1. Structural Insights into the Generation of Single-Base Deletions by the Y Family DNA Polymerase Dbh 
Ryan C. Wilson, Janice D. Pata 
Molecular Cell 
Volume 29, Issue 6, Pages (March 2008)
DOI: /j.molcel
Copyright © 2008 Elsevier Inc. Terms and Conditions

2. Figure 1
Crystal Structure of Dbh Extending DNA Synthesis beyond a Single-Base Deletion in a Repetitive Sequence
(A) Ternary complex structure. The polymerase and primer-template DNA are shown in ribbons representation, the incoming dNTP is shown as a ball-and-stick model, and the Ca2+ ion (bound in the metal B position) is shown as a sphere (green). Residues Arg333 and Tyr249 that interact with the bulged template cytosine at position −3 are shown in ball-and-stick representation. Coloring is as follows: palm (magenta), thumb (green), fingers (blue), C-terminal domain (orange), linker between polymerase and C-terminal domains (pale yellow); and DNA (white). The template strand single-base deletion hot spot sequence (5′-GCCC-3′) is labeled and highlighted in red.
(B) Protein-DNA interactions. Hydrogen bonds and van der Waals contacts are indicated by dotted lines; residues that contact the DNA with side-chain atoms are labeled in boldface; other residues that contact main-chain atoms are labeled in plain type. Residues are colored according to domain, as in (A). Nucleotides are numbered relative to the templating base (position 0). The complete sequence of the DNA oligonucleotides used is shown; the template-strand hot spot sequence is highlighted in red. Unpaired nucleotides at the 5′ and 3′ ends of the template strand (positions +2 and −12) were not clearly visible in the electron density maps and were thus not modeled. Residues in the flexible loop that are close enough to contact the backbone of the template in positions −1, −2, and −3 are boxed and labeled in italic font; precise contacts are not identified, because of the weak electron density in this part of the protein. The Ca2+ ion bound at the active site in the metal B position is shown as a circle labeled B.
(C–E) The active sites and primer-template DNA sequences are shown for (C) preinsertion binary complex, (D) insertion ternary complex, and (E) postinsertion binary complex. For each structure, a final refined 2Fo − Fc electron density map, calculated using the same resolution limits (Table 1) as used for refining each structure and contoured at 1.2 σ (gray mesh), is shown for the DNA substrates, calcium, and Dbh residues Asp7, Asp105, Glu106, Tyr10, Tyr48, and Arg51 and is superimposed on the final refined model. The incoming nucleotide (ddGTP), DNA, and selected residues in the active site pocket are shown in stick representation; other portions of the protein are shown in ribbons representation. The 3′ primer terminus (dG) of each structure is labeled. Atoms are colored by element: oxygen (red), nitrogen (blue), phosphate (yellow), carbon (white), and calcium (green). Coordination of the Ca2+ ion in the ternary complex is shown with dotted lines (dark gray).
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 Extrahelical Conformation of the Skipped Template Base
(A) Stereo view of the single-base deletion hot spot, showing the template cytosine in position −3 (C-3) stacking on Tyr249 and hydrogen bonding (dark gray dots) with Arg333. A final refined 2Fo − Fc electron density map, calculated using data from 20−2.7 Å resolution and contoured at 1.3 σ (gray mesh), is superimposed on the final model.
