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The Escherichia coli DNA polymerase III and ␥ subunits are single-strand DNA-dependent ATPases (the latter requires the ␦ and ␦ subunits for significant ATPase activity) involved in loading processivity clamp ␤. They are homologous to clamp-loading proteins of many organisms from phages to humans. Alignment of 27 prokaryotic /␥ homologs and 1 eukaryotic /␥ homolog has refined the sequences of nine previously defined identity and functional motifs. Mutational analysis has defined highly conserved residues required for activity in vivo and in vitro. Specifically, mutations introduced into highly conserved residues within three of those motifs, the P loop, the DExx region, and the SRC region, inactivated complementing activity in vivo and clamp loading in vitro and reduced ATPase catalytic efficiency in vitro. Mutation of a highly conserved residue within a fourth motif, VIc, inactivated clamp-loading activity and reduced ATPase activity in vitro, but the mutant gene, on a multicopy plasmid, retained complementing activity in vivo and the mutant gene also supported apparently normal replication and growth as a haploid, chromosomal allele.The dnaX polymerization gene of Escherichia coli encodes two DNA polymerase III components, and ␥. is the fulllength translational product of the DnaX reading frame. The shorter ␥ is identical to the first 430 residues of , but its C terminus is generated by a programmed Ϫ1 ribosomal frameshift which results in the incorporation of a glutamate as the 431st amino acid followed by a stop codon (3,19,63).functions as the replisome organizer, dimerizing the core polymerase (30,33,43,58) and interacting with and stimulating the replicative DnaB helicase and primase (31,73). also contributes to processivity by stabilizing the processivity clamp (32) and the holoenzyme (73) on the leading strand.␥ functions in a five-subunit complex (␥ 2-4 -␦-␦Ј--) (12,20,42,49,51,62) to load and unload the processivity clamp ␤ (2,5,25,26,45,47,59,67). The binding of two or three ATP molecules by the ␥ subunit of the complex alters the conformation of the complex, allowing ␦ to bind directly to and open the clamp and allowing assembly of a primed DNA-open clamp-␥ complex structure (25,26,45). Hydrolysis of the ATP, required for closing the clamp around primed DNA, occurs in two sequential steps. The first might release ␤ from the ␥ complex; the second might then release DNA (enclosed within the clamp). Alternatively, the first hydrolysis might release DNA from the ␥ complex into the open clamp and the second would then release ␤ (encircling the DNA). Another possibility for the second hydrolysis might be resetting of the ␥ complex for the next cycle (26).The N-terminal region of is identical to ␥, except for the 431st residue, and is capable of all the known activities of ␥ in vitro, including loading ␤ (with ␦ or ␦-␦Ј) (59) and assembly in vitro or in vivo (from an artificial -complex operon) into clamp-loading complexes ( 2-4 -␦-␦Ј--) (13, 49, 54). The ␥ complex is often thought to be the principal clam...

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SummaryTemperature sensitivity of DNA polymerization and growth, resulting from mutation of the t and g subunits of Escherichia coli DNA polymerase III, are suppressed by Cs,Sx mutations of the initiator gene, dnaA. These mutations simultaneously cause defective initiation at 208C. Efficient suppression, defined as restoration of normal growth rate at 398C to essentially all the cells, depends on functional oriC. Increasing DnaA activity in a strain capable of suppression, by introducing a copy of the wild-type allele, increasing the suppressor gene dosage or introducing a seqA mutation, reversed the suppression. This suggests that the suppression mechanism depends on reduced activity of DnaACs,Sx. Models that assume that suppression results from an initiation defect or from DnaACs,Sx interaction with polymerization proteins during nascent strand synthesis are proposed.

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