The replicative polymerase of Escherichia coli, DNA polymerase HI, consists of a three-subunit core polymerase plus seven accessory subunits. Of these seven, r and fy are products of one replication gene, dnaX. The shorter 'y is created from within the 'r reading frame by a programmed ribosomal -1 frameshift over codons 428 and 429 followed by a stop codon in the new frame. Two temperature-sensitive mutations are available in dnaX. The 2016(Ts) mutation altered both 'r and y by changing codon 118 from glycine to aspartate; the 36(Ts) mutation affected the activity only of r because it altered codon 601 (from glutamate to lysine). Evidence which indicates that, of these two proteins, only the longer is essential includes the following.(i) The 36(Ts) mutation is a temperature-sensitive lethal allele, and overproduction of wild-type y cannot restore its growth. (ii) An allele which produced r only could be substituted for the wild-type chromosomal gene, but a -y-only allele could not substitute for the wild-type dnaX in the haploid state. Thus, the shorter subunit 'y is not essential, suggesting that r can substitute for the usual function(s) of 'y. Consistent with these results, we found that a functional polymerase was assembled from nine pure subunits in the absence of the fy subunit. However, the possibility that, in cells growing without -y, proteolysis of r to form a -y-like product in amounts below the Western blot (immunoblot) sensitivity level cannot be excluded.Escherichia coli DNA polymerase III (Pol III) holoenzyme consists of a three-subunit core polymerase (a, £, 0) plus seven accessory subunits (13, T, -y, 8, 8', X, q) (20,31,33,54). Study of subassemblies and pure subunits has advanced the understanding of accessory subunit function. In an ATP-dependent reaction, the -y complex (-y, 8, 8', X, 4I) catalyzes the transfer of 1 to the primed template to form a preinitiation complex (10,27,37,39,52). This preinitiation complex consists of a 1 dimer which completely encircles the DNA and slides freely along the duplex (19,47). The core or individual a (DNA polymerase) (25, 44) and a (proofreading exonuclease) (42) subunits then bind and polymerize with high processivity, tethered to the template by the 1 dimer clamped around the duplex DNA behind a (11,19,47).Both T and y are produced from one gene, dnaX (8,13,18,35,62). The 71.1-kDa T is the full-length 643-amino-acid translational product of the dnaXmessenger. The 47.5-kDa y is terminated within the reading frame by a programmed ribosomal -1 frameshift over codons 428 and 429 (2, 9, 48, 49, 51). The shifted ribosomes incorporate one unique amino acid and then encounter a stop codon. The result is that -y is identical to the first 430 residues of X plus a unique C-terminal residue. The frameshift signal is so efficient that the T/y ratio in nonoverproducing strains is about 1 (2, 9, 22, 51).Although not required for processive synthesis in reconstituted systems which included the other nine subunits, addition of T stimulated total synthesis (27,28). M...

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Chromosome replication and cell division of Escherichia coli are coordinated with growth such that wild-type cells divide once and only once after each replication cycle. To investigate the nature of this coordination, the effects of inhibiting replication on Z-ring formation and cell division were tested in both synchronized and exponentially growing cells with only one replicating chromosome. When replication elongation was blocked by hydroxyurea or nalidixic acid, arrested cells contained one partially replicated, compact nucleoid located mid-cell. Cell division was strongly inhibited at or before the level of Z-ring formation. DNA cross-linking by mitomycin C delayed segregation, and the accumulation of about two chromosome equivalents at mid-cell also blocked Z-ring formation and cell division. Z-ring inhibition occurred independently of SOS, SlmA-mediated nucleoid occlusion, and MinCDE proteins and did not result from a decreased FtsZ protein concentration. We propose that the presence of a compact, incompletely replicated nucleoid or unsegregated chromosome masses at the normal mid-cell division site inhibits Z-ring formation and that the SOS system, SlmA, and MinC are not required for this inhibition. Bacterial DNA replication and cell division are coordinated with growth so that a single, timely division follows each genome duplication. This rule, however, is complicated by the fact that cells can divide rapidly, with the replication and division machinery operating continuously and concurrently (e.g., see reference 1). Division at mid-cell, which occurs with high precision (2, 3), begins with polymerization of the FtsZ protein into a circumferential ring on the cytoplasmic membrane inner surface (4). Although division frequency is ultimately determined by growth rate (5, 6), regulatory mechanisms control both the location and timing of FtsZ ring assembly (for recent reviews, see the works of de Boer [7], Chien et al. [8], Lutkenhaus et al. [9], and Egan and Vollmer [10]).Placement of Z rings mid-cell depends on negative activities of the Min proteins and nucleoid occlusion. The MinC protein of Escherichia coli binds to FtsZ, preventing Z-ring assembly at all positions except at mid-cell, where its time-averaged concentration is lowest (11-13). The nucleoid occlusion (14) proteins SlmA and Noc, of E. coli and Bacillus subtilis, respectively (15, 16), bind DNA at specific sequences and prevent division over replicating nucleoids in rapidly growing cells. SlmA prevents Z-ring formation by also binding FtsZ (17, 18); the Noc protein functions similarly but has not been shown to bind FtsZ directly (19). Because the SlmA and Noc DNA binding sites are absent from the terminus domain, which is replicated last and in the cell center (20-23), nucleoid occlusion contributes to both spatial and temporal control of Z-ring formation (17)(18)(19)). An slmA null mutant which is also min null frequently forms Z-ring-like structures over nucleoids when grown in LB medium (15). However, the SlmA protein might not be the ...

