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Structure of Polc Reveals Unique DNA Binding and Fidelity Determinants

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Structure of Polc Reveals Unique DNA Binding and Fidelity Determinants Structure of PolC reveals unique DNA binding SEE COMMENTARY and fidelity determinants Ronald J. Evansa,1, Douglas R. Daviesb,1, James M. Bullarda, Jeffrey Christensenb, Louis S. Greena, Joseph W. Guilesa, Janice D. Patac, Wendy K. Ribblea, Nebojsa Janjica, and Thale C. Jarvisa,2 aReplidyne, Inc., Louisville, CO 80027; bdeCODE biostructures, Bainbridge Island, WA 98110; and cWadsworth Center, New York State Department of Health, Albany, NY 12201 Edited by John Kuriyan, University of California, Berkeley, CA, and approved November 3, 2008 (received for review October 7, 2008) PolC is the polymerase responsible for genome duplication in many provides limited insight into critical interactions governing PolC Gram-positive bacteria and represents an attractive target for anti- substrate binding and inhibition. bacterial development. We have determined the 2.4-Å resolution A significant body of literature is available on substrate-bound crystal structure of Geobacillus kaustophilus PolC in a ternary complex and apoenzyme structures of viral and bacteriophage replicative with DNA and dGTP. The structure reveals nascent base pair interac- DNA polymerases (9, 10). These polymerases have specialized tions that lead to highly accurate nucleotide incorporation. A unique features geared toward genome duplication within the context of a ␤-strand motif in the PolC thumb domain contacts the minor groove, host infection. Among cellular replicative polymerases, substrate allowing replication errors to be sensed up to 8 nt upstream of the ternary structure information has only recently become available active site. PolC exhibits the potential for large-scale conformational with a low-resolution structure of Thermus aquaticus (Taq) DnaE flexibility, which could encompass the catalytic residues. The struc- (11). However, at 4.6-Å resolution, that TaqDnaE ternary complex ture suggests a mechanism by which the active site can communicate did not reveal much detail regarding key DNA interactions. The with the rest of the replisome to trigger proofreading after nucleotide present work describes a much higher-resolution structure of a misincorporation, leading to an integrated model for controlling the cellular replicative polymerase, PolC, bound to nucleotide substrate dynamic switch between replicative and repair polymerases. This and DNA, thus providing insights into critical DNA interactions for ternary complex of a cellular replicative polymerase affords insights this unique class of DNA polymerase. into polymerase fidelity, evolution, and structural diversity. Results and Discussion DNA polymerase III ͉ DNA replication ͉ Gram-positive polymerase ͉ Three-Dimensional Structure of PolC. We determined the structure of polymerase and histidinol phosphatase (PHP) ͉ ternary complex Geobacillus kaustophilus PolC (GkaPolC) to 2.4-Å resolution in a ternary complex with primer-template DNA and incoming nucle- NA polymerases are the enzymes responsible for DNA syn- otide (Fig. 1). To protect the DNA from degradation during Ј Ј thesis. Cellular organisms typically use multiple DNA poly- crystallization, we deleted the 3 –5 proofreading exonuclease D BIOCHEMISTRY merase types. The ‘‘replicative’’ polymerase performs the bulk of domain. We also removed a poorly conserved N-terminal domain genome duplication, whereas various specialty polymerases repair from the construct. Importantly, these truncations did not com- damaged DNA and resolve Okazaki fragments. Across every promise core polymerase function (Fig. 1B). The structure spans kingdom of life, replicative polymerases exhibit certain hallmarks residues 233-1444, with the only disordered regions of the complex Ј Ј such as high fidelity, speed, and processivity (1). Polymerase being the linker region that replaces the 3 –5 exonuclease domain, holoenzyme accessory proteins play an integral role in achieving the 2 nearby loops, and the distal 4 bp of the DNA. We refined 3 extraordinary efficiency and accuracy of the replicative polymerase complexes [supporting information (SI) Table S1] that differ in the divalent metal ions included during the crystallization (Mg2ϩ/Zn2ϩ, complex. These include a ‘‘sliding clamp’’ that encircles the DNA 2ϩ 2ϩ 2ϩ and increases processivity (2). Mn only, and Mn /Zn ). Except where noted, we describe the Mg-bound structure because the 3 structures are nearly identical Bacterial replicative polymerases comprise the C family of DNA Ј polymerases (3) and differ significantly from the replicative poly- apart from the metal-binding sites. In all cases, the 3 end of the merases of eukaryotes, bacteriophage, and archaea, which belong primer strand DNA was