(B) Comparison of extrahelical template bases from Dbh polymerase (white) and Pol λ (blue; PDB code 2BCS [Garcia-Diaz et al., 2006]). Amino acids that stabilize the respective bulged bases (C-3 in Dbh and C-2 in Pol λ) are shown. Superposition of the two structures is based on the backbone atoms (ribose and phosphate) of the bulged base and the 5′- and 3′-flanking nucleotides (rmsd 0.78 over 21 atoms). Atoms are colored as in Figure 1.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
Orientation of the C-Terminal Domains of Dbh and Dpo4 and Their Interactions with DNA
(A) Comparison of the structures of apo-Dbh (left; PDB code 1K1Q, chain A [Silvian et al., 2001]), Dbh ternary complex (middle), and Dpo4 ternary complex (right; Ab-2A, PDB code 1S0O, chain B [Ling et al., 2004]). Arrows indicate direction and magnitude of movement needed to move the C-terminal domain of one structure to the orientation found in the Dbh ternary complex structure. Rotation axes are shown as black lines. Structures were aligned based on the polymerase domains; Dbh (apo) and Dbh (ternary), rmsd 1.2 Å, 232 Cα atoms; Dbh(ternary) and Dpo4 (Ab-2A ternary), rmsd 1.7 Å, 240 Cα atoms.
(B) Residues of the C-terminal domain of Dbh that contact the DNA. Atoms in the C-terminal domain of Dbh that are located within 3.8 Å of the DNA are highlighted in white.
(C) Comparison of DNA binding by the C-terminal domains in the ternary complexes of the Dbh (top) and Dpo4 Ab-2a (bottom). The bulged base in Dbh (C-3) and the bulged ribose in Dpo4 (Ab-2A) are highlighted in red. The structures are oriented identically, by superimposition of the polymerase domains as in (A), and are viewed from the same direction. For clarity, only the DNA and C-terminal domains are shown. Structures are colored as in Figure 1.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Nucleotide Incorporation by Dbh on Primer-Template DNAs Containing Single-Base Deletion Hot Spot Sequences, Assayed under Single-Turnover Conditions
Primer was 5′ end labeled (∗) with 33P and annealed to each of the templates shown. Dbh polymerase assays were performed as described in the Experimental Procedures.
(A) Incorporation of dCTP by Dbh on primer-template DNAs containing variations of the 5′-GCCC-3′ hot spot sequence (gray boxes) that change each C individually to a T at positions −1 (T-1), −2 (T-2), and −3 (T-3) upstream of the G positioned at the active site.
(B) Incorporation of dCTP or dGTP by Dbh on primer-template DNA containing an unmodified hot spot sequence (gray box) to yield single-base deletion (left) or correct (right) nucleotide incorporation products, respectively.
(C) Fraction of primer DNA extended by one nucleotide, as a function of time, for the reaction products shown in (A) and (B). The fraction of input primer (primer, 10 nt) extended by one nucleotide (primer +1, 11 nt) was quantitated for each of the reactions: T-3 (closed triangles), T-2 (closed squares), T-1 (closed circles), single-base deletion (open triangles), and correct (open diamonds). Marker lanes (M) in (A) and (B) contain 5′ end-labeled DNA oligonucleotides of lengths 9, 10 (identical to the primer), and 11 nt.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
Theoretical Model of Dbh Skipping Over a Template Base on a Repetitive Hot Spot Sequence that Can Adopt Multiple Conformations
The deletion hot spot sequence 5′-GCCC-3′ is positioned with the 5′ G templating the incorporation of an incoming dCTP. Three models of the template strand (superimposed on one another) have been constructed, showing in succession each of the three Cs in the hot spot sequence bulging out of the DNA duplex, while the remaining two Cs pair with two Gs at the 3′ end of the primer. See text for additional information. (Top panel) View looking down onto the active site. (Bottom panel) View looking along the DNA axis toward the active site. The hot spot sequence is colored red, orange, and yellow in the three successive models. The protein domains are colored as in Figure 1.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6 Proposed Mechanisms for Initiation of Single-Base Deletions
(A) Streisinger template-slippage.
(B) dNTP-stabilized misalignment.
(C) Misincorporation-misalignment. Each mechanism is shown using the Dbh single-base deletion hot spot sequence (boxed), with the relative rate at which Dbh uses each mechanism indicated, based on the experiments shown in Figure 4 and on previous results (Potapova et al., 2002). The base pair positioned at the Dbh polymerase active site is highlighted in gray.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions