Chromosomal replicases are multiprotein machines comprised of a DNA polymerase, a sliding clamp, and a clamp loader. This study examines replicase components for their ability to be switched between Grampositive and Gram-negative organisms. These two cell types diverged over 1 billion years ago, and their sequences have diverged widely. Yet the Escherichia coli ␤ clamp binds directly to Staphylococcus aureus PolC and makes it highly processive, confirming and extending earlier results (Low, R. L., Rashbaum, S. A., and Cozzarelli, N. R. (1976) J. Biol. Chem. 251, 1311-1325). We have also examined the S. aureus ␤ clamp. The results show that it functions with S. aureus PolC, but not with E. coli polymerase III core. PolC is a rather potent polymerase by itself and can extend a primer with an intrinsic speed of 80 -120 nucleotides per s. Both E. coli ␤ and S. aureus ␤ converted PolC to a highly processive polymerase, but surprisingly, ␤ also increased the intrinsic rate of DNA synthesis to 240 -580 nucleotides per s. This finding expands the scope of ␤ function beyond a simple mechanical tether for processivity to include that of an effector that increases the intrinsic rate of nucleotide incorporation by the polymerase.Numerous proteins cooperate to perform the complicated task of replicating duplex DNA. At the heart of this process lies the replicative DNA polymerase machinery. The chromosomal replicase of Escherichia coli is a large particle, DNA polymerase III holoenzyme, which consists of 10 different proteins in stoichiometries that range from one to four (reviewed in Refs. 1-3). Within the holoenzyme are two copies of the three subunit core polymerase (␣, DNA polymerase (4); ⑀, proofreading 3Ј-5Ј-exonuclease (5); and ) (6 -8). The ␤ subunit of DNA polymerase III holoenzyme is a protein ring that encircles DNA and slides along the duplex (9, 10). The ␤ ring binds the ␣ subunit of core and tethers the holoenzyme to DNA for high processivity in DNA synthesis (9). The five subunits that comprise ␥ complex (␥␦␦Ј) act as a clamp loader to open and close the ␤ ring around DNA in an ATP-driven reaction (11,12).The main bacterial system for study of cellular DNA replication is the Gram-negative E. coli. However, there is a great deal of diversity in the eubacterial kingdom. Perusal of bacterial genome data bases reveals homologs to the E. coli ␤ clamp, ␣ subunit of core polymerase, and at least two (␥ and ␦Ј) of the five subunits of ␥ complex, suggesting that the overall strategy of using a clamp and clamp loader generalizes among this diverse group of organisms (eukaryotes also use a clamp loader replication factor C) and clamp (proliferating cell nuclear antigen)) (13,14).The evolutionary split between Gram-positive and Gramnegative bacteria occurred over 1 billion years ago, and the sequences of the replicative polymerase and ␤ clamp have diverged considerably. Purification of the replicative DNA polymerase, encoded by polC, from a variety of Gram-positive cells yields only a single polypeptide, in contrast to the...

This report takes a proteomic/genomic approach to characterize the DNA polymerase III replication apparatus of the extreme thermophile, Aquifex aeolicus. Genes (dnaX, holA, and holB) encoding the subunits required for clamp loading activity (, ␦, and ␦) were identified. The dnaX gene produces only the full-length product, , and therefore differs from Escherichia coli dnaX that produces two proteins (␥ and ). Nonetheless, the A. aeolicus proteins form a ␦␦ complex. The dnaN gene encoding the ␤ clamp was identified, and the ␦␦ complex is active in loading ␤ onto DNA. A. aeolicus contains one dnaE homologue, encoding the ␣ subunit of DNA polymerase III. Like E. coli, A. aeolicus ␣ and interact, although the interaction is not as tight as the ␣؊ contact in E. coli. In addition, the A. aeolicus homologue to dnaQ, encoding the ⑀ proofreading 3-5-exonuclease, interacts with ␣ but does not form a stable ␣⅐⑀ complex, suggesting a need for a brace or bridging protein to tightly couple the polymerase and exonuclease in this system. Despite these differences to the E. coli system, the A. aeolicus proteins function to yield a robust replicase that retains significant activity at 90°C. Similarities and differences between the A. aeolicus and E. coli pol III systems are discussed, as is application of thermostable pol III to biotechnology.Chromosomal replicases of all cellular organisms studied thus far are composed of three components, the DNA polymerase, a ring-shaped DNA sliding clamp, and a clamp loader that uses ATP to assemble the sliding clamp onto DNA (1-3). In bacteria, the sliding clamp is a homodimer called ␤ (4). The ring-shaped ␤ dimer completely encircles DNA and slides along the duplex (5). The ␤ clamp also binds the DNA polymerase III, thereby tethering it to DNA for high processivity (5).This report on the Aquifex aeolicus pol III 1 replicase is part of our continuing study of comparing and contrasting replicases from a variety of bacteria. Most knowledge of bacterial DNA polymerase III (pol III) structure and function has been obtained from studies of the Escherichia coli replicase, DNA polymerase III holoenzyme (reviewed in Ref. 6). Therefore, a brief overview of its structure and function is instructive for the comparisons to be made in this report. In E. coli, the catalytic subunit of DNA polymerase III is the ␣ subunit (129.9 kDa) encoded by dnaE; it lacks a proofreading exonuclease (7). The proofreading 3Ј-5Ј-exonuclease activity is contained in the ⑀ (27.5 kDa) subunit (dnaQ) that forms a 1:1 complex with ␣ (8, 9). The pol III ␣Ϫ⑀ complex is found tightly associated to a third subunit, called , to form the heterotrimeric E. coli DNA polymerase III core (10). The subunit (holE, 8.6 kDa) is not essential for growth and is generally not conserved in bacteria (11).The E. coli pol III ␣ subunit and pol III core subassembly act distributively on primed ssDNA and have only low activity; they are even further inhibited by the presence of SSB (7, 12). However, after the ␤ clamp has been assembled onto a primed s...

