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• W2008017844 abstract "The crystal structure of Thermus aquaticus DNA polymerase III α subunit reveals that the structure of the catalytic domain of the eubacterial replicative polymerase is unrelated to that of the eukaryotic replicative polymerase but rather belongs to the Polβ-like nucleotidyltransferase superfamily. A model of the polymerase complexed with both DNA and β-sliding clamp interacting with a reoriented binding domain and internal β binding site was constructed that is consistent with existing biochemical data. Within the crystal, two C-terminal domains are interacting through a surface that is larger than many dimer interfaces. Since replicative polymerases of eubacteria and eukaryotes/archaea are not homologous, the nature of the replicative polymerase in the last common ancestor is unknown. Although other possibilities have been proposed, the plausibility of a ribozyme DNA polymerase should be considered. The crystal structure of Thermus aquaticus DNA polymerase III α subunit reveals that the structure of the catalytic domain of the eubacterial replicative polymerase is unrelated to that of the eukaryotic replicative polymerase but rather belongs to the Polβ-like nucleotidyltransferase superfamily. A model of the polymerase complexed with both DNA and β-sliding clamp interacting with a reoriented binding domain and internal β binding site was constructed that is consistent with existing biochemical data. Within the crystal, two C-terminal domains are interacting through a surface that is larger than many dimer interfaces. Since replicative polymerases of eubacteria and eukaryotes/archaea are not homologous, the nature of the replicative polymerase in the last common ancestor is unknown. Although other possibilities have been proposed, the plausibility of a ribozyme DNA polymerase should be considered. Replicative DNA polymerases function at the heart of the replication fork to faithfully duplicate chromosomal DNA. In both bacteria and eukaryotes, the replicative polymerase forms the core of a large complex macromolecular assembly termed the replicase. The eubacterial replicase, or DNA polymerase III (PolIII) holoenzyme, is composed of fifteen subunits that can be divided into three functional elements: the core polymerase, the clamp loader complex, and the β-sliding clamp (Kornberg and Baker, 1992Kornberg A. Baker T. DNA Replication.Second Edition. Freeman, New York1992Google Scholar). In Escherichia coli, the PolIII core is a heterotrimer composed of α, ɛ, and θ subunits. The α subunit is the replicative DNA polymerase (PolIIIα). The ɛ subunit of the complex is a 3′-5′ proofreading exonuclease which ensures the fidelity of replication. The final subunit, θ, has no known function except a minor stimulation of the ɛ subunit (Johnson and O'Donnell, 2005Johnson J. O'Donnell M. Cellular DNA replicases: components and dynamics at the replication fork.Annu. Rev. Biochem. 2005; 2005: 283-315Crossref Scopus (420) Google Scholar). Association of PolIIIα with the clamp (β subunit), a ring-shaped molecule believed to topologically encircle the duplex DNA product (Kong et al., 1992Kong X.P. Onrust R. O'Donnell M. Kuriyan J. Three-dimensional structure of the beta subunit of E. coli DNA polymerase III holoenzyme: A sliding DNA clamp.Cell. 1992; 69: 425-437Abstract Full Text PDF PubMed Scopus (607) Google Scholar), makes the complex extremely processive. Due to the antiparallel structure of the double helix and because polymerases can only synthesize DNA with a 5′ to 3′ polarity, the replicase utilizes two PolIII core complexes to synthesize one strand of DNA continuously and the other discontinuously. The continuous strand is synthesized in the same direction as replication fork movement and is termed the leading strand, whereas the discontinuous strand is synthesized in the opposite direction, in 1–3 kb Okazaki fragments, and is termed the lagging strand. The clamp loader complex, a multisubunit ATPase (subunit composition of γτ2δδ′χψ) functions to load the clamp onto RNA primed initiation sites (Johnson and O'Donnell, 2005Johnson J. O'Donnell M. Cellular DNA replicases: components and dynamics at the replication fork.Annu. Rev. Biochem. 