Cross-utilization of the β Sliding Clamp by Replicative Polymerases of Evolutionary Divergent Organisms

Abstract

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...

Structure of a small-molecule inhibitor of a DNA polymerase sliding clamp

“…In fact, the C-terminal regions of the Pol C subunits of these Gram-positive replicases also inhibit ␤-dependent synthesis by E. coli Pol III core, and E. coli ␤ even functions with the Pol C replicase of these Grampositive organisms (22). These observations support functional conservation, in addition to sequence conservation of this important polymerase/␤-clamp connection and underscore the possibility that a chemical compound may inhibit proteinprotein interactions from diverse organisms.…”

A genome‐wide strategy for the identification of essential genes in Staphylococcus aureus

“…For example, Gram-positive organisms have two essential genes, polC and dnaE, homologous to the E. coli dnaE gene encoding the a-subunit of DNA polymerase III holoenzyme. Purified PolC contains both polymerase and 3¢-5¢-exonuclease activity, whereas DnaE has only polymerase activity (Sanjanwala and Ganesan, 1991;Bruck and O´Donnell, 2000;Klemperer et al, 2000). In our screen, we isolated antisense inhibitors to both genes.…”

A universal protein–protein interaction motif in the eubacterial DNA replication and repair systems

“…For example, PolC subunits from Staphylococcus aureus, Streptococcus pyogenes, and Bacillus subtilis can use E. coli ␤ as their processivity subunit (1,10,11). In contrast, E. coli DnaE cannot use ␤ from the other species (11), the E. coli clamp loader complex cannot load S. aureus ␤ (11), and the S. pyogenes clamp loader complex cannot load E. coli ␤ (1).…”