Localization of the exonuclease and polymerase domains of Bacillus subtilis DNA polymerase III

Barnes, Marjorie H.; Hammond, Russell A.; Kennedy, Christopher C.; Mack, Susan L.; Brown, Neal C.

doi:10.1016/0378-1119(92)90601-k

Cited by 33 publications

(28 citation statements)

References 17 publications

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“…Mutants of B. subtilis polC have been described previously (11) and were available for use as a reference. Growth of wild-type and resistant S. aureus and E. faecalis (4). This region of AU-resistant B. subtilis isolates contains at least four amino acids whose DNA sequences are known to be altered (4).…”

Section: Discussionmentioning

confidence: 99%

“…Pol IIIC enzymes were purified from native organisms by a modified version of a previously described method (2). Wild-type and HBEMAU-resistant B. subtilis isolates were included in this study because purification and analysis of the enzymes from these organisms have been well characterized (4,11). Briefly, cultures were grown at 37°C in BHI medium to an optical density of 1 (600 nm; path length, 1 cm) and harvested by centrifugation at 4,600 ϫ g (15,000 ϫ g for S. aureus) for 15 min at 4°C.…”

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confidence: 99%

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Low Frequencies of Resistance among Staphylococcus and Enterococcus Species to the Bactericidal DNA Polymerase Inhibitor N ³ -Hydroxybutyl 6-(3′-Ethyl-4′-Methylanilino) Uracil

Butler

Skow

Stephenson

et al. 2002

Antimicrob Agents Chemother

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The 6-anilinouracils (AUs) constitute a new class of bactericidal antibiotics selective against gram-positive (Gr ؉ ) organisms. The AU family of compounds specifically inhibits a novel target, replicative DNA polymerase Pol IIIC. Like other antibiotics, AUs can be expected to engender the development of resistant bacteria. We have used a representative AU and clinically relevant strains of Staphylococcus aureus and Enterococcus to determine the frequency and mechanism(s) of resistance development. The frequency of resistance was determined by using N 3 -hydroxybutyl 6-(3-ethyl-4-methylanilino) uracil (HBEMAU) and commercially available antibiotics at eight times the MICs. For all five Gr ؉ organisms tested, the frequency of resistance to HBEMAU ranged from 1 ؋ 10 ؊8 to 3 ؋ 10 ؊10 . The frequencies of resistance to the antibiotics tested, including rifampin, gentamicin, and ciprofloxacin, were either greater than or equal to those for HBEMAU. In order to understand the mechanism of resistance, HBEMAU-resistant organisms were isolated. MIC assays showed that the organisms had increased resistance to AU inhibitors but not to other families of antibiotics. Inhibition studies with DNA polymerases from HBEMAU-sensitive and -resistant strains demonstrated that the resistance was associated with Pol IIIC. DNA sequence analysis of the entire polC genes from both wild-type and resistant organisms revealed that the resistant organisms had a sequence change that mapped to a single amino acid codon in all strains examined.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Low Frequencies of Resistance among Staphylococcus and Enterococcus Species to the Bactericidal DNA Polymerase Inhibitor N ³ -Hydroxybutyl 6-(3′-Ethyl-4′-Methylanilino) Uracil

Butler

Skow

Stephenson

et al. 2002

Antimicrob Agents Chemother

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“…The PolC chromosomal polymerase of Gram-positive cells is homologous to the E. coli ␣ subunit and also contains a region of homology to ⑀. Accordingly, PolC (164 kDa) contains both enzymatic activities of ␣ and ⑀ on one polypeptide chain (35)(36)(37)(38)(39)(40)(41)(42)(43)(44). The PolC polymerase contains some unique sequence regions not found in the Gram-negative ␣ subunit, including a zinc finger and possibly a second nucleotide-binding site (45,46).…”

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Conserved Interactions in the Staphylococcus aureus DNA PolC Chromosome Replication Machine

Bruck

Georgescu

O'Donnell

2005

Journal of Biological Chemistry

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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...