2005; 2005: 283-315Crossref Scopus (420) Google Scholar). Upon completion of an Okazaki fragment, PolIIIα must be released from the DNA so that synthesis of the next fragment can begin. This cycling of PolIIIα, also known as the lagging strand processivity switch, is facilitated by the τ subunit of the clamp loader complex (Leu et al., 2003Leu F.P. Georgescu R. O'Donnell M.F. Mechanism of the E. coli τ processivity switch during lagging-strand synthesis.Mol. Cell. 2003; 11: 315-327Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The interactions between PolIIIα, clamp, and the τ subunit have been studied best in E. coli. Two distinct clamp binding sites on PolIIIα have been identified: one internal and one C-terminal. The internal β binding site is absolutely required for binding clamp both in vitro and in vivo (Dohrmann and McHenry, 2005Dohrmann P.R. McHenry C.S. A bipartite polymerase-processivity Ffactor interaction: only the internal β binding site of the α subunit is required for processive replication by the DNA polymerase III holoenzyme.J. Mol. Biol. 2005; 350: 228-239Crossref PubMed Scopus (68) Google Scholar). The role of the C-terminal binding site is less clear. Peptides corresponding to the C-terminal twenty amino acids of PolIIIα bind to both the clamp and the τ subunit in a competitive reaction (Lopez de Saro et al., 2003Lopez de Saro F.J. Georgescu R.E. O'Donnell M. A peptide switch regulates DNA polymerase processivity.Proc. Natl. Acad. Sci. USA. 2003; 100: 14689-14694Crossref PubMed Scopus (64) Google Scholar). However, a C-terminal deletion mutant of PolIIIα has only a 4-fold reduction in clamp binding but a 400-fold reduction in τ subunit binding. This implies that the majority of the energy for binding clamp comes from the internal β binding site. Therefore, upon encountering a nick, the τ subunit must disrupt the internal β binding site in order to displace the polymerase from clamp (Leu et al., 2003Leu F.P. Georgescu R. O'Donnell M.F. Mechanism of the E. coli τ processivity switch during lagging-strand synthesis.Mol. Cell. 2003; 11: 315-327Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar); the mechanism through which this disruption occurs is unknown. Six families of DNA-dependent DNA polymerases (A, B, C, D, X, and Y) have been identified on the basis of amino acid sequence comparisons (Filée et al., 2002Filée J. Forterre P. Sen-Lin T. Laurent J. Evolution of DNA polymerase families: evidence for multiple gene exchange between cellular and viral proteins.J. Mol. Evol. 2002; 54: 763-773Crossref PubMed Scopus (178) Google Scholar). Polymerases from the first three families play critical roles in replication. PolIIIα is a member of family C, which is found exclusively in eubacteria, whereas all archaeal and eukaryotic replicative polymerases belong to family B. Family A includes DNA polymerase I, a eubacterial polymerase that converts the Okazaki RNA primer into DNA. Family D polymerases are currently restricted to archaea, and their biological role is unknown. The last two families, families X and Y, contain specialized polymerases that are involved in DNA repair (Filée et al., 2002Filée J. Forterre P. Sen-Lin T. Laurent J. Evolution of DNA polymerase families: evidence for multiple gene exchange between cellular and viral proteins.J. Mol. Evol. 2002; 54: 763-773Crossref PubMed Scopus (178) Google Scholar). Crystal structures have been determined for members of all the major families of DNA polymerase except family C. Although no structure is known for a true eukaryotic replicative polymerase, those of distant viral homologs of these enzymes have been determined (Wang et al., 1997Wang J. Sattar A.K. Wang C.C. Karam J.D. Konigsberg W.H. Steitz T.A. Crystal structure of a pol α family replication DNA polymerase from bacteriophage RB69.Cell. 1997; 89: 1087-1099Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar). These structures have revealed that all DNA polymerases share a common overall architecture likened to that of a right hand, consisting of three domains: the fingers, palm, and thumb. The fingers domain interacts with the incoming nucleotide and the ssDNA template, while the thumb domain binds the duplex DNA product. The palm domain contains the catalytic residues that bind the magnesium ions needed for the phosphoryl transfer reaction. The structures of the fingers and thumb domains are unique in each family, whereas the palm domain can be assigned to one of two folds. Families A, B, and Y all share the classic palm domain first seen in the structure of the Klenow fragment of DNA polymerase I (Ollis et al., 1985Ollis D.L. Brick P. Hamlin R. Xuong N.G. Steitz T.A. Structure of large fragment of Escherichia coli DNA polymerase I complexed with dTMP.Nature. 