terminated with a dideoxynucleoside to to the B family. The major C family replicative polymerases are prevent catalysis. DnaE (PolIII), found primarily in Gram-negative bacteria, and PolC exhibits the canonical polymerase configuration resembling PolC (PolIIIC), found primarily in Gram-positive bacteria (4). a right hand. The central region of PolC forms the polymerase core Apoenzyme crystal structures of DnaE have revealed surprising (residues 828-1293), with fingers, palm, and thumb domains that are structural differences in the catalytic center of the enzyme com- defined by their interactions with the DNA substrate. The DNA pared with B family polymerases, suggesting a separate evolution- duplex is held between the thumb and the C-terminal domain of ary origin for the C family (5, 6). As the core component of the replicative polymerase complex in Author contributions: R.J.E., D.R.D., N.J., and T.C.J. designed research; R.J.E., D.R.D., J.M.B., Gram-positive pathogens such as Staphylococcus aureus, PolC has J.C., L.S.G., and W.K.R. performed research; J.W.G. contributed new reagents/analytic tools; received considerable attention as a potential target for antibacte- J.D.P. analyzed data; and D.R.D., J.D.P., and T.C.J. wrote the paper. rial drug discovery (7, 8). No currently marketed antibiotics target The authors declare no conflict of interest. the central replication apparatus, making PolC a novel target for This article is a PNAS Direct Submission. antibacterial development. Gram-negative and Gram-positive bac- Data deposition: The atomic coordinates and structure factors have been deposited in the teria are separated by Ͼ1 billion years of evolution, and PolC and Protein Data Bank, www.pdb.org (PDB ID codes 3F2B, 3F2C, and 3F2D). DnaE share Ͻ20% sequence identity; PolC is further differentiated See Commentary on page 20565. from DnaE by domain rearrangements and by the presence of an 1R.J.E. and D.R.D. contributed equally to this work. intrinsic 3Ј–5Ј proofreading exonuclease domain. Notably, PolC 2To whom correspondence should be addressed. E-mail: [email protected]. and DnaE frequently exhibit differential sensitivity to inhibition by This article contains supporting information online at www.pnas.org/cgi/content/full/ nucleotide analogs (8), suggesting significant differences in active- 0809989106/DCSupplemental. site structure. Thus, the available structural information on DnaE © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0809989106 PNAS ͉ December 30, 2008 ͉ vol. 105 ͉ no. 52 ͉ 20695–20700 Downloaded by guest on September 29, 2021 Fig. 1. PolC structure and domain organization. (A) Ribbon representation of PolC structure showing DNA primer strand (white) and DNA template strand (orange). dGTP is shown in sphere representation, as are bound Mg2ϩ (green) and Zn2ϩ (gray) ions. (B) Domains are shown for full-length PolC and for the crystallized truncation. Relative polymerase primer extension and 3Ј–5Ј exonuclease activities are indicated. (C) PolC surface representation with domains colored as in B and modeling ssDNA (pink) binding to the OB fold in PolC based on the alignment of RPA70-ssDNA (1JMC) to the PolC OB fold (Fig. S4). PolC, which we have termed the duplex-binding (DB) domain. The 1C). PolC OB plays an important role in intrinsic polymerase DNA single-stranded DNA (ssDNA) template enters the polymerase function; truncation to remove PolC OB results in an elevated Km active site through a crevice formed between the fingers and DB and a 20-fold reduction in polymerase activity (data not shown). domain. An oligonucleotide/oligosaccharide-binding (OB) domain SSBs play a critical role in melting template secondary structure in (Pfam PF01336) and a polymerase and histidinol phosphatase advance of the lagging-strand DNA polymerase. N-terminal fusion (PHP) domain (12) (Pfam PF02811) are located N-terminal to the of RB69 SSB to polymerase enhances intrinsic processivity (14). polymerase domain. Thus, it seems quite plausible that C family polymerases could Despite having limited sequence similarity, individual domains of benefit from an intrinsic SSB-like function. Interestingly, few other PolC and DnaE (5, 6) superimpose with rmsds ranging from 1.19 DNA polymerases exhibit OB folds. Å for the OB domain to 2.55 Å for the index finger domain (Fig. S1). One unique feature of the PolC palm domain is that it contains PolC PHP Domain Has a Metal-Binding Cluster Yet Lacks Catalytic a zinc finger (Fig. S2) consisting of a tetrahedral cluster of highly Activity. The PHP motif is universally found in C family poly- conserved cysteines that are essential for activity (13). Although the merases, but rarely in other polymerases (3). The diverse PHP zinc finger points away from the polymerization active site and is family is associated with a range of hydrolase activities, with many unlikely
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