Genetic identification of two distinct DNA polymerases, DnaE and PolC, that are essential for chromosomal DNA replication in Staphylococcus aureus

Inoue, Ryotaro; Kaito, Chikara; Tanabe, Mikio; Kamura, Koushirou; Akimitsu, Nobuyoshi; Sekimizu, Kazuhisa

doi:10.1007/s004380100564

Cited by 67 publications

(73 citation statements)

References 26 publications

(39 reference statements)

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“…Interestingly, DnaE orthologs from proteobacteria such as Escherichia coli lack several of the key residues involved in metal binding (12) and thus appear unlikely to harbor an active exonuclease. Nonetheless, genetic evidence points to a functional importance of this domain in both PolC and DnaE, with mutations adjacent to metal-chelating resi- dues exhibiting slow-stop and template-slippage phenotypes (15,16). These phenotypes may reflect a role for the PHP domain in holoenzyme interactions or in facilitating efficient conformational changes necessary for polymerase fidelity.…”

Section: Polc Php Domain Has a Metal-binding Cluster Yet Lacks Catalyticmentioning

confidence: 99%

Structure of PolC reveals unique DNA binding and fidelity determinants

Evans

Davies²,

Bullard

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

124

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PolC is the polymerase responsible for genome duplication in many Gram-positive bacteria and represents an attractive target for antibacterial development. We have determined the 2.4-Å resolution crystal structure of Geobacillus kaustophilus PolC in a ternary complex with DNA and dGTP. The structure reveals nascent base pair interactions that lead to highly accurate nucleotide incorporation. A unique ␤-strand motif in the PolC thumb domain contacts the minor groove, allowing replication errors to be sensed up to 8 nt upstream of the active site. PolC exhibits the potential for large-scale conformational flexibility, which could encompass the catalytic residues. The structure suggests a mechanism by which the active site can communicate with the rest of the replisome to trigger proofreading after nucleotide misincorporation, leading to an integrated model for controlling the dynamic switch between replicative and repair polymerases. This ternary complex of a cellular replicative polymerase affords insights into polymerase fidelity, evolution, and structural diversity.DNA polymerase III ͉ DNA replication ͉ Gram-positive polymerase ͉ polymerase and histidinol phosphatase (PHP) ͉ ternary complex D NA polymerases are the enzymes responsible for DNA synthesis. Cellular organisms typically use multiple DNA polymerase types. The ''replicative'' polymerase performs the bulk of genome duplication, whereas various specialty polymerases repair damaged DNA and resolve Okazaki fragments. Across every kingdom of life, replicative polymerases exhibit certain hallmarks such as high fidelity, speed, and processivity (1). Polymerase holoenzyme accessory proteins play an integral role in achieving the extraordinary efficiency and accuracy of the replicative polymerase complex. These include a ''sliding clamp'' that encircles the DNA and increases processivity (2).Bacterial replicative polymerases comprise the C family of DNA polymerases (3) and differ significantly from the replicative polymerases of eukaryotes, bacteriophage, and archaea, which belong to the B family. The major C family replicative polymerases are DnaE (PolIII), found primarily in Gram-negative bacteria, and PolC (PolIIIC), found primarily in Gram-positive bacteria (4). Apoenzyme crystal structures of DnaE have revealed surprising structural differences in the catalytic center of the enzyme compared with B family polymerases, suggesting a separate evolutionary origin for the C family (5, 6).As the core component of the replicative polymerase complex in Gram-positive pathogens such as Staphylococcus aureus, PolC has received considerable attention as a potential target for antibacterial drug discovery (7,8). No currently marketed antibiotics target the central replication apparatus, making PolC a novel target for antibacterial development. Gram-negative and Gram-positive bacteria are separated by Ͼ1 billion years of evolution, and PolC and DnaE share Ͻ20% sequence identity; PolC is further differentiated from DnaE by domain rearrangements and by the presence of an ...

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Section: Polc Php Domain Has a Metal-binding Cluster Yet Lacks Catalyticmentioning

confidence: 99%

Structure of PolC reveals unique DNA binding and fidelity determinants

Evans

Davies²,

Bullard

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

124

View full text Add to dashboard Cite

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“…Why Is an Error-prone Polymerase Essential for Cell Viability?-Several studies in closely related Gram-positive bacteria have demonstrated that the dnaE gene is essential for viability (5,7). Further, studies by Dervyn et al (4) suggest that DnaE may be responsible for lagging strand synthesis, although alternative explanations may exist.…”

Section: Does Dnae Function At the Replicationmentioning

confidence: 99%

“…The dnaE gene is essential in Gram-positive bacteria that are closely related to Streptococcus pyogenes, such as Enterococcus faecalis, Staphylococcus aureus, and Bacillus subtilis (4,5,7). Furthermore, at nonpermissive temperature, a ts B. subtilis dnaE mutant is disrupted in lagging strand synthesis but not leading strand synthesis of plasmid DNA (4).…”

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confidence: 99%

The Essential C Family DnaE Polymerase Is Error-prone and Efficient at Lesion Bypass

Bruck

Goodman

O'Donnell

2003

Journal of Biological Chemistry

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DnaE-type DNA polymerases belong to the C family of DNA polymerases and are responsible for chromosomal replication in prokaryotes. Like most closely related Gram-positive cells, Streptococcus pyogenes has two DnaE homologs Pol C and DnaE; both are essential to cell viability. Pol C is an established replicative polymerase, and DnaE has been proposed to serve a replicative role. In this report, we characterize S. pyogenes DnaE polymerase and find that it is highly error-prone. DnaE can bypass coding and noncoding lesions with high efficiency. Error-prone extension is accomplished by either of two pathways, template-primer misalignment or direct primer extension. The bypass of abasic sites is accomplished mainly through "dNTP-stabilized" misalignment of template, thereby generating (؊1) deletions in the newly synthesized strand. This mechanism may be similar to the dNTP-stabilized misalignment mechanism used by the Y family of DNA polymerases and is the first example of lesion bypass and error-prone synthesis catalyzed by a C family polymerase. Thus, DnaE may function in an error-prone capacity that may be essential in Gram-positive cells but not Gram-negative cells, suggesting a fundamental difference in DNA metabolism between these two classes of bacteria.

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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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confidence: 99%

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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Genetic identification of two distinct DNA polymerases, DnaE and PolC, that are essential for chromosomal DNA replication in Staphylococcus aureus

Cited by 67 publications

References 26 publications

Structure of PolC reveals unique DNA binding and fidelity determinants

Structure of PolC reveals unique DNA binding and fidelity determinants

The Essential C Family DnaE Polymerase Is Error-prone and Efficient at Lesion Bypass

Conserved Interactions in the Staphylococcus aureus DNA PolC Chromosome Replication Machine

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