1985; 313: 762-766Crossref PubMed Scopus (727) Google Scholar). This fold is also observed in the palm domain of reverse transcriptase (Kohlstaedt et al., 1992Kohlstaedt L.A. Wang J. Friedman J.M. Rice P.A. Steitz T.A. Crystal structure at 3.5Å resolution of HIV-1 reverse transcriptase complexed with an inhibitor.Science. 1992; 256: 1783-1790Crossref PubMed Scopus (1730) Google Scholar) and T7 RNA polymerase (Sousa et al., 1993Sousa R. Chung Y.J. Rose J.P. Wang B.C. Crystal structure of bacteriophage T7 RNA polymerase at 3.3Å resolution.Nature. 1993; 364: 593-599Crossref PubMed Scopus (328) Google Scholar). In contrast, the palm domains of the family X polymerases (Sawaya et al., 1994Sawaya M.R. Pelletier H. Kumar A. Wilson S.H. Kraut J. Crystal structure of rat DNA polymerase beta: evidence for a common polymerase mechanism.Science. 1994; 264: 1930-1935Crossref PubMed Scopus (389) Google Scholar, Davies et al., 1994Davies II, J.F. Almassy R.J. Hostomska Z. Ferre R.A. Hostomsky Z. 2.3 Å crystal structure of the catalytic domain of DNA polymerase β.Cell. 1994; 76: 1123-1133Abstract Full Text PDF PubMed Scopus (171) Google Scholar) belong to the Polβ-like nucleotidyltransferase (βNT) superfamily (Aravind and Koonin, 1999Aravind L. Koonin E.V. DNA polymerase β-like nucleotidyltransferase superfamily: identification of these new families, classification and evolutionary history.Nucleic Acids Res. 1999; 27: 1609-1618Crossref PubMed Scopus (268) Google Scholar). It is generally accepted that there is no relationship between the classic and the βNT palm folds, and they may represent a case of convergent evolution to a common catalytic mechanism (Steitz et al., 1994Steitz T.A. Smerdon S.J. Jager J. Joyce C.M. A unified polymerase mechanism for nonhomologous DNA and RNA polymerases.Science. 1994; 266: 2022-2025Crossref PubMed Scopus (266) Google Scholar). Regardless of the fold of their palm domains, all known polynucleotide polymerases utilize the same two-metal-ion mechanism for nucleotide addition (Steitz, 1998Steitz T.A. A mechanism for all polymerases.Nature. 1998; 391: 231-232Crossref PubMed Scopus (474) Google Scholar). Further, superposition of the DNA substrates bound to the classic and βNT palm folds shows a similar orientation of the catalytic metal ions and nonhomologous “thumb” and “fingers” domains carrying out similar functions (Steitz et al., 1994Steitz T.A. Smerdon S.J. Jager J. Joyce C.M. A unified polymerase mechanism for nonhomologous DNA and RNA polymerases.Science. 1994; 266: 2022-2025Crossref PubMed Scopus (266) Google Scholar), which lead to the renaming of these domains from that given (Pelletier et al., 1994Pelletier H. Sawaya M.R. Kumar A. Wilson S.H. Kraut K. Structures of ternary complexes of rat DNA polymerase β, a DNA template-primer, and ddCTP.Science. 1994; 264: 1891-1903Crossref PubMed Scopus (741) Google Scholar). Despite decades of research on DNA polymerases, relatively little detail is known of the polymerase actually responsible for replicating the genome of eubacteria, PolIIIα. The expected polymerase domains of PolIIIα (Kim et al., 1997Kim D.R. Pritchard A.E. McHenry C.S. Localization of the active site of the α subunit of the escherichia coli DNA polymerase III holoenzyme.J. Bacteriol. 1997; 179: 6721-6728PubMed Google Scholar) share no recognizable sequence similarity with any polymerase of known structure. It has therefore been unclear whether family C polymerases have the same architecture as the other DNA polymerase families and whether their catalytic domain is related to that of the family B eukaryotic replicative polymerases. Furthermore, unlike the majority of polymerases whose structures are known, PolIIIα functions as part of a larger macromolecule machine. Within this machine, PolIIIα is known to interact directly with four other subunits: ɛ, θ, β, and τ (Kornberg and Baker, 1992Kornberg A. Baker T. DNA Replication.Second Edition. Freeman, New York1992Google