Insights into the Replisome from the Structure of a Ternary Complex of the DNA Polymerase III α-Subunit

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

doi:10.1016/j.jmb.2008.07.058

Cited by 85 publications

(122 citation statements)

References 53 publications

(71 reference statements)

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“…The first of these HhH motifs contacts the backbone phosphates of the primer strand at 8 and 9 residues upstream of the nascent base pair, whereas the second HhH motif contacts backbone phosphates of the template strand 12 and 13 residues upstream of the nascent base pair. These interactions are conserved in the DnaE complex (11). Even a 17-aa C-terminal truncation of GkaPolC, which would partially disrupt the second HhH motif, results in a detectable decrease in polymerase activity (data not shown); C-terminal truncation of Bacillus subtilis PolC by 44 aa (corresponding to residue 1400 in GkaPolC) results in complete loss of activity (20), consistent with disruption of both HhH motifs.…”

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

confidence: 70%

“…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: 75%

“…S4). In the DnaE ternary complex (11), the OB domain appears to form a track guiding the template strand into the active site. In the PolC structure, the short ssDNA downstream template occupies the channel formed by the DB and fingers domain but does not reach the OB domain.…”

Section: Resultsmentioning

confidence: 99%

“…S5), suggesting a functional importance for metal binding. PHP domains found in DnaEs of thermophilic origin exhibit 3Ј-5Ј exonuclease activity (11). In contrast, PolC PHP lacks detectable nuclease activity (Fig.…”

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

confidence: 94%

“…These polymerases have specialized features geared toward genome duplication within the context of a host infection. Among cellular replicative polymerases, substrate ternary structure information has only recently become available with a low-resolution structure of Thermus aquaticus (Taq) DnaE (11). However, at 4.6-Å resolution, that TaqDnaE ternary complex did not reveal much detail regarding key DNA interactions.…”

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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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: 70%

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

confidence: 75%

Section: Resultsmentioning

confidence: 99%

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

confidence: 94%

mentioning

confidence: 99%

See 3 more Smart Citations

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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Inhibition of DNA synthesis facilitates expansion of low‐complexity repeats

Kuzminov

2013

BioEssays

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Summary Simple DNA repeats (trinucleotide repeats, micro- and minisatellites) are prone to expansion/contraction via formation of secondary structures during DNA synthesis. Such structures both inhibit replication forks and create opportunities for template-primer slippage making these repeats unstable. Yet, certain aspects of simple repeat instability suggest additional mechanisms of replication inhibition dependent on the primary DNA sequence, rather than on secondary structure formation. I argue that expanded simple repeats, due to their lower DNA complexity, should transiently inhibit DNA synthesis by locally depleting specific DNA precursors. Such transient inhibition should promote formation of secondary structures and should stabilize these structures, facilitating strand slippage. Thus, replication problems at simple repeats could be explained by potentiated toxicity, where the secondary structure-driven repeat instability is enhanced by DNA polymerase stalling at the low complexity template DNA.

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Optical tweezers experiments resolve distinct modes of DNA‐protein binding

McCauley¹,

Williams²

2008

Biopolymers

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Optical tweezers are ideally suited to perform force microscopy experiments that isolate a single biomolecule, which then provides multiple binding sites for ligands. The captured complex may be subjected to a spectrum of forces, inhibiting or facilitating ligand activity. In the following experiments, we utilize optical tweezers to characterize and quantify DNA binding of various ligands. High mobility group type B (HMGB) proteins, which bind to double‐stranded DNA, are shown to serve the dual purpose of stabilizing and enhancing the flexibility of double stranded DNA. Unusual intercalating ligands are observed to thread into and lengthen the double‐stranded structure. Proteins binding to both double‐ and single‐stranded DNA, such as the alpha polymerase subunit of E. coli Pol III, are characterized, and the subdomains containing the distinct sites responsible for binding are isolated. Finally, DNA binding of bacteriophage T4 and T7 single‐stranded DNA (ssDNA) binding proteins is measured for a range of salt concentrations, illustrating a binding model for proteins that slide along double‐stranded DNA, ultimately binding tightly to ssDNA. These recently developed methods quantify both the binding activity of the ligand as well as the mode of binding. © 2008 Wiley Periodicals, Inc. Biopolymers 91: 265–282, 2009.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

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Insights into the Replisome from the Structure of a Ternary Complex of the DNA Polymerase III α-Subunit

Cited by 85 publications

References 53 publications

Structure of PolC reveals unique DNA binding and fidelity determinants

Structure of PolC reveals unique DNA binding and fidelity determinants

Inhibition of DNA synthesis facilitates expansion of low‐complexity repeats

Optical tweezers experiments resolve distinct modes of DNA‐protein binding

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