Which enzymatic activity provides proofreading during DNA replication to improve fidelity?

• A. 3'→5' exonuclease activity of DNA polymerase.
• B. 5'→3' exonuclease activity of DNA polymerase.
• C. Endonuclease activity alone.
• D. Polymerase activity without proofreading.

Answer: (A)

Proofreading during DNA replication is provided by the 3'→5' exonuclease activity of DNA polymerase. When a wrong nucleotide is added, the polymerase can pause and switch to this exonuclease mode, removing the mispaired nucleotide from the 3' end of the growing strand. After the incorrect base is excised, synthesis resumes in the correct direction, restoring the proper sequence. This backtracking and removal at the 3' end directly corrects errors as they occur, dramatically increasing fidelity.

The 5'→3' exonuclease activity is used to remove nucleotides from the 5' end of a preceding fragment (such as RNA primers or during nick translation), not to proofread the nascent strand. Endonuclease activity cuts within DNA but doesn’t selectively correct misincorporated bases during replication. Polymerase activity without proofreading would just add nucleotides without removing mistakes, leading to higher error rates.