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“…It has been shown that the primary sequence of the E. coli dnaQ protein has weak but significant homology with that of the N-terminal region of Bacillus subtilis pol III [4,5]. Subsequently, site-directed mutagenesis has localized the 3' to 5' exonuclease domain within a 200 amino acid segment in the N-terminal of B. subtilis DNA pol III [11]. Our interest in understanding the evolutionary relationships between Gram-positive and Gram»Corresponding author.…”

Section: Introductionmentioning

confidence: 99%

Evolution of dnaQ, the gene encoding the editing 3′ to 5′ exonuclease subunit of DNA polymerase III holoenzyme in Gram‐negative bacteria

1997

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The nucleotide sequences of the dnaQ genes from Salmonella typhimurium and Buchnera aphidicola, encoding the ε-subunit of the DNA polymerase III holoenzyme, have been determined. The Salmonella typhimurium dnaQ protein consists of 243 amino acid residues with a calculated molecular weight of 27224. The Buchnera aphidicola dnaQ protein contains 233 amino acid residues with a calculated molecular weight of 27170. A multiple sequence alignment of the amino acid sequences of the dnaQ proteins and those of DNA polymerase ms from Grampositive bacteria produced six homologous segments. These homologous segments contain highly conserved amino acid sequence motifs involved in catalytically important metal ion bindings (ligands 1,2 and 3). However, metal ligand 4 is found to be altered in the 3'-5' exonuclease domain of the family C DNA polymerases and dnaQ proteins in Gram-negative bacteria. From these results, we propose that the last common ancestor of the dnaQ gene of Gram-negative bacteria and the DNA polymerase ΙΠ gene (pol C gene) of Gram-positive bacteria was a single gene containing both 3'-5' exonuclease and DNA polymerase domains and then the dnaQ gene separated from the polymerase gene in Gram-negative bacteria.

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Localization of the exonuclease and polymerase domains of Bacillus subtilis DNA polymerase III

Cited by 33 publications

References 17 publications

Low Frequencies of Resistance among Staphylococcus and Enterococcus Species to the Bactericidal DNA Polymerase Inhibitor N ³ -Hydroxybutyl 6-(3′-Ethyl-4′-Methylanilino) Uracil

Low Frequencies of Resistance among Staphylococcus and Enterococcus Species to the Bactericidal DNA Polymerase Inhibitor N ³ -Hydroxybutyl 6-(3′-Ethyl-4′-Methylanilino) Uracil

Conserved Interactions in the Staphylococcus aureus DNA PolC Chromosome Replication Machine

Evolution of dnaQ, the gene encoding the editing 3′ to 5′ exonuclease subunit of DNA polymerase III holoenzyme in Gram‐negative bacteria

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Localization of the exonuclease and polymerase domains of Bacillus subtilis DNA polymerase III

Cited by 33 publications

References 17 publications

Low Frequencies of Resistance among Staphylococcus and Enterococcus Species to the Bactericidal DNA Polymerase Inhibitor N 3 -Hydroxybutyl 6-(3′-Ethyl-4′-Methylanilino) Uracil

Low Frequencies of Resistance among Staphylococcus and Enterococcus Species to the Bactericidal DNA Polymerase Inhibitor N 3 -Hydroxybutyl 6-(3′-Ethyl-4′-Methylanilino) Uracil

Conserved Interactions in the Staphylococcus aureus DNA PolC Chromosome Replication Machine

Evolution of dnaQ, the gene encoding the editing 3′ to 5′ exonuclease subunit of DNA polymerase III holoenzyme in Gram‐negative bacteria

Contact Info

Product

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Low Frequencies of Resistance among Staphylococcus and Enterococcus Species to the Bactericidal DNA Polymerase Inhibitor N ³ -Hydroxybutyl 6-(3′-Ethyl-4′-Methylanilino) Uracil

Low Frequencies of Resistance among Staphylococcus and Enterococcus Species to the Bactericidal DNA Polymerase Inhibitor N ³ -Hydroxybutyl 6-(3′-Ethyl-4′-Methylanilino) Uracil