This report identifies the dnaX homolog from Thermus thermophilis. Replicases from bacteria to humans contain subunits that are homologous to one another. These homologs are subunits of a clamp loading apparatus that loads sliding clamps onto DNA, which in turn act as mobile tethers for the replication machinery. In Escherichia coli, two of these subunits (␥ and ) are encoded by one gene (dnaX) in nearly equal amounts by way of an efficient translational frameshift. The ␥ and subunits form the central touchpoint that holds together two DNA polymerases with one clamp loading apparatus to form the E. coli chromosomal replicase, DNA polymerase III holoenzyme. The E. coli holoenzyme is an efficient replication machine that simultaneously replicates both strands of duplex DNA. The T. thermophilis dnaX homolog also contains a frameshift signature and produces both -and ␥-like proteins. Recombinant T. thermophilis -and ␥-like proteins, expressed in E. coli, have an oligomeric state similar to that of their E. coli counterparts and display ATPase activity that is stimulated by DNA. These results imply that T. thermophilis utilizes a DNA polymerase III holoenzyme replication machinery similar to that of E. coli.Chromosomal replicases are composed of several subunits in all organisms (1). In keeping with the need to replicate long chromosomes, these multicomponent replicases are rapid and highly processive. Cellular chromosomal replicases derive their processivity from a protein subunit that is shaped like a ring and completely encircles DNA (2, 3). This "sliding clamp" protein acts as a mobile tether for the polymerase machinery (4). The sliding clamp does not assemble onto DNA by itself but requires a complex of several proteins, called a "clamp loader," that couples ATP hydrolysis to the assembly of sliding clamps onto DNA (5). The three components of the Escherichia coli replicase, DNA polymerase III holoenzyme, are the three protein DNA polymerase III core (␣⑀ ), the ␤ subunit DNA sliding clamp, and the five protein ␥ complex clamp loader (␥␦␦Ј ) (for review, see Ref. 6). In eukaryotes, from yeast to humans, the three components are the DNA polymerase ␦, the PCNA sliding clamp, and the five protein Replication Factor C (RFC) clamp loader (for review, see Ref.3).The crystal structure of the circular sliding clamps from a variety of organisms have been determined (7-9), as have the crystal structures of the ␦Ј subunit (10) and complex of the clamp loader apparatus from E. coli. 1 With the aim to crystalize larger complexes, we have started to isolate the genes encoding the replicase subunits from a thermophile as heat stabile proteins are often more amenable to crystal structure analysis.As a beginning to identify and characterize the replicase of a thermophile, we started by looking for a homolog to the E. coli dnaX gene that encodes two protein subunits (␥ and ) of the DNA polymerase III holoenzyme through an efficient translational frameshifting mechanism (11-13). The dnaX gene has another homolog, holB, which encod...

The microbial flora of the upper vagina and cervix was examined in 38 adult baboons at various stages of the menstrual cycle. The mean number of different species isolated from each baboon was 9.5, with species of Bacteroides, Corynebacterium and group D streptococci predominating. Lactobacilli and mycoplasmas were found in 47.4 and 44.7% of the animals, respectively. No ureaplasmas were isolated. Cyclical variations in the microbial flora were minimal.