Volume 13, Issue 4, Pages 561-572 (February 2004) A Structural Solution for the DNA Polymerase λ-Dependent Repair of DNA Gaps with Minimal Homology  Miguel Garcia-Diaz, Katarzyna Bebenek, Joseph M Krahn, Luis Blanco, Thomas A Kunkel, Lars C Pedersen  Molecular Cell  Volume 13, Issue 4, Pages 561-572 (February 2004) DOI: 10.1016/S1097-2765(04)00061-9

Figure 1 Structure-Based Amino Acid Alignment of the Four Human Family X DNA Polymerases Amino acid identities with human Pol λ are shown in white over a black background. Conserved residues are indicated in bold black letters. Residue numbers indicate position with respect to the N-terminal Met. The secondary structure elements of human Pol λ are indicated on top of the alignment. The color of the background corresponds to the structural subdomains: 8 kDa domain (purple), fingers (blue), palm (red), and thumb (green). Molecular Cell 2004 13, 561-572DOI: (10.1016/S1097-2765(04)00061-9)

Figure 2 Biochemical Properties of the Catalytic Core of Pol λ (A) Photographs of an SDS-PAGE gel with purified Pol λ (lane 1). Protein molecular-weight markers are shown in lane 2. (B) Substrates used for polymerase assays: template/primer (1), five nucleotide gap without a 5′ phosphate (2), and five nucleotide gap with a 5′ phosphate (3). P, T, and DP indicate the upstream primer for polymerase extension, the template, and the downstream primer strands, respectively. (C) Polymerization by Pol λ in a five nucleotide gap. Reactions were carried out using 50 nM Pol λ and 200 nM DNA for 10 min at 37°C using a template/primer (lane 1), a five nucleotide gap (lane 2), or a five nucleotide gap with a 5′ phosphate (lane 3), and processed as described in Experimental Procedures. (D) Error rates in the M13 assay. The assay was carried out as described in Experimental Procedures. The error rates for −1 frameshift (black) and base substitution (gray) errors are expressed for full-length Pol λ, the 39 kDa fragment of Pol λ, and Pol β. Data for full-length Pol λ and Pol β are from Bebenek et al. (2003). (E) dRP lyase assay. The reactions were carried out as described in Experimental Pro-cedures. The dRP-containing substrate was incubated with 50 nM 39 kDa (lane 1) or full-length (lane 2) Pol λ for 10 min at 37°C. Molecular Cell 2004 13, 561-572DOI: (10.1016/S1097-2765(04)00061-9)

Figure 3 Pol λ Bound to a Two Nucelotide Gap (A) Ribbon representation of human Pol λ bound to a two nucleotide gap. The different subdomains are colored purple (8 kDa), blue (fingers), red (palm), and green (thumb). The C terminus and N terminus of the protein are labeled in black (C and N, respectively). The template strand (T) is shown in gray, while the primer (P) and downstream primer (D) strands are shown in yellow. The secondary structure elements of human Pol λ (Figure 2) are labeled in yellow. (B) Pol λ active site. Electron density plot showing a number of interactions in the active site of the enzyme. The template strand is gray, the primer strand is yellow, and Pol λ residues are red. Hydrogen bonding is shown for the terminal base pair. (C) Protein-DNA contacts in Pol λ. Stick diagram of the Pol λ-DNA interactions. Red arrows represent hydrogen bonding, and blue arrows represent other protein-DNA contacts. For hydrogen bonds, the atom establishing the bond is indicated. Water molecules involved in hydrogen bonding are indicated with an encircled W. Three sugar rings are labeled 0, −1, and +1, indicating the first available template nucleotide and its 3′ and 5′ template neighbors, respectively. (D) The 8 kDa of Pol λ. Relevant residues are shown in red. Van der Waals surfaces are transparent. Molecule A of the PDB file was used to generate the figures using Molscript (Kraulis, 1991), Grasp (Nicholls et al., 1991), and Raster3D (Merritt and Bacon, 1997). Molecular Cell 2004 13, 561-572DOI: (10.1016/S1097-2765(04)00061-9)

Figure 4 Superimposition of Pol λ with the Open and Closed Conformations of Pol β Superimposition of the α carbon trace of human Pol λ (red) with the structures of human Pol β in an open (1BPX; yellow) and closed (1BPY; blue) conformation. The roman numerals refer to different regions of the Pol λ structure as indicated in the text. The rms deviation was 1.4 Å for 112 C-α atoms for 1BPX and 1.4 Å for 113 C-α atoms for 1BPY. Molecular Cell 2004 13, 561-572DOI: (10.1016/S1097-2765(04)00061-9)

Figure 5 Closed Conformation of Pol λ (A) Superimposition of the thumb and palm subdomains of Pol λ (red) and Pol β in an open (yellow; 1BPX) and closed (blue; 1BPY) conformation. The thumb motion during the conformational change in Pol β is indicated with a yellow arrow. The three catalytic carboxylates are shown in stick representation for all three proteins. Also shown are the two metal ions from 1BPY. (B) Overlay of the active site of Pol λ (red) and Pol β in an open (yellow) and closed (blue) conformation. The incoming ddCTP in the Pol β 1BPY structure is shown in green. The distance between the 3′ C of the terminal primer nucleotide and the α-phosphate of the incoming ddCTP is 7.27 Å (as compared to 4.47 Å in Pol β 1BPY). (C) Palm-thumb interactions in Pol λ. The ribbon is colored according to the structural subdomains. Relevant Pol λ residues are shown in stick representation (gold). (D) Thumb-8 kDa interactions in Pol λ (red) and Pol β (1BPY, blue). Relevant residues are shown in red (Pol λ) and blue (Pol β) in a stick representation. The van der Waals surface of the residues is transparent. Molecular Cell 2004 13, 561-572DOI: (10.1016/S1097-2765(04)00061-9)

Figure 6 Electrostatic Surface Potential of Pol λ and Pol β In this view, the molecules are rotated 180° relative to those in Figure 7B. The incoming dNTP (in yellow) is bound in a positively charged channel. The image in the left panel was created by ovelaying the Pol λ structure with that of Pol β 1BPY. Only the incoming dNTP from the Pol β structure is shown. Molecular Cell 2004 13, 561-572DOI: (10.1016/S1097-2765(04)00061-9)

Figure 7 Biological Implications of the Pol λ Structure (A) Stereo view of an overlay of the active site of Pol λ with that of Pol β in a closed conformation (1BPY). The DNA corresponds to the Pol λ structure, while the incoming ddCTP (green) and metal ions (gray balls) correspond to Pol β 1BPY. Relevant residues are shown in red (Pol λ) and blue (Pol β). The template strand is gray and the primer terminus is yellow. (B) Electrostatic surface potential of Pol λ and Pol β. The DNA for each of the structures is shown in gray (template) and yellow (primer and downstream primer). The potential ranges from −8 kT/e (red) to 8 kT/e (blue). Molecular Cell 2004 13, 561-572DOI: (10.1016/S1097-2765(04)00061-9)