ø29 DNA polymerase is a multifunctional enzyme, able to incorporate and to proofread misinserted nucleotides, maintaining a very high replication fidelity. Since both activities are functionally separated, a mechanism is needed to guarantee proper coordination between synthesis and degradation, implying movement of the DNA primer terminus between polymerization and 3′-5′ exonuclease active sites. Using single-turnover conditions, we have demonstrated that ø29 DNA polymerase edits the polymerization errors using an intramolecular pathway; that is, the primer terminus travels from one active site to the other without dissociation from the DNA. On the other hand, by using chemical tags, we could infer a difference in length of only one nucleotide to contact the primer strand when it is in the polymerization mode versus the editing mode. Using the same approach, it was estimated that ø29 DNA polymerase covers a DNA region of ten nucleotides, as has been measured in other polymerases using different techniques.

This investigation has been aided by research grant 2R01 GM27242-20 from the National Institutes of Health, by grant no. PB93-0173 from Dirección General de Investigación Cientı́fica y Técnica, by grant ERBFMX CT97 0125 from European Union, and by an Institutional grant from Fundación Ramón Areces.

Peer reviewed