucleotides, excising the incorrect base. DNA polymerase then incorporates the correct nucleotide. This activity occurs in both prokaryotic and eukaryotic systems, with prokaryotic DNA polymerase I, II, and III all possessing exonuclease functions.
• Mismatch Repair After Replication:
  • If a mismatch is not corrected during replication, it can still be repaired post-replication. Specialized proteins, such as MutS and MutL, recognize errors and signal for correction. MutS identifies the mismatch and binds to the error site, while MutL creates a nick in the DNA to facilitate repair.
  • The exonuclease function removes the mismatched nucleotide, and DNA polymerase inserts the correct base. The process concludes with DNA ligase sealing the gap by forming a new phosphodiester bond.
• Methylation in Prokaryotes for Error Detection:
  • In prokaryotes like E. coli, methylation helps distinguish the parental strand from the newly synthesized strand. The parental DNA is methylated, while the daughter strand is not, allowing the polymerase to detect and correct errors on the unmethylated, newly formed strand.
• Strand Discrimination in Eukaryotes:
  • Unlike in prokaryotes, methylation does not occur in eukaryotic systems. Instead, the PCNA protein signals the new strand, allowing proteins like MutS and MutL to locate errors. These proteins then facilitate the repair process by coordinating exonuclease activity and DNA polymerase insertion of the correct nucleotide.
• Nucleotide Excision Repair:
  • In cases where external factors, such as UV light, cause thymine dimers or other types of damage, nucleotide excision repair mechanisms take over. This involves cleaving the damaged section of DNA and replacing it with the correct sequence. DNA polymerase fills in the gap, and ligase seals the strand.