Scholar). The molecular detail of how these subunits function with PolIIIα in replicating the genome is poorly understood. The crystal structure of full-length PolIIIα described here provides a framework for interpreting the existing biochemical information, as well as a foundation for future biochemical and structural studies aimed at understanding bacterial replication. The crystal structure of full-length Thermus aquaticus PolIIIα was determined to 3.0 Å resolution and represents the first crystal structure of a cellular replicative polymerase. The amino acid sequence of the Taq enzyme is 39% identical to that of its E. coli homolog (see Figure S1 in the Supplemental Data available with this article online). Indeed, replication of the closely related organism Thermus thermophilus has been reconstituted in vitro and shown to not differ significantly from that of E. coli (Bullard et al., 2002Bullard J.M. Williams J.C. Acker W.K. Jacobi C. Janjic N. McHenry C.S. DNA Polymerase III holoenzyme from Thermus thermophilus identification, expression, purification of components, and the use to reconstitute a processive replicase.J. Biol. Chem. 2002; 277: 13401-13408Crossref PubMed Scopus (20) Google Scholar). The high degree of similarity allows us to confidently identify the corresponding residues in E. coli PolIIIα (throughout the manuscript, residue numbering in E. coli will have the prefix Eco). Orthorhombic crystals in space group C2221 were grown by vapor diffusion and contained one polymerase molecule per asymmetric unit. Initial phases were obtained by the single isomorphous replacement method using crystals soaked in mercury. These phases were improved by cross crystal averaging using a second crystal form (space group P212121), which diffracted to 3.4 Å. After temperature factor sharpening of the amplitudes the resulting maps gave readily interpretable side-chain density across the majority of the molecule. A typical portion of electron density is shown in Figure 1. The structures of the enzyme complexed with dATP that had been soaked into the crystals were determined in the C2221 crystal form by difference Fourier techniques. Both crystal forms were obtained in the presence of primer/template DNA; however, no density for this substrate was observed. Recently, it has become apparent that this maybe due to the presence of an active nuclease domain (see below) that presumably digested the DNA. The final model, refined against the C2221 crystal form, has good geometry and an R factor and free R factor of 22.7% and 27.5%, respectively (Table 1).Table 1Crystallographic Data and Refinement Statistics of PolIIIαApodATP SoakApoSpace groupC2221C2221P212121Unit cell dimensions (Å)175.1 × 186.9 × 125.8175.1 × 186.9 × 125.8116.2 × 119.9 × 241.5Resolution (Å)50–3.050–3.750–3.4Rsym (%)aNumbers in parentheses correspond to the highest resolution shell., bRsym = Σ|I − <I>|/ΣI where I = observed intensity and <I> = average intensity of multiple observations of symmetry-related reflections.8.9 (70.4)20.9 (100.00)14.4 (100.00)I/σaNumbers in parentheses correspond to the highest resolution shell.24.4 (1.6)6.9 (1.2)7.4 (1.1)Completeness (%)aNumbers in parentheses correspond to the highest resolution shell.97.8 (80.9)100.0 (100.0)93.8 (94.2)Unique reflections40,36121,99784,203RedundancyaNumbers in parentheses correspond to the highest resolution shell.12.9 (7.3)4.9 (4.9)3.5 (3.5)Copies in AU112Phasing resolution (Å)50–3.250–6.0Phasing power (acentric)0.9060.797Figure of merit0.2890.396Rmsd bond length (Å)0.0090.013Rmsd bond angle (°)1.1691.380Rcryst (%)22.723.9Rfree (%)27.530.2PDB ID2HPI2HPMa Numbers in parentheses correspond to the highest resolution shell.b Rsym = Σ|I − <I>|/ΣI where I = observed intensity and <I> = average intensity of multiple observations of symmetry-related reflections. Open table in a new tab The 140 kDa polymerase is organized into six domains that form an irregular pyramid around a central cavity (Figure 2). Four domains in the N-terminal two-thirds of the molecule form the characteristic hand-shaped cleft previously found in other polymerases. The structure of the polymerase complexed with dATP locates the polymerase active site region and, therefore, by analogy to other polymerases, the fingers (residues 623–835; Eco560–778) and the palm (residues 286–492 and 575–622; Eco272–430 and Eco511–559), which together make up the base and one wall of the cleft. The remainder of the cleft is formed by an N-terminal polymerase and histidinol phosphatase (PHP) domain (residues 1–285; Eco1–272) and a small four helix bundle which is most likely the thumb (residues 493–574; Eco431–510). The localization of the polymerase domains to the N-terminal two-thirds of the protein is consistent with previous deletion mutagenesis studies (Kim et al., 1997Kim D.R. Pritchard A.E. McHenry C.S. Localization of the active site of the α subunit of the escherichia coli DNA polymerase III holoenzyme.J. Bacteriol. 