Volume 16, Issue 4, Pages 609-618 (November 2004) Insights into Strand Displacement and Processivity from the Crystal Structure of the Protein-Primed DNA Polymerase of Bacteriophage φ29  Satwik Kamtekar, Andrea J. Berman, Jimin Wang, José M. Lázaro, Miguel de Vega, Luis Blanco, Margarita Salas, Thomas A. Steitz  Molecular Cell  Volume 16, Issue 4, Pages 609-618 (November 2004) DOI: 10.1016/j.molcel.2004.10.019

Figure 1 Ribbon Representation of the Domain Organization of φ29 DNA Polymerase The exonuclease domain is shown in red, the palm in pink, TPR1 in gold, the fingers in blue, TPR2 in cyan, and the thumb in green. D249 and D458, which provide the catalytic carboxylates of the polymerase active site, are shown using space-filling spheres. Molecular Cell 2004 16, 609-618DOI: (10.1016/j.molcel.2004.10.019)

Figure 2 Interactions of ssDNA with the Exonuclease Site (A) For clarity, the polymerase domain and two 5′ bases are omitted; a segment (residues 560–575) of the thumb backbone is shown as a green ribbon. The side chains of residues T15, N62, F65, Y148, P129 (in yellow), and L567 (green stick representation) contact the DNA. Metal ion A is in position to coordinate the pro-S nonbridging oxygen of the scissile phosphate, E14, and D169 (the position of metal ion A was determined as described in the Experimental Procedures). The DNA residues are labeled T3–T5. (B) A difference electron density map calculated at 2.7 Å between the cocrystallized ssDNA complex and the native protein. The map is calculated with phases from the native structure and contoured at 2.5σ to provide an unbiased view of the electron density corresponding to bound DNA. The structure of the ssDNA complex is superimposed, with the exonuclease domain shown in red and the thumb subdomain in green. The DNA residues are labeled T1–T5. Molecular Cell 2004 16, 609-618DOI: (10.1016/j.molcel.2004.10.019)

Figure 3 Homology Modeling of DNA from the Structure of a RB69 DNA Polymerase Ternary Complex onto the Structure of φ29 DNA Polymerase (A) A superposition of the RB69 DNA polymerase (Franklin et al., 2001) and φ29 DNA polymerase palms. The catalytic carboxylates are shown in space-filling representation. (B) The DNA from the RB69 ternary complex (Franklin et al., 2001) modeled onto the φ29 DNA polymerase structure using the superposition shown in (A) without any further adjustment. The positions of the modeled primer (gray), template (black), and incoming nucleotide (yellow, space-filling spheres) are indicated; the polymerase is colored as in Figure 1. Molecular Cell 2004 16, 609-618DOI: (10.1016/j.molcel.2004.10.019)

Figure 4 Structures of TPR1 and TPR2, Domains that Are Specific to Protein-Primed DNA Polymerases (A) TPR1 forms a compact domain. This region is an insertion between the palm and the fingers subdomains. The motif, identified on the basis of sequence analysis (residues 302–358, gold), can be extended to include residues 261–301 as well (brown), thereby forming a subdomain with no homology to the palm subdomains of other B family polymerases. (B) Structural analogy between TPR2 (cyan) and the specificity loop (gold) of T7 RNA polymerase. The fragments of both palms used for superposition are colored in pink (φ29 DNA polymerase) and gray (T7 RNA polymerase). The atoms of the residues containing the catalytic carboxylates are shown as space-filling spheres. Molecular Cell 2004 16, 609-618DOI: (10.1016/j.molcel.2004.10.019)

Figure 5 Structural Basis of Processivity and Strand Displacement (A and B) Homology-modeled DNA from the RB69 DNA polymerase ternary complex is shown in the context of a space-filling representation of φ29 DNA polymerase in two different orientations. The polymerase is colored as in Figure 1: exonuclease, red; palm, pink; TPR1, gold; fingers, blue; TPR2, cyan; thumb, green. The primer strand of the DNA is colored gray, and the template is colored yellow. The orientation in (A) is similar to that in Figure 1 and shows topological encirclement of modeled upstream duplex product DNA by the thumb, palm and TPR2. The polymerase orientation in (B) is rotated approximately 75° from (A) and shows modeled downstream template passing through a narrow tunnel made by the exonuclease domain, palm subdomain, and TPR2 before entering the polymerase active site. (C) An electrostatic surface representation of the polymerase. This view shows that positively charged (blue) protein surface would contact downstream template. The electrostatic surface is contoured with saturating values of blue and red set at ±15 kT respectively. Molecular Cell 2004 16, 609-618DOI: (10.1016/j.molcel.2004.10.019)

Figure 6 Paths Leading to the Active Site of φ29 DNA Polymerase (A) A surface representation of the polymerase with homology-modeled DNA (primer, red; template, yellow; incoming dNTP, space-filling purple) sliced into two halves to show three paths leading into the active site. A narrow tunnel allows the modeled uncopied downstream template into the active site while a large pore provides a path for incoming dNTP. Modeled upstream product duplex exits from the polymerase active site through a tunnel of intermediate dimensions. (B) Schematic representation of φ29 DNA polymerase with DNA substrate. The protein is diagramed in two levels. The upper level contains the exonucleolytic domain, TPR2 subdomain, and thumb subdomain (outlined in red, cyan, and green, respectively). The rest of the protein is indicated in gray. An asterisk marks the polymerase active site position. Molecular Cell 2004 16, 609-618DOI: (10.1016/j.molcel.2004.10.019)