Conserved Interactions in the Staphylococcus aureus DNA PolC Chromosome Replication Machine

Abstract

The PolC holoenzyme replicase of the Gram-positive Staphylococcus aureus pathogen has been reconstituted from pure subunits. We compared individual S. aureus replicase subunits with subunits from the Gram-negative Escherichia coli polymerase III holoenzyme for activity and interchangeability. The central organizing subunit, , is smaller than its Gram-negative homolog, yet retains the ability to bind single-stranded DNA and contains DNA-stimulated ATPase activity comparable with E. coli . S. aureus also stimulates PolC, although they do not form as stabile a complex as E. coli polymerase III⅐. We demonstrate that the extreme C-terminal residues of PolC bind to and function with ␤ clamps from different bacteria. Hence, this polymerase-clamp interaction is highly conserved. Additionally, the S. aureus ␦ wrench of the clamp loader binds to E. coli ␤. The S. aureus clamp loader is even capable of loading E. coli and Streptococcus pyogenes ␤ clamps onto DNA. Interestingly, S. aureus PolC lacks functionality with heterologous ␤ clamps when they are loaded onto DNA by the S. aureus clamp loader, suggesting that the S. aureus clamp loader may have difficulty ejecting from heterologous clamps. Nevertheless, these overall findings underscore the conservation in structure and function of Gram-positive and Gram-negative replicases despite >1 billion years of evolutionary distance between them.The Escherichia coli chromosomal replicase, DNA polymerase III holoenzyme, is extremely rapid (ϳ1 kb/s) and extends DNA by thousands of nucleotides without dissociating from the DNA template (1-3). These special features require accessory proteins, as the polymerase subunit alone is only weakly active in synthesis and lacks high processivity. The accessory proteins act as a clamp loader ATPase complex and a ring-shaped subunit that functions as a DNA sliding clamp (␤). The ␤ clamp encircles the DNA duplex and binds the polymerase, tethering it to DNA for rapid and processive chain extension. The clamp loader uses energy derived from ATP hydrolysis to pry open the ␤ clamp and to close it around a primed template.The E. coli ␥/ clamp loader consists of five proteins required for clamp loading ( 2 ␥ 1 ␦ 1 ␦Ј 1 ) and also the attached and ancillary subunits (4 -7). In E. coli, the / subunits connect the replicase to single-stranded DNA-binding protein (SSB), 1 but are not required for clamp loading (8 -10). The / subunits also contribute to the stability of the ␥/ complex in vitro (11). In E. coli, the dnaX gene produces two polypeptides, and ␥.(71 kDa) is the full-length product of the gene, whereas ␥ (47 kDa) is a truncated version of , produced by a translational frameshift (12)(13)(14). Both and ␥ are capable of functioning with ␦ and ␦Ј in clamp loading action (15). However, the unique C-terminal 24-kDa C-terminal region of provides extra functions relative to ␥. For example, (but not ␥) binds to the polymerase directly (16). Hence, the presence of multiple subunits within the clamp loader enables it to cross-link two polymerases, ther...

Essential Biological Processes of an Emerging Pathogen: DNA Replication, Transcription, and Cell Division in Acinetobacter spp

“…The hydrophobic groove on the ␤ sliding clamp and its binding motifs in other proteins are strongly conserved across bacterial species (27,260). An analogous mechanism is also shared by the PCNA sliding clamp of archaea and eukaryotes (209), although the consensus sequence recognized by PCNA (QxxLxxFF) is different from that of the bacterial ␤-binding motifs (55).…”

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

“…The E. coli Pol III C-terminal peptide inhibits ␤-dependent DNA synthesis by the Gram-positive Pol C replicases of S. pyogenes and Staphylococcus aureus (21). 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).…”

Conservation of Eubacterial Replicases

“…This enzyme possesses its own proofreading activity, is highly processive, and is essential in organisms containing the gene. The interaction of PolC with t would appear to be weaker than the binding between the a and t subunits (4,8,9), suggesting that t does not secure this polymerase as tightly to the replisome. In E. coli, it appears that the t subunit might sequester the a subunit from b 2 until it 'senses' the correct DNA context for its binding to b 2 (12).…”

“…The assembly of the d, d' and g/t subunits into clamp loaders has been demonstrated for an expanding number of organisms -Streptococcus pyogenes and Staphylococcus aureus, both Gram positive organisms (4,9), the two Gram negative organisms, Helicobacter pylori (Kongsuwan, unpublished) and Pseudomonas aeruginosa (8), and two thermophiles, Thermus thermophilus (6) and Aquifex aeolicus (5). The distribution of the g subunit (a truncated version of t which is also encoded by the dnaX gene) is variable.…”