1997; 179: 6721-6728PubMed Google Scholar). The domain that contains the internal binding site for the β-clamp, which we have termed the β binding domain (residues 836–1012; Eco779–973), extends from the active site via the fingers and completes the base of the pyramid. The C-terminal domain, or CTD, (residues 1013–1220; Eco974–1160) lies loosely seated on top of the β binding domain and caps the pyramid. The core of the palm domain, which contains the metal ion binding carboxylates that are indispensable for catalysis, consists of a five stranded mixed β sheet connected by two α helices. The topology and connectivity of this core is identical to that of the βNT fold (Figure 3A), placing the replicative polymerases of eubacteria (family C DNA polymerases) into the βNT superfamily. In contrast, the replicative polymerases of eukaryotes belong to the classic palm superfamily (Figure 3A), as do the majority of DNA polymerases. In fact, the only other DNA polymerase family to utilize the βNT fold is family X, whose founding member, Polβ, acts in base-excision repair (Davies et al., 1994Davies II, J.F. Almassy R.J. Hostomska Z. Ferre R.A. Hostomsky Z. 2.3 Å crystal structure of the catalytic domain of DNA polymerase β.Cell. 1994; 76: 1123-1133Abstract Full Text PDF PubMed Scopus (171) Google Scholar, Sawaya et al., 1994Sawaya M.R. Pelletier H. Kumar A. Wilson S.H. Kraut J. Crystal structure of rat DNA polymerase beta: evidence for a common polymerase mechanism.Science. 1994; 264: 1930-1935Crossref PubMed Scopus (389) Google Scholar). Other βNT polymerases have more specialized functions. Poly(A) polymerase is a template-independent RNA polymerase that polyadenylates pre-mRNA in eukaryotes, and the CCA adding enzyme ensures maturation and repair of the 3′ end of tRNA by means of the template independent addition of the sequence CCA (Aravind and Koonin, 1999Aravind L. Koonin E.V. DNA polymerase β-like nucleotidyltransferase superfamily: identification of these new families, classification and evolutionary history.Nucleic Acids Res. 1999; 27: 1609-1618Crossref PubMed Scopus (268) Google Scholar). The structure of the palm domain of rat Polβ (Davies et al., 1994Davies II, J.F. Almassy R.J. Hostomska Z. Ferre R.A. Hostomsky Z. 2.3 Å crystal structure of the catalytic domain of DNA polymerase β.Cell. 1994; 76: 1123-1133Abstract Full Text PDF PubMed Scopus (171) Google Scholar, Sawaya et al., 1994Sawaya M.R. Pelletier H. Kumar A. Wilson S.H. Kraut J. Crystal structure of rat DNA polymerase beta: evidence for a common polymerase mechanism.Science. 1994; 264: 1930-1935Crossref PubMed Scopus (389) Google Scholar) is most closely related in the structural database to the palm domain of PolIIIα. The two palm domains superimpose with a root-mean-square deviation (rmsd) of 2.6 Å over 77 Cα atoms (Figure 3B). The degree of similarity between the βNT palm domains of polymerase families C and X is remarkably similar to that observed between the classic palm domains of polymerase families A and B. Superposition of the palm domain of the family A Klenow fragment (Ollis et al., 1985Ollis D.L. Brick P. Hamlin R. Xuong N.G. Steitz T.A. Structure of large fragment of Escherichia coli DNA polymerase I complexed with dTMP.Nature. 1985; 313: 762-766Crossref PubMed Scopus (727) Google Scholar) on the palm domain of the family B RB69 polymerase (Wang et al., 1997Wang J. Sattar A.K. Wang C.C. Karam J.D. Konigsberg W.H. Steitz T.A. Crystal structure of a pol α family replication DNA polymerase from bacteriophage RB69.Cell. 1997; 89: 1087-1099Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar) gives an rmsd of 2.6 Å over 82 Cα atoms. All βNT family members contain three conserved acidic residues that bind the catalytic magnesium ions (Pelletier et al., 1994Pelletier H. Sawaya M.R. Kumar A. Wilson S.H. Kraut K. Structures of ternary complexes of rat DNA polymerase β, a DNA template-primer, and ddCTP.Science. 