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Structures of phi29 DNA polymerase complexed with substrate: the mechanism of translocation in B-family polymerases

2007; Springer Nature; Volume: 26; Issue: 14 Linguagem: Inglês

10.1038/sj.emboj.7601780

1460-2075

Andrea J. Berman, Satwik Kamtekar, Jessica L. Goodman, José M. Lázaro, Miguel de Vega, Luis Blanco, Margarita Salas, Thomas A. Steitz,

Bacteriophages and microbial interactions

Article5 July 2007free access Structures of phi29 DNA polymerase complexed with substrate: the mechanism of translocation in B-family polymerases Andrea J Berman Andrea J Berman Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA Search for more papers by this author Satwik Kamtekar Satwik Kamtekar Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USAPresent address: Pfizer Inc., 700 Chesterfield Parkway West, Chesterfield, MO 63017, USA Search for more papers by this author Jessica L Goodman Jessica L Goodman Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA Search for more papers by this author José M Lázaro José M Lázaro Centro de Biología Molecular 'Severo Ochoa' (CSIC-UAM), Universidad Autónoma, Canto Blanco, Madrid, Spain Search for more papers by this author Miguel de Vega Miguel de Vega Centro de Biología Molecular 'Severo Ochoa' (CSIC-UAM), Universidad Autónoma, Canto Blanco, Madrid, Spain Search for more papers by this author Luis Blanco Luis Blanco Centro de Biología Molecular 'Severo Ochoa' (CSIC-UAM), Universidad Autónoma, Canto Blanco, Madrid, Spain Search for more papers by this author Margarita Salas Margarita Salas Centro de Biología Molecular 'Severo Ochoa' (CSIC-UAM), Universidad Autónoma, Canto Blanco, Madrid, Spain Search for more papers by this author Thomas A Steitz Corresponding Author Thomas A Steitz Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA Department of Chemistry, Yale University, New Haven, CT, USA Howard Hughes Medical Institute, Yale University, New Haven, CT, USA Search for more papers by this author Andrea J Berman Andrea J Berman Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA Search for more papers by this author Satwik Kamtekar Satwik Kamtekar Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USAPresent address: Pfizer Inc., 700 Chesterfield Parkway West, Chesterfield, MO 63017, USA Search for more papers by this author Jessica L Goodman Jessica L Goodman Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA Search for more papers by this author José M Lázaro José M Lázaro Centro de Biología Molecular 'Severo Ochoa' (CSIC-UAM), Universidad Autónoma, Canto Blanco, Madrid, Spain Search for more papers by this author Miguel de Vega Miguel de Vega Centro de Biología Molecular 'Severo Ochoa' (CSIC-UAM), Universidad Autónoma, Canto Blanco, Madrid, Spain Search for more papers by this author Luis Blanco Luis Blanco Centro de Biología Molecular 'Severo Ochoa' (CSIC-UAM), Universidad Autónoma, Canto Blanco, Madrid, Spain Search for more papers by this author Margarita Salas Margarita Salas Centro de Biología Molecular 'Severo Ochoa' (CSIC-UAM), Universidad Autónoma, Canto Blanco, Madrid, Spain Search for more papers by this author Thomas A Steitz Corresponding Author Thomas A Steitz Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA Department of Chemistry, Yale University, New Haven, CT, USA Howard Hughes Medical Institute, Yale University, New Haven, CT, USA Search for more papers by this author Author Information Andrea J Berman1, Satwik Kamtekar1, Jessica L Goodman1, José M Lázaro2, Miguel de Vega2, Luis Blanco2, Margarita Salas2 and Thomas A Steitz 1,3,4 1Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA 2Centro de Biología Molecular 'Severo Ochoa' (CSIC-UAM), Universidad Autónoma, Canto Blanco, Madrid, Spain 3Department of Chemistry, Yale University, New Haven, CT, USA 4Howard Hughes Medical Institute, Yale University, New Haven, CT, USA *Corresponding author. Department of Molecular Biophysics and Biochemistry, Yale University, Room 418, Bass Center, 266 Whitney Avenue, New Haven, CT 06520-8114, USA. Tel.: +1 203 432 5617/5619; Fax: +1 203 432 3282; E-mail: [email protected] The EMBO Journal (2007)26:3494-3505https://doi.org/10.1038/sj.emboj.7601780 