Abstract
The alpha subunit of the replicase of all bacteria contains a php domain, initially identified by its similarity to histidinol phosphatase but of otherwise unknown function (Aravind, L., and Koonin, E. V. (1998) Nucleic Acids Res. 26, 3746-3752). Deletion of 60 residues from the NH2 terminus of the alpha php domain destroys epsilon binding. The minimal 255-residue php domain, estimated by sequence alignment with homolog YcdX, is insufficient for epsilon binding. However, a 320-residue segment including sequences that immediately precede the polymerase domain binds epsilon with the same affinity as the 1160-residue full-length alpha subunit. A subset of mutations of a conserved acidic residue (Asp43 in Escherichia coli alpha) present in the php domain of all bacterial replicases resulted in defects in epsilon binding. Using sequence alignments, we show that the prototypical gram+ Pol C, which contains the polymerase and proofreading activities within the same polypeptide chain, has an epsilon-like sequence inserted in a surface loop near the center of the homologous YcdX protein. These findings suggest that the php domain serves as a platform to enable coordination of proofreading and polymerase activities during chromosomal replication.
Highlights
The multisubunit core of the E. coli Pol III replicase was found to contain the polymerase subunit ␣ bound to subunits ⑀ and [8]. ⑀ is the product of the mutD gene and was initially identified as conferring high levels of spontaneous mutagenesis when defective [9]
Alignments of the Pol C sequences with the Pol III ␣ php domain sequences and ⑀ sequences show that the php domain is conserved within Pol C sequences, even though the proofreading exonuclease does not need a protein-binding site to secure it to the polymerase (Fig. 4)
We plan to exploit the existence of the proofreading exonuclease of the E. coli Pol III holoenzyme as a separate subunit to establish the communication channels required for the coordination of these activities in a prototypical multisubunit replicase
Summary
The multisubunit core of the E. coli Pol III replicase was found to contain the polymerase subunit ␣ bound to subunits ⑀ and [8]. ⑀ is the product of the mutD gene and was initially identified as conferring high levels of spontaneous mutagenesis when defective [9]. Editing activity was found in the same polypeptide chain as Pol2 I and required exchange of the 3Ј-primer terminus between the polymerase and proofreading exonuclease sites, which are separated by ϳ30 Å [2].
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