1994; 264: 1891-1903Crossref PubMed Scopus (741) Google Scholar). Superposition of the PolIIIα and rat Polβ palm domains aligns the three catalytic aspartates of Polβ (D190, D192, and D256) with the three absolutely conserved aspartate residues of PolIIIα (D463, D465, and D618; EcoD401, EcoD403, and EcoD555), identified by Pritchard and McHenry, 1999Pritchard A.E. McHenry C.S. Identification of the acidic residues in the active site of DNA polymerase III.J. Mol. Biol. 1999; 285: 1067-1080Crossref PubMed Scopus (39) Google Scholar as the catalytic residues of PolIIIα by mutagenesis (Figure 3B). Thus, it is expected that PolIIIα will utilize the same two-metal-ion catalytic mechanism as Polβ and all other known polymerases (Steitz, 1998Steitz T.A. A mechanism for all polymerases.Nature. 1998; 391: 231-232Crossref PubMed Scopus (474) Google Scholar). In fact, examination of the PolIIIα active site reveals a single magnesium ion located between the catalytic residues D463 (EcoD401) and D465 (EcoD403). This ion was assigned as magnesium after inspection of anomalous difference Fourier maps derived from crystals soaked in manganese. Three other conserved residues in Polβ (G179, S180, and R254) are also conserved in PolIIIα (G425, S426, and K616; EcoG363, EcoS364, and EcoK553). The glycine and serine lie in a loop which forms part of the incoming nucleotide binding pocket and are conserved across the βNT superfamily (although in some sequences the serine is replaced by a second glycine). The arginine/lysine forms a salt bridge with the phosphate moiety of the terminal 3′ base of the primer in the ternary complex of Polβ (Sawaya et al., 1994Sawaya M.R. Pelletier H. Kumar A. Wilson S.H. Kraut J. Crystal structure of rat DNA polymerase beta: evidence for a common polymerase mechanism.Science. 1994; 264: 1930-1935Crossref PubMed Scopus (389) Google Scholar) and is absolutely conserved as a positive residue in both family C and family X polymerases. The fingers of PolIIIα are a large crescent-shaped structure composed exclusively of α helices (Figure 2). The face of the fingers domain that is adjacent to the palm domain is formed by a network of highly conserved residues, dominated by solvent-exposed aromatic and arginine residues. Indeed, mapping of an alignment of 150 PolIIIα sequences onto the surface of PolIIIα shows that most of the conserved residues cluster at the interface between palm and fingers domains (Figure S2). The conservation in this region and the chemical nature of the inside face of the fingers domain is consistent with DNA polymerases from all families. Inspection of difference electron density maps generated using observed amplitudes from native and crystals that had been soaked in 5 mM dATP shows strong electron density features on the inside face of the fingers domain consistent with the triphosphate moiety of a bound dATP molecule (Figure S3). The triphosphate interacts with a cluster of four highly conserved arginine residues (R452 and R458 from the palm and R766 and R767 from the fingers domain; EcoR390, EcoR396, EcoR709, and EcoR710 respectively), and lies approximately 10 Å away from the catalytic aspartates on the palm domain. The position of the dATP in the binary complex is not consistent with the ternary position necessary for catalytic insertion, but rather may correspond to a preinsertion site analogous to that observed in T7 RNA polymerase (Temiakov et al., 2004Temiakov D. Patlan V. Anikin M. McAllister W.T. Yokoyama S. Vassylyev D.G. Structural basis for substrate selection by T7 RNA polymerase.Cell. 2004; 116: 351-353Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar) and Klenow fragment (Beese et al., 1993Beese L.S. Friedman J.M. Steitz T.A. Crystal structures of the Klenow fragment of DNA polymerase I complexed with deoxynucleoside triphosphate and pyrophosphate.Biochemistry. 