The NH2-terminal php Domain of the α Subunit of theEscherichia coliReplicase Binds the ϵ Proofreading Subunit

Abstract The α 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 α php domain destroys ϵ binding. The minimal 255-residue php domain, estimated by sequence alignment with homolog YcdX, is insufficient for ϵ binding. However, a 320-residue segment including sequences that immediately precede the polymerase domain binds ϵ with the same affinity as the 1160-residue full-length α subunit. A subset of mutations of a conserved acidic residue (Asp43 in Escherichia coli α) present in the php domain of all bacterial replicases resulted in defects in ϵ 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 ϵ-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.