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Replicative DNA polymerases (DNAPs) move along template DNA in a processive manner. The structural basis of the mechanism of translocation has been better studied in the A-family of polymerases than in the B-family of replicative polymerases. To address this issue, we have determined the X-ray crystal structures of phi29 DNAP, a member of the protein-primed subgroup of the B-family of polymerases, complexed with primer-template DNA in the presence or absence of the incoming nucleoside triphosphate, the pre- and post-translocated states, respectively. Comparison of these structures reveals a mechanism of translocation that appears to be facilitated by the coordinated movement of two conserved tyrosine residues into the insertion site. This differs from the mechanism employed by the A-family polymerases, in which a conserved tyrosine moves into the templating and insertion sites during the translocation step. Polymerases from the two families also interact with downstream single-stranded template DNA in very different ways. Introduction Single-subunit replicative polymerases contain a polymerase domain divided into functional subdomains arranged in a gross common architecture likened to a right hand (Kohlstaedt et al, 1992). The thumb and fingers subdomains form the sides of a 'U'-shaped cleft, at the bottom of which is the catalytic palm subdomain that utilizes a two-metal ion mechanism for catalyzing phosphodiester bond formation (Steitz et al, 1993). The thumb subdomain stabilizes the primer-template duplex product and the fingers subdomain contains basic residues that bind the triphosphate moiety of the incoming nucleotide and the pyrophosphate product of the phosphoryl transfer reaction (Beese et al, 1993; Doublié et al, 1998). The coordinated movements of these subdomains have been extensively studied in polymerase families, including family A (bacterial repair polymerases, most bacteriophage replicative polymerases, and T7 RNA polymerase (RNAP)) and family B (viral and eukaryotic genome replicating enzymes) (Rothwell and Waksman, 2005). Structural studies have led to the suggestion that after binding a primer-template DNA substrate, A-family polymerases bind an incoming nucleoside triphosphate at a pre-insertion site located near the fingers subdomain before escorting it into the insertion site (Beese et al, 1993; Li et al, 1998a; Temiakov et al, 2004), whereas it has been proposed from biochemical studies that B-family polymerases bind the incoming nucleoside triphosphate directly in the insertion site at the base of the fingers (Yang et al, 2002b). Structural studies of A-family polymerases have also proposed a pre-insertion site for the templating base in the replication cycle of this family (Johnson et al, 2003; Temiakov et al, 2004; Yin and Steitz, 2004); no evidence for a templating pre-insertion site in the B-family exists. Following the phosphoryl transfer reaction, the newly incorporated nucleotide moves from the insertion site to the priming site, allowing the next incoming nucleotide to bind. This last step, known as translocation, facilitates processive movement of a polymerase along template DNA and is therefore a critical feature of the nucleotide addition cycle of replicative polymerases (Figure 1). Figure 1.The polymerization cycle. Polymerase (pale blue circle) binds a primer-template substrate (blue and red) and then the incoming dNTP (green). In some polymerases, the incoming dNTP binds a pre-insertion site before binding the insertion site (yellow) opposite the templating nucleotide. The polymerase then catalyzes the incorporation of the dNTP into the primer strand, resulting in a primer extended by one nucleotide and a molecule of pyrophosphate bound near the active site. Release of the pyrophosphate is associated with translocation of the new primer terminus out of the insertion site and into the priming site (Yin and Steitz, 2004). Boxes indicate the states captured in our crystal structures. For consistency, template strand numbering refers to the base positions in the initial binary complex. Download figure Download PowerPoint