Characterization and overexpression of the gene encoding Staphylococcus aureus DNA polymerase III

Pacitti, Diane Frances; Barnes, Marjorie H.; Li, Dong H.; Brown, Neal C.

doi:10.1016/0378-1119(95)00377-i

Cited by 21 publications

(17 citation statements)

References 19 publications

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“…For example, the SOS regulon of S. aureus is much more limited than that of E. coli (44), which could decrease the range of DNA damage signals to which the SOS regulon of S. aureus responds. In addition, the replicative DNA polymerases in S. aureus (PolC) and E. coli (DnaE) are significantly different in primary amino acid sequence (Ͻ20% identity) (45), domain arrangements, the presence of an intrinsic 3=-5= proofreading exonuclease domain in PolC (but not DnaE), and differential sensitivity to inhibition by nucleotide analogs (46).…”

Section: As It Was Too Weak) These Values Indi-mentioning

confidence: 99%

DNA Targeting as a Likely Mechanism Underlying the Antibacterial Activity of Synthetic Bis-Indole Antibiotics

Opperman

Kwasny

et al. 2016

Antimicrob Agents Chemother

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bWe previously reported the synthesis and biological activity of a series of cationic bis-indoles with potent, broad-spectrum antibacterial properties. Here, we describe mechanism of action studies to test the hypothesis that these compounds bind to DNA and that this target plays an important role in their antibacterial outcome. The results reported here indicate that the bis-indoles bind selectively to DNA at A/T-rich sites, which is correlated with the inhibition of DNA and RNA synthesis in representative Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) organisms. Further, exposure of E. coli and S. aureus to representative bis-indoles resulted in induction of the DNA damage-inducible SOS response. In addition, the bis-indoles were found to be potent inhibitors of cell wall biosynthesis; however, they do not induce the cell wall stress stimulon in S. aureus, suggesting that this pathway is inhibited by an indirect mechanism. In light of these findings, the most likely basis for the observed activities of these compounds is their ability to bind to the minor groove of DNA, resulting in the inhibition of DNA and RNA synthesis and other secondary effects.

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Section: As It Was Too Weak) These Values Indi-mentioning

confidence: 99%

DNA Targeting as a Likely Mechanism Underlying the Antibacterial Activity of Synthetic Bis-Indole Antibiotics

Opperman

Kwasny

et al. 2016

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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“…The 6-anilinouracils (AUs) illustrated in Fig. 1 are selective inhibitors of DNA polymerase III-c2 (pol III), an enzyme product of the polC gene (2,8,12). This enzyme is essential for the replication of the chromosome in gram-positive bacteria (7,9,17) and is found in low-GϩC-content eubacteria, including staphylococci, enterococci, streptococci, Listeria species, Bacillus species, and clostridia (1,8).…”

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In Vitro Antimicrobial Activities of Novel Anilinouracils Which Selectively Inhibit DNA Polymerase III of Gram-Positive Bacteria

Daly

Giehl

Brown³

et al. 2000

Antimicrob Agents Chemother

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The 6-anilinouracils are novel dGTP analogs that selectively inhibit the replication-specific DNA polymerase III of gram-positive eubacteria. Two specific derivatives, IMAU (6-[3-iodo-4-methylanilino]uracil) and EMAU (6-[3-ethyl-4-methylanilino]uracil), were substituted with either a hydroxybutyl (HB) or a methoxybutyl (MB) group at their N3 positions to produce four agents: HB-EMAU, MB-EMAU, HB-IMAU, and MB-IMAU. These four new agents inhibited Staphylococcus aureus, coagulase-negative staphylococci, Enterococcus faecalis, and Enterococcus faecium. Time-kill assays and broth dilution testing confirmed bactericidal activity. These anilinouracil derivatives represent a novel class of antimicrobials with promising activities against gram-positive bacteria that are resistant to currently available agents, validating replication-specific DNA polymerase III as a new target for antimicrobial development.New antibacterial agents are needed to combat the multiply resistant gram-positive bacteria endemic in modern health care facilities (15). The 6-anilinouracils (AUs) illustrated in Fig. 1 are selective inhibitors of DNA polymerase III-c2 (pol III), an enzyme product of the polC gene (2,8,12). This enzyme is essential for the replication of the chromosome in gram-positive bacteria (7,9,17) and is found in low-GϩC-content eubacteria, including staphylococci, enterococci, streptococci, Listeria species, Bacillus species, and clostridia (1, 8). Briefly, the AUs, inhibitors of the DNA pol III enzyme, act through their capacity to mimic the guanine moiety of dGTP by forming three hydrogen bonds with cytosine at one of the two active domains (6,7,14). This leaves the second active site on the inhibitor, the aryl domain, available to bind to DNA pol III, which sequesters the enzyme into a nonproductive complex with template primer DNA (19).Structure-activity relationships of these AUs have been described previously (18, 19). The prototypic AUs, which have either weak antimicrobial activities or unacceptably low aqueous solubility (3-6, 13, 17), have now been substituted in their N3 positions and aryl rings to produce a series of more potent and more soluble molecules (13,16,19). The latest generation of these soluble forms (19) includes the N 3 -hydroxybutyl (HB) and N 3 -methoxybutyl (MB) derivatives of 6-[3Ј-ethyl-4Ј-methylanilino]uracil (EMAU) and 6-[3Ј-iodo-4Ј-methylanilino]uracil (IMAU) shown in Fig. 1. In this study, we describe the in vitro activities of HB-IMAU, HB-EMAU, MB-IMAU, and MB-EMAU against staphylococci and enterococci, bacteria that are pathogenic in humans and are difficult to treat with currently available and investigational antimicrobial agents.(This work was presented in part at the 39th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, Calif., 1999 [J. S. Daly, T. Giehl, N. C. Brown, C.Zhi, G. E. Wright, and R. T. Ellison III, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 1808, 1999].) Materials and methods. Bacterial strains used in this study were un...

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Characterization and overexpression of the gene encoding Staphylococcus aureus DNA polymerase III

Cited by 21 publications

References 19 publications

DNA Targeting as a Likely Mechanism Underlying the Antibacterial Activity of Synthetic Bis-Indole Antibiotics

DNA Targeting as a Likely Mechanism Underlying the Antibacterial Activity of Synthetic Bis-Indole Antibiotics

Conserved Interactions in the Staphylococcus aureus DNA PolC Chromosome Replication Machine

In Vitro Antimicrobial Activities of Novel Anilinouracils Which Selectively Inhibit DNA Polymerase III of Gram-Positive Bacteria

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