THE 3'-5'-EXONUCLEASE SITE OF DNA-POLYMERASE-III FROM GRAM-POSITIVE BACTERIA - DEFINITION OF A NOVEL MOTIF STRUCTURE

The primary structure of the 3'-5' exonuclease (Exo) site of the Gram(+) bacterial DNA polymerase III (Pol III) was examined by site-directed mutagenesis of Bacillus subtilis Pol III (BsPol III). It was found to differ significantly from the conventional three-motif substructure established for the fro site of DNA polymerase I of Escherichia coli (EcPol I) and the majority of other DNA polymerase-exonucleases. Motifs I and II were conventionally organized and anchored functionally by the predicted carboxylate residues. However, the conventional downstream motif, motif III, was replaced by motif ms, a novel 55-amino-acid (aa) segment incorporating three essential aa (His(565), Asp(533) and Asp(570)) which are strictly conserved in three Gram(+) Pol III and in the Ec fro epsilon (epsilon). Despite its unique substructure, the Gram(+) Pol III-specific fro site was conventionally independent of Pol, the site of 2'-deoxyribonucleoside 5-triphosphate (dNTP) binding and polymerization. The entire fro site, including motif III epsilon, could be deleted without profoundly affecting the enzyme's capacity to polymerize dNTPs. Conversely, Pol and all other sequences downstream of the fro site could be deleted with little apparent effect on fro activity. Whether the three essential aa within the unique motif III epsilon substructure participate in the conventional two-metal-ion mechanism elucidated for the model fro site of EcPol I, remains to be established.