Polymerases are molecular machines that convert chemical energy into mechanical energy, and two models, the power stroke and Brownian-ratchet mechanisms, have been used to explain the energetics of translocation (Hanson and Huxley, 1955; Simon et al, 1992). In the context of the polymerization cycle, the power stroke mechanism derives the energy for translocation from the dissociation of the pyrophosphate product of the nucleotidyl transfer reaction, whereas the Brownian-ratchet mechanism utilizes the kinetic energy of primer-template diffusion to facilitate the unidirectional movement of the polymerase along the template strand (Guajardo and Sousa, 1997). Each model has testable predictions, and it is possible that a combination of the two occurs. Despite a wealth of studies on processive RNAPs and bacterial DNA polymerases (DNAPs), little is known about the translocation step in eukaryotic-like replicative DNAPs. The B-family DNAP of Bacillus subtilis bacteriophage phi29 is an appealing system in which to study the structural biology of B-family replication, because it is small and biochemically well characterized (Blanco and Salas, 1996). In addition to the general features of polymerase and exonuclease activities shared by B-family polymerases, phi29 DNAP has a strand displacement capacity and high processivity. It can also initiate replication from a protein primer, terminal protein (TP), a characteristic that is shared by polymerases from several pathogenic viruses, such as adenovirus, poliovirus, and hepatitis virus (Salas, 1991). Initial structural studies provided insights into the intrinsic strand displacement, processivity, and protein priming activities of phi29 DNAP. The structure of the apo phi29 DNAP exhibited two globular domains, an N-terminal exonuclease domain and a C-terminal polymerase domain. This structure contains three tunnels, one of which is formed by the exonuclease domain and the palm and TP-interacting region 2 (TPR2) subdomains of the polymerase domain. Homology modeling of a substrate complex using the primer-template DNA and incoming nucleotide substrates from the ternary complex of the B-family DNAP from bacteriophage RB69 (Franklin et al, 2001) identified this tunnel at the location where phi29 DNAP would bind the downstream 5′ region of single-stranded template DNA and suggested mechanisms for processivity and strand displacement (Kamtekar et al, 2004); truncation of the TPR2 subdomain reduced processivity and strand displacement, consistent with the proposed mechanism (Rodríguez et al, 2005). These structures also showed that the two subdomains, TPR1 and TPR2, which are only present in protein-primed polymerases, interact with the intermediate and priming domains of TP, respectively (Kamtekar et al, 2006). Here, we present four crystal structures of complexes of phi29 DNAP with substrates. These include the structure of polymerase bound to a primer-template substrate (binary complex) in the post-translocated state, before the next incoming nucleotide binds the polymerase, as well as the structures of complexes of polymerase bound to two different primer-templates and their complementary incoming nucleotides (ternary complexes). Finally, we describe the structure of polymerase bound to single-stranded DNA (ssDNA) (ssDNA complex). Comparison of the structures of these complexes allows us to understand ssDNA and double-stranded DNA binding in B-family DNAPs and to propose a mechanism of translocation in this family. Results Four views of substrate binding: binary, ternary, exonuclease, and ssDNA template complexes The structures described here represent different stages of the replication cycle. One of them contains a molecule of polymerase bound to ssDNA in a tunnel that lies downstream of the active site, a complex that is relevant to understanding protein-primed initiation. This structure also contains ssDNA bound at the exonuclease active site. The binary complex structure contains polymerase bound to a primer-template DNA substrate that is in the post-translocated position. The two ternary complexes contain primer-template DNA and an incoming nucleotide (dNTP) that is complementary to the templating base (0 position) (Figure 1). The sequences of the substrates differ at several positions in these ternary complexes, facilitating sequence-specific comparisons (Table I). Table 1. Data collection and refinement statistics for phi29 DNAP-substrate complexes Ternary1 Ternary2 Binary ssDNA complex Substrates Primera 5′-GACTGCTTACAT 5′-GACTGCTTACG 5′-GACTGCTTAC Templateb 3′-CTGACGAATGTACA 3′-CTGACGAATGCACA 3′-CTGACGAATGCACAATC ssDNA 5′GGACTTT Data collection Resolution limit (Å) 50.0–2.2 (2.28–2.20) 50.0–2.03 (2.10–2.03) 50.0–2.6 (2.69–2.6) 50.0–1.6 (1.66–1.6) Space group P212121 P21 C2 P21 Copies in the AU Protein 1 2 4 2 DNA 1 3 2/2c 5 Cell parameters a, b, c (Å) 58.9, 78.2, 157.8 72.8, 114.7, 104.8 216.6, 146.3, 115.1 54.2, 200.2, 67.0 α, β, γ (deg) 90, 90, 90 90, 94.1, 90 90, 117.9, 90 90, 109.4, 90 Unique reflections 36 737 107 450 95 762 172 311 I/σ(I) 8.9 (1.2) 16.3 (2.4) 5.8 (1.0) 49.9 (3.7) Redundancy 4.8 (2.5) 3.5 (2.3) 2.1 (1.9) 6.9 (5.5) Completeness (%) 97 (83.7) 98 (83.9) 98.4 (95.1) 97.8 (92.2) Rmerge 0.166 (0.85) 0.076 (0.353) 0.152 (0.773) 0.045 (0.44) Refinement statistics Data range 40–2.20 (2.26–2.20) 40–2.03 (2.09–2.03) 50–2.6 (2.67–2.60) 50–1.6 (1.64–1.60) Rwork/Rfree 19.5/25.4 (26.8/34.0) 18.7/23.4 (23.5/32.3) 21.9/27.3 (36.6/39.7) 16.1/19.4 (22.2/27.5) RMSD Bond lengths (Å) 0.010 0.009 0.009 0.011 Bond angles (deg) 1.3 1.3 1.2 1.4 Number of atoms Protein 4680 9367 18 488 9267 Nucleic acid 534 1493 1207 553 Ions 2 4 0 0 Water 357 969 89 1494 Average B-factors (Å2) Protein 32.9 22.1 27.3 20.7 Nucleic acid 39.7 23.6 29.3 23.9 PDBID 2PYL 2PYJ 2PZS 2PY5 a The incoming dNTP of each ternary complex is indicated in bold. b The 0 position on the template strand is underlined. c In the binary crystal form, there are two copies of primer-template substrate and two copies of single-stranded template. Although the exonuclease and polymerase domains move slightly with respect to each other, the global structures of the polymerases complexed with substrate remain largely unchanged when compared to the apo structures of polymerase (PDBID 1XI1 and 1XHX). The root mean squared deviations (RMSD) calculated over all pairs of polymerases in the apo and complex structures range from 0.7 to 2.8 Å over 572 Cα. The catalytic palm subdomains (residues 190–260 and 427–530) of all of these copies of polymerase are very similar, with an RMSD range of 0.3–1.0 Å over 173 Cα. Substrate binding Single-stranded DNA complex, Phi29 DNAP bound to ssDNA crystallizes in space group P21 and diffracts to better than 1.6 Å resolution (Table I). The two non-crystallographically related copies in the asymmetric unit are very well ordered, except for amino acid residues 305–311 in copy A and residues 305–313 in copy B. These residues are part of a mobile loop in TPR1 that is only well ordered in the presence of TP (Kamtekar et al, 2006) or duplex DNA product. The two copies of polymerase are very similar, with an RMSD of 0.8 Å over the 561 Cα atoms, but exhibit significant differences in exonuclease residues 140–145 and at residue Y165 that may have functional implications for exonuclease activity (Supplementary Figure S1). Each copy of polymerase in this crystal form binds the 3′ end of one ssDNA emerging from the downstream template tunnel at the polymerase active site, and the 3′ end of another ssDNA at the exonuclease active site. One copy of polymerase binds a third ssDNA in a biologically non-relevant location (Supplementary Figure S3c). Binary complex. The crystals of the binary complex diffract to 2.6 Å resolution and contain four copies of polymerase per asymmetric unit related by pseudo-222 non-crystallographic symmetry (Table I). Two of the copies were modeled with a primer-template substrate (Supplementary Figure S3a). The other two copies of polymerase in this crystal form have density for a single-stranded 5′ template overhang in the downstream template tunnel, but are missing amino acids 306–314 and the duplex region of the primer-template due to disorder. Averaged electron density maps indicated the presence of one of the two missing primer-template duplex regions, but the quality of this density was poor and it was therefore not included in the final model. The binary complex is representative of a post-translocation state (Figure 1), as the primer terminus occupies the priming site. The phosphate moiety of the priming nucleotide interacts with the invariant Y500 in motif KxY as predicted (Figure 2D) (Blasco et al, 1995). Two residues, Y254 and Y390, occupy the insertion site. The former (Y254) is called the steric gate residue, because its location in the active site would lead to a steric clash with the 2′-hydroxyl of a ribonucleotide (Gao et al, 1997; Franklin et al, 2001), thereby preventing the incorporation of ribonucleotides into the primer strand (Bonnin et al, 1999). The latter amino acid, Y390, is from a B-family conserved sequence motif at the base of the fingers subdomain. Neither of the catalytic metal ions is observed in this complex, and, similar to the binary complexes from the A-family (Li et al, 1998b; Johnson et al, 2003), one of the catalytic aspartates (D249) is not properly oriented for catalysis (Figure 2A and C). Figure 2.Comparison of the binary and ternary complexes. The binary complex is shown in yellow and the ternary complex in green. Metals are indicated as magenta spheres. The incoming dNTP from the ternary complex is shown as magenta sticks. The fingers subdomain rotates 14° in going from the opened binary complex to the closed ternary complex. (A) Binary complex. The residues that form the nascent base pair-binding site in the ternary complex are shown as spheres and the active site carboxylates are shown as sticks. The fingers subdomain is shown in cartoon representation. Two conserved tyrosine residues occupy the insertion site. (B) Ternary complex. The conserved lysine residues that interact with the phosphates are also shown. The density from a simulating annealing omit map using phases calculated from a model with the nascent-base pair omitted and amplitudes from the ternary2 data contoured at 2.5 σ is shown as gray mesh for the nascent base pair. (C) Comparison of the binary and ternary complex structures. All of the mechanistically significant amino-acid movements are indicated. Black dashed lines represent interactions. Red dashed lines indicate steric clashes. The distances indicated are in Å. The density shown for metal ion B (manganese) is from an anomalous difference Fourier map calculated using data from 50.0–2.03 Å resolution and contoured at 6 σ. (D) The propagated shift of the DNA base pair planes between the binary and ternary complexes. When the fingers close, residues S388 and N387 (shown as spheres) stack against the templating nucleotide and incoming dNTP, respectively, completing the nascent base pair-binding pocket. Y500 interacts with the phosphate moiety of the priming nucleotide. Download figure Download PowerPoint Ternary complexes. The ternary1 and ternary2 complexes crystallize in space groups P212121 and P21, and diffract to 2.2 and 2.0 Å resolution, respectively (Table I). The orthorhombic crystal form contains one copy of polymerase bound to primer-template DNA and incoming nucleotide per asymmetric unit. The monoclinic crystal form contains two ternary complexes and a third copy of primer-template DNA bound in a biologically non-relevant manner (Supplementary Figure S3b). The RMSDs over the 173 Cα in the catalytic palm subdomain among the three copies of ternary complex range from 0.6–0.8 Å. We have chosen copy A of the ternary2 complex, except where noted, as a representative ternary complex in all of the following discussion, because it is well ordered in electron density maps (Figure 2B). In the ternary complexes, the dNTP is bound at the insertion site, poised for catalysis (Figure 1) and the primer terminus occupies the priming site. The priming nucleotide in each of the ternary complexes interacts with Y500 of motif KxY (Figure 2D) (Blasco et al, 1995). In each complex, the base moiety of the dNTP forms a Watson–Crick base pair with the templating nucleotide and its deoxyribose ring stacks on the phenolic side chain of the steric gate residue, Y254. This steric gate side chain occupies a less favorable rotameric state, which is also observed in other ternary complexes (Huang et al, 1998; Franklin