Structure of PolC reveals unique DNA binding and fidelity determinants

Abstract

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 ...

Structural evidence for the rare tautomer hypothesis of spontaneous mutagenesis

“…The interactions between the water molecule and the base, and the three anchoring protein residues are conserved in all three complexes. (B) A member of the C-family DNA polymerase, Geobacillus kaustophilus DNA polymerase PolC (3F2B) (43). A water molecule makes similar interaction with the incoming nucleotide base which is coordinated by a single histidine instead of the anchoring side chains.…”

Replicative DNA Polymerases

“…The most recent structure is of a ternary complex containing primer-template DNA and incoming dNTP bound to Pol C from Geobacillus kaustophilus (Gka) (Evans et al 2008). The Taq and Eco structures without DNA are very similar in the regions that can be compared (Fig.…”

“…These latter enzymes have the proofreading and polymerase activities in a single polypeptide, and a different domain organization, with putative t-binding and OB-fold domains preceding a discontinuous PHP domain that incorporates the proofreader, and the b-binding motif being right at the carboxyl terminus (Evans et al 2008). Although it was for some time believed that Pol C was dedicated to leading-strand synthesis and DnaE to the lagging strand (Dervyn et al 2001), recent work suggests that most chromosomal DNA synthesis in these bacteria is performed by Pol C. However, because only the DnaE polymerase can extend RNA primers like those made by the DnaG primase, it has a critical role in lagging-strand synthesis.…”

“…The Gka Pol C ternary complex is also similar to the Taq a -DNA structure in this respect. Interestingly, the structure of a b 2 -DNA complex (Georgescu et al 2008a) can be docked neatly into both the a and Pol C -DNA structures to give plausible models of the a or Pol C -b 2 -DNA replicases in the polymerization mode (Evans et al 2008;Wing et al 2008), and it has been suggested that the open structures might mimic the replicase structure in the editing mode ( Fig. 2A).…”

A consensus view of DNA binding by the C family of replicative DNA polymerases

“…C family polymerases are quite large multidomain proteins, larger than polymerases of other families, which may explain why structural representatives of this class have been solved only recently. In this issue of PNAS, Evans et al (2) present the structure of Geobacillus kaustophilus Pol C bound to a DNA substrate at 2.4-Å resolution. This structure provides a high-resolution analysis of a C family replicative DNA polymerase bound to DNA and dNTP.…”