Extragenic suppressor mutations which had the ability to suppress a dnaX2016(Ts) DNA polymerization defect and which concomitantly caused cold sensitivity have been characterized within the dnaA initiation gene. When these alleles (designated Cs, Sx) were moved into dnaX ؉ strains, the new mutants became cold sensitive and phenotypically were initiation defective at 20؇C (J. R. Walker, J. A. Ramsey, and W. G. Haldenwang, Proc. Natl. Acad. Sci. USA 79:3340-3344, 1982). Detailed localization by marker rescue and DNA sequencing are reported here. One mutation changed codon 213 from Ala to Asp, the second changed Arg-432 to Leu, and the third changed codon 435 from Thr to Lys. It is striking that two of the three spontaneous mutations occurred in codons 432 and 435; these codons are within a very highly conserved, 12-residue region (K. Skarstad and E. Boye, Biochim. Biophys. Acta 1217:111-130, 1994; W. Messer and C. Weigel, submitted for publication) which must be critical for one of the DnaA activities. The dominance of wild-type and mutant alleles in both initiation and suppression activities was studied. First, in initiation function, the wild-type allele was dominant over the Cs, Sx alleles, and this dominance was independent of location. That is, the dnaA ؉ allele restored growth to dnaA(Cs, Sx) strains at 20؇C independently of which allele was present on the plasmid. The dnaA(Cs, Sx) alleles provided initiator function at 39؇C and were dominant in a dnaA(Ts) host at that temperature. On the other hand, suppression was dominant when the suppressor allele was chromosomal but recessive when it was plasmid borne. Furthermore, suppression was not observed when the suppressor allele was present on a plasmid and the chromosomal dnaA was a null allele. These data suggest that the suppressor allele must be integrated into the chromosome, perhaps at the normal dnaA location. Suppression by dnaA(Cs, Sx) did not require initiation at oriC; it was observed in strains deleted of oriC and which initiated at an integrated plasmid origin.The Escherichia coli dnaX gene encodes the and ␥ subunits of DNA polymerase III holoenzyme (20,38,51,86). , the larger protein (71.1 kDa), results from translation of the complete 643-codon message. ␥, the shorter product (47.5 kDa), results from a programmed Ϫ1 ribosomal frameshift over messenger codons 428 to 430 (10, 21, 72, 73, 75). This shift results in the translation of one unique amino acid, as residue 431, which is followed by a stop codon. The net result is that and ␥ are identical over the N-terminal 430 residues. The dnaX gene was defined by two temperature-sensitive (Ts) mutations, dnaX2016(Ts) (19) and dnaX36(Ts) (32). The dnaX2016(Ts) mutation changes codon 118 from glycine (GGT) to aspartate (GAT) and affects both and ␥ (7). Shifting the dnaX2016(Ts) mutant from the permissive temperature of 30ЊC to 42ЊC causes an immediate stop in DNA synthesis, as expected for a defect in polymerization, and growth gradually ceases (16). The dnaX36(Ts) mutation changes codon 601 from glutamat...

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...