et al, 2001), suggesting that the stacking interaction with a sugar moiety of a nucleotide stabilizes its unusual conformation. Along with Y254, the side chain of the tyrosine from conserved sequence motif 2a (Y390) forms part of the nascent base pair-binding pocket. Y390 also interacts with the hydroxyl of Y226 through a hydrogen bond. Both aspartates that bind the catalytic magnesium ions participate in the active site (Figure 2B and C). The phosphates of the incoming dNTP interact with the basic side chains of residues K371 and K383 from the fingers subdomain. Consistent with mutational data, the conserved sequence motif B residue K383 (Saturno et al, 1997) interacts with the α- and γ-phosphates of the incoming dNTP, and the pre-B motif residue K371 (Truniger et al, 2002) interacts with the γ-phosphate (Figure 2B). It is possible that these residues comprise part of a pre-insertion-binding site for the nucleotide, as, in the apo structure, they were observed to interact with sulfate ions which are sterically and electrostatically similar to the phosphate groups of a nucleotide (PDBID: 1XHX) (Kamtekar et al, 2004), although kinetics experiments with the B-family DNAP from bacteriophage RB69 have been interpreted to indicate the absence of a pre-insertion site in that system (Yang et al, 2002b). Biochemical experiments (Truniger et al, 2004) and sequence alignments with a conserved arginine from the A-family (Doublié et al, 1998) have implicated a third lysine residue, K379, in dNTP binding. The structure shows that K379 interacts with the γ-phosphate indirectly through a network of water molecules. The ternary complex structure contains both metal ions, A and B, which have respectively been assigned as a magnesium ion and a manganese ion in the ternary2 complex based on an anomalous difference Fourier map (Figure 2C). The α- and γ-phosphates of the incoming dNTP, the catalytic aspartate residues (D249 and D458), and the carbonyl of V250 of the palm subdomain coordinate the catalytic metals. A 2′, 3′-dideoxynucleotide was incorporated at the primer terminus to facilitate the formation of a ternary complex, and the absence of the 3′ hydroxyl results in a slightly skewed coordination geometry of metal ion A. While the structure of this ternary complex confirms the general substrate positioning in phi29 DNAP that was predicted from our previous homology modeling using the ternary complex of RB69 DNAP, some features are clearly different. The presence of the TPR2 subdomain in phi29 DNAP that is absent in RB69 DNAP results in a slight shift in the position of the DNA from its homology model placement (Franklin et al, 2001; Kamtekar et al, 2004). As predicted, the upstream duplex is topologically encircled by the thumb and TPR2 subdomains, but the base pairs distal to the active site interact with subdomain TPR2, resulting in their displacement of ∼5 Å off the active site relative to the DNA in the homology-modeled complex. The structure also resolves the minor clashes between the thumb and the upstream duplex observed in the modeling. Likewise, the single-stranded downstream template enters the active site through the downstream template tunnel formed from the exonuclease domain and the TPR2, palm, and fingers subdomains, as predicted, but the interactions within the tunnel were unpredicted, and have implications for sequence-independent recognition of template DNA as well as for the mechanism of translocation. Comparison of the pre-translocation ternary and post-translocation binary complexes. The binding of the incoming dNTP triggers a 14° rotation of the fingers subdomain toward the polymerase active site (Figure 2), corresponding to a ∼7 Å movement of the tip of the fingers. As in other polymerases, the triphosphate moiety of the incoming nucleotide acts as an electrostatic crosslink between conserved residues of the fingers and the catalytic metal ions chelated to the conserved carboxylates, thereby keeping the fingers closed (Doublié et al, 1998; Huang et al, 1998; Li et al, 1998b; Franklin et al, 2001; Yin and Steitz, 2004). Once closed, the fingers complete the nascent base pair-binding pocket (Figure 2B and C). The structure of the