What is the proofreading function of DNA polymerase and how does it improve fidelity?

What is the proofreading function of DNA polymerase and how does it improve fidelity?

• A. Polymerase possesses 3'→5' exonuclease activity that removes misincorporated nucleotides during replication, increasing accuracy
• B. Polymerase has 5'→3' exonuclease activity that removes mismatches
• C. Proofreading occurs only after replication by ligase
• D. Fidelity is determined solely by base pairing

Answer: (A)

The main idea is that many DNA polymerases double as quality inspectors during replication. When the polymerase adds a wrong nucleotide, the newly formed 3' end doesn’t pair properly with the template. Instead of quietly continuing, the enzyme shifts the mispaired end into a separate 3'→5' exonuclease site built into the polymerase. There, the incorrect nucleotide is removed, the primer terminus is realigned in the polymerase’s active site, and DNA synthesis resumes with the correct base being incorporated. This in-line editing dramatically improves fidelity, lowering the error rate by orders of magnitude compared with base pairing alone. This proofreading function is not the same as the 5'→3' exonuclease activity that some polymerases use to remove RNA primers during lagging-strand synthesis, nor is replication fidelity determined solely by base pairing geometry. The proofreading step happens during replication itself, before ligase seals the final backbone, and it works in concert with correct base pairing to keep error rates very low.

The main idea is that many DNA polymerases double as quality inspectors during replication. When the polymerase adds a wrong nucleotide, the newly formed 3' end doesn’t pair properly with the template. Instead of quietly continuing, the enzyme shifts the mispaired end into a separate 3'→5' exonuclease site built into the polymerase. There, the incorrect nucleotide is removed, the primer terminus is realigned in the polymerase’s active site, and DNA synthesis resumes with the correct base being incorporated. This in-line editing dramatically improves fidelity, lowering the error rate by orders of magnitude compared with base pairing alone.

This proofreading function is not the same as the 5'→3' exonuclease activity that some polymerases use to remove RNA primers during lagging-strand synthesis, nor is replication fidelity determined solely by base pairing geometry. The proofreading step happens during replication itself, before ligase seals the final backbone, and it works in concert with correct base pairing to keep error rates very low.