1993; 32: 14095-14101Crossref PubMed Scopus (166) Google Scholar). The PHP domain is found in all PolIIIα sequences and was originally identified by Aravind and Koonin, 1998Aravind L. Koonin E.V. Phosphoesterase domains associated with DNA polymerases of diverse origins.Nucleic Acids Res. 1998; 26: 3746-3752Crossref PubMed Scopus (191) Google Scholar for its similarity to histidinol phosphatase. Recently, T. the PolIIIα was found to exhibit a zinc ion-dependent 3′-5′ exonuclease activity, which was attributed to its PHP domain (Stano et al., 2006Stano N.M. Chen J. McHenry C.S. A Co-proofreading Zn++-dependent exonuclease within a bacterial replicase.Nat. Struct. Mol. Biol. 2006; (in press)PubMed Google Scholar). This surprising result implies that PolIIIα may posses two proofreading activities, one residing in cis with the PHP domain and the other in trans with the ɛ subunit. PHP domains are also found associated with some family X polymerases, providing a further link between these two polymerase families (Aravind and Koonin, 1998Aravind L. Koonin E.V. Phosphoesterase domains associated with DNA polymerases of diverse origins.Nucleic Acids Res. 1998; 26: 3746-3752Crossref PubMed Scopus (191) Google Scholar); no classic palm polymerase is known to contain a PHP domain. The structures of only two other members of the PHP family are known, those of E. coli YcdX (Teplyakov et al., 2003Teplyakov A. Obmolova G. Khil P.P. Howard A.J. Camerini-Otero R.D. Gilliland G.L. Crystal structure of the Escherichia coli YcdX protein reveals a trinuclear zinc active site.Proteins. 2003; 51: 315-318Crossref PubMed Scopus (38) Google Scholar) and the Thermotoga maritime protein TM0559 (PDB ID, 2ANU), both of which are isolated PHP proteins of unknown function. All three structures are formed from an α7β7 barrel (Figure 4A). However, the β strands of YcdX are all parallel, whereas in TM0559 and the PHP domain the direction of the fourth β strand is reversed creating an unusual mixed β barrel structure. The presumed PHP active site sits in a cleft at the C-terminal side of the β barrel and contains two metal ions held in place by protein imidazole and carboxylate groups (Figure 4A). With the exception of the PolIIIα sequences from Proteobacteria (including E. coli) and some gram-positive bacteria, the residues involved in metal binding are conserved across the entire PHP family (Aravind and Koonin, 1998Aravind L. Koonin E.V. Phosphoesterase domains associated with DNA polymerases of diverse origins.Nucleic Acids Res. 1998; 26: 3746-3752Crossref PubMed Scopus (191) Google Scholar). The proteins that lack conservation appear to have residue substitutions which would compromise one or more of their metal binding sites. In agreement with biochemical analysis (Stano et al., 2006Stano N.M. Chen J. McHenry C.S. A Co-proofreading Zn++-dependent exonuclease within a bacterial replicase.Nat. Struct. Mol. Biol. 2006; (in press)PubMed Google Scholar), X-ray data collected at wavelengths near the zinc absorption edge establish that the two metal ions at the PHP active site are zinc. The two zinc ions are separated by a distance of 4.6 Å rather than a distance of 4.0 Å that would be expected for efficient catalysis by the two-metal-ion mechanism (Steitz, 1998Steitz T.A. A mechanism for all polymerases.Nature. 1998; 391: 231-232Crossref PubMed Scopus (474) Google Scholar). Comparison with YcdX and TM0559 reveals a potential third zinc binding site in PolIIIα formed by residues E72 (EcoD69), H95 (EcoH83), and C145 (EcoG134). In the current structure this site is occupied by a water molecule (Figure 4A). However, a zinc ion at this site would be 4.0 Å away from the nearest zinc ion observed in the current structure and hence more consistent with catalysis. We have tentatively assigned the four helix bundle located above the PHP domain as the thumb (Figure 2). Although the structure of the thumb of PolIIIα is unrelated to any structure in the structural database, many polymerases, including Polβ, reverse transcriptase and the bypass polymerase," @default.
• W2008017844 created "2016-06-24" @default.
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• W2008017844 creator A5066661430 @default.
• W2008017844 creator A5089839411 @default.
• W2008017844 date "2006-09-01" @default.
• W2008017844 modified "2023-10-18" @default.
• W2008017844 title "The Structure of T. aquaticus DNA Polymerase III Is Distinct from Eukaryotic Replicative DNA Polymerases" @default.
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