duplex DNA in the binary complex is distorted compared to its structure in the ternary complex. The nucleotide bases in the binary structure are substantially displaced, with the entire nucleotide at the –1 position of the template strand lifted almost 2 Å off the active site, whereas the positions of the phosphate backbones shift with an RMSD of less than 1 Å. The distortion of the duplex DNA appears to be a consequence of the position of the templating nucleotide. When the fingers are closed, the nascent base pair-binding pocket holds the templating nucleotide in position and the upstream bases of the template strand stack accordingly. However, in the binary complex, where the fingers are opened, the residues completing the nascent base pair binding pocket are too far away to stabilize the nucleotide in the templating position. This results in the displacement of the templating nucleotide by ∼1.5 Å upstream from its position in the ternary complex; the stacking of the upstream nucleotides follows, slightly distorting the duplex (Figure 2D). Similar systematic shifts are observed in comparing the binary and ternary complexes of the A-family polymerases from B. stearothermophilus (Johnson et al, 2003) and Thermus aquaticus (Li et al, 1998b), and the X-family polymerase β from rat (Pelletier et al, 1994, 1996), suggesting that this could be the more stable DNA conformation in the absence of an incoming dNTP. Despite these shifts in the binary and ternary complexes, an extensive water network mediating most of the protein–nucleic acid interactions is conserved among different complexes. In both the binary and ternary complexes, the polymerase makes contacts with the sugar-phosphate backbone of duplex DNA through a few direct interactions and through multiple water-mediated hydrogen bonds (Figure 3 and Supplementary Figure S2). The only direct side chain contact with the minor groove of the duplex product is made by a highly conserved lysine (K498) that interacts with the N3 of a purine or the O2 of a pyrimidine at the primer strand –2 position (Figure 3). More than thirty ordered water molecules facilitate hydrogen bonds between conserved and nonconserved amino acids and the DNA duplex in each of the ternary complexes and in the binary complex to maintain flexibility in duplex binding (Figure 3 and Supplementary Figure S2). Several of these water molecules are also present in the apo polymerase structures. These water molecules thus act as surrogate side chains, as the entropic penalty for their immobilization is independent of the binding of DNA duplex. Figure 3.Water-mediated interactions maintain sequence nonspecific binding. The C:G base pair is from the ternary1 complex, and the A:T base pair is from the ternary2 complex. Red spheres are water molecules and black dashes are hydrogen bonds. Amino acids are colored by subdomain as in Kamtekar et al (2004). Download figure Download PowerPoint The opening of the fingers that occurs in the transition from the ternary complex to the binary complex is accompanied by several mechanistically significant changes. When the fingers open, the side chain of Y390 from conserved sequence motif 2a moves into the insertion site, such that the newly incorporated nucleotide can no longer reside there. This observation is consistent with biochemical data suggesting that Y390 interacts either directly or indirectly with the incoming dNTP (Blasco et al, 1992). If no nucleotide occupies the insertion site, the steric gate residue (Y254) can flip to its most favorable rotamer. This rotamer places the phenolic ring of the steric gate residue directly in the insertion site, stacking on the conserved tyrosine at the base of the fingers (Y390), one of the most energetically stable tyrosine–tyrosine interactions (Chelli et al, 2002) (Figure 2A and C). The positions of both of these tyrosine residues in the insertion site preclude the primer terminus from binding at the insertion site while the fingers are opened. Therefore, the primer terminus must move to the priming site, resulting in translocation of the DNA by one nucleotide. The rotation of Y390 breaks its hydrogen bond with Y226 (Figure 2C), a residue in