The Structure of T. aquaticus DNA Polymerase III Is Distinct from Eukaryotic Replicative DNA Polymerases

Bailey, Scott; Wing, Richard A.; Steitz, Thomas A.

doi:10.1016/j.cell.2006.07.027

Cited by 123 publications

(168 citation statements)

References 48 publications

(77 reference statements)

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“…The PolC palm domain has the same topology as the catalytic domain of human DNA polymerase ␤ (Fig. 4 A and B), providing further evidence that the C family bacterial replicative polymerases are not evolutionarily related to the B family eukaryotic and phage replicative polymerases (5,6,19) typified by RB69 polymerase (Fig. 4C).…”

Section: Structural Clues To the Evolution Of The 3 -5 Exonuclease Inmentioning

confidence: 66%

“…Because DNA passes through ␤ at an angle, there is sufficient space between ␤ and the PHP domain to accommodate the 3Ј-5Ј exonuclease domain in the intact PolC enzyme. Similar models have been proposed for DnaE (5,6,11). These models provide a static view of the holoenzyme, but do not suggest a dynamic mechanism for the enzyme to switch from polymerization mode to either exonuclease proofreading or translesion synthesis modes.…”

Section: Structural Clues To the Evolution Of The 3 -5 Exonuclease Inmentioning

confidence: 92%

“…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).…”

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confidence: 99%

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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: Structural Clues To the Evolution Of The 3 -5 Exonuclease Inmentioning

confidence: 66%

Section: Structural Clues To the Evolution Of The 3 -5 Exonuclease Inmentioning

confidence: 92%

mentioning

confidence: 99%

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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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“…Unlike Y-family polymerases whose structures are adapted to specialist lesion bypass (20), sequence analysis reveals few clues to DnaE2 function. All major DNA polymerase IIIα structural/functional domains (23,24) are readily identified in DnaE2, except for the very C-terminal region which in E. coli has been implicated in the interaction of α with the clamploader subunit, τ (28,29). A strong α-τ interaction enables simultaneous leading and lagging-strand synthesis by the DNA polymerase III holoenzyme (13), and the absence of this region in DnaE2 and all other nonessential dnaE-type α-subunits (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…All three DNA polymerase IIIα active-site acidic residues (23,24) are present in Mtb DnaE2 (D 439 , D 441 , D 579 ; Fig. S3).…”

Section: Catalytic Activity Of Dnae2 Is Required For Induced Mutagenementioning

confidence: 99%

Essential roles for imuA ′- and imuB -encoded accessory factors in DnaE2-dependent mutagenesis in Mycobacterium tuberculosis

Warner

Ndwandwe

Abrahams

et al. 2010

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

120

205

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In Mycobacterium tuberculosis (Mtb), damage-induced mutagenesis is dependent on the C-family DNA polymerase, DnaE2. Included with dnaE2 in the Mtb SOS regulon is a putative operon comprising Rv3395c, which encodes a protein of unknown function restricted primarily to actinomycetes, and Rv3394c, which is predicted to encode a Y-family DNA polymerase. These genes were previously identified as components of an imuA-imuB-dnaE2-type mutagenic cassette widespread among bacterial genomes. Here, we confirm that Rv3395c (designated imuA′) and Rv3394c (imuB) are individually essential for induced mutagenesis and damage tolerance. Yeast two-hybrid analyses indicate that ImuB interacts with both ImuA′ and DnaE2, as well as with the β-clamp. Moreover, disruption of the ImuB-β clamp interaction significantly reduces induced mutagenesis and damage tolerance, phenocopying imuA′, imuB, and dnaE2 gene deletion mutants. Despite retaining structural features characteristic of Y-family members, ImuB homologs lack conserved active-site amino acids required for polymerase activity. In contrast, replacement of DnaE2 catalytic residues reproduces the dnaE2 gene deletion phenotype, strongly implying a direct role for the α-subunit in mutagenic lesion bypass. These data implicate differential protein interactions in specialist polymerase function and identify the split imuA′-imuB/dnaE2 cassette as a compelling target for compounds designed to limit mutagenesis in a pathogen increasingly associated with drug resistance. (1), a Cfamily DNA polymerase implicated in error-prone bypass of DNA lesions. Loss of DnaE2 activity renders Mtb hypersensitive to DNA damage and eliminates induced mutagenesis. Moreover, dnaE2 deletion attenuates virulence and reduces the frequency of drug resistance in vivo. Mtb contains two DnaE-type polymerases; the other, DnaE1, provides essential, high-fidelity replicative polymerase function (1). However, the basis for the functional specialization of the DnaE subunits remains unclear (2, 3). Although structural determinants such as active-site architecture contribute significantly to inherent fidelity, it is possible that differential interactions with other DNA metabolic proteins modulate polymerase function.Bacterial genomes containing a DnaE2-type DNA polymerase almost invariably encode a homolog of ImuB (4-6), a putative Yfamily polymerase that is usually present in a LexA-regulated imuA-imuB-dnaE2 gene cassette (5). In Caulobacter crescentus, both ImuB and ImuA are required for induced mutagenesis and damage tolerance (6) whereas plasmid-encoded DnaE2 and ImuB mediate UV-induced mutagenesis in Deinococcus deserti (7). Although distributed widely across the bacterial domain, the imuA-imuB-dnaE2 cassette is not found in organisms possessing umuDC homologs (5). This suggests that the encoded proteins perform an analogous function to DNA polymerase V (8), the Y-family member required for damage-induced mutagenesis in Escherichia coli (9).Mtb contains a putative SOS-inducible operon, Rv3395c-Rv3394c (1, 10), locat...

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BacterialDNAPolymerases, Chemistry of

Pomerantz

O’Donnell

2008

Wiley Encyclopedia of Chemical Biology

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All DNA polymerases share a common two‐metal ion catalyzed chemistry of nucleotide incorporation. Structure analysis, however, suggests that DNA polymerases share one of two different ancestors, which converged to employ the same mechanism. Escherichia coli , the prototypical bacterium, encodes five different DNA polymerases. The chromosomal replicase functions closely with clamps, clamp‐loaders, and other proteins. Oxidative damage to DNA during normal cell growth requires interplay among the several distinct DNA polymerases, which enable the replicase to circumvent these obstacles and complete chromosomal replication. Additional processes involving DNA polymerases are brought into action during heightened levels of DNA damage. We review here DNA polymerase structure, catalytic mechanism, and several pathways in which various bacterial DNA polymerases act.

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The Structure of T. aquaticus DNA Polymerase III Is Distinct from Eukaryotic Replicative DNA Polymerases

Cited by 123 publications

References 48 publications

Structure of PolC reveals unique DNA binding and fidelity determinants

Structure of PolC reveals unique DNA binding and fidelity determinants

Essential roles for imuA ′- and imuB -encoded accessory factors in DnaE2-dependent mutagenesis in Mycobacterium tuberculosis

BacterialDNAPolymerases, Chemistry of

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