On-off Regulation of 3′ Exonuclease Excision to DNA Polymerization by Exo+ Polymerase

Abstract

The role of 3′ exonuclease excision in DNA polymerization was evaluated in primer extensions using 3′ allele-specific primers that had exonuclease-digestible and exonuclease-resistant 3′ termini. With exonuclease-digestible unmodified 3′ mismatched primers, the exo+ polymerase yielded template-dependent products. Using exonuclease-resistant 3′ mismatched primers, no primer-extended product resulted from exo+ polymerase. As a control, polymerase without proofreading activity yielded primer-dependent products from 3′ mismatched primers. These data indicated that a successful removal of the mismatch is required for DNA polymerization from the 3′ mismatched primers by exo+ polymerase. In addition to the well-known proofreading from this mismatch removal, the premature termination in DNA polymerization, due to the failure of the efficient removal of the mismatched nucleotides, worked as an off-switch in maintaining the high fidelity in DNA replication from exo+ polymerase.