LOCALIZATION OF THE ACTIVE-SITE OF THE ALPHA-SUBUNIT OF THE ESCHERICHIA-COLI DNA-POLYMERASE-III HOLOENZYME

Using a deletion approach on the alpha subunit of DNA polymerase III f rom Escherichia call, we show that there is an N-proximal polymerase d omain which is distinct from a more C-proximal tau and beta binding do main, Although deletion of 60 residues from the alpha N terminus aboli shes polymerase activity, deletions of 48, 169, and 342 amino acids fr om the C terminus progressively impair its catalytic efficiency but pr eserve an active site, Deletion of 342 C-terminal residues reduces k(c at) 46-fold, increases the K-m for gapped DNA 5.5-fold, and increases the K-m for deoxynucleoside triphosphates (dNTPs) twofold. The 818-res idue protein with polymerase activity displays typical Michaelis-Mente n behavior, catalysing a polymerase reaction that is saturable with su bstrate and linear with time. With the aid of newly acquired sequences of the polymerase III alpha subunit from a variety of organisms, cand idates for two key aspartate residues in the active site are identifie d at amino acids 401 and 403 of the E. coli sequence by inspection of conserved acidic amino acids. The motif Pro-Asp-X-Asp, where X is a hy drophobic amino acid, is shown to be conserved among all known DnaE pr oteins, including those from Bacillaceae, cyanobacteria, Mycoplasma, a nd mycobacteria, The E. coli DnaE deletion protein with only the N-ter minal 366 amino acids does not have polymerase activity, consistent wi th the proposed position of the active-site residues.