Results: 5

1.
Figure 1

Figure 1. Structural overview of wild-type pol β. From: Structures of dNTP Intermediate States During DNA Polymerase Active Site Assembly.

(A) Structural overlay of the open binary pol β/DNA complex (3ISB) and the closed ternary pol β/DNA/dNTP complex (2FMS). The portions of pol β that undergo a structural change during the open to closed transition are show in salmon (open) and yellow (closed). The incoming nucleotide and templating base are shown in yellow with the Mg2+ ions in red. The DNA backbone of the upstream duplex is represented as an orange ribbon. (B) Close-up of the closed ternary pol β active site. Key amino acids, templating base, and incoming nucleotide are shown in yellow and important interactions are indicated (dashed lines). Mgc and Mgn represent the catalytic and nucleotide binding magnesium ions, respectively.

Bret D. Freudenthal, et al. Structure. 2012 November 7;20(11):1829-1837.
2.
Figure 3

Figure 3. Comparison of the intermediate nucleotide binding states during metal binding. From: Structures of dNTP Intermediate States During DNA Polymerase Active Site Assembly.

The location of the active site aspartate residues, incoming nucleotide, and templating base (tn) are shown for each structure. The metal free, one metal, and two metal structures are shown in tan, cyan, and green, respectively. The Mg2+ ion from the one metal pol β structure is cyan and the Mn2+ ions from the two metal pol β structure are shown in green (See also Figure S4). (A) Overlay of the metal free and one metal pol β structures. (B) Overlay of the one metal and two metal pol β structures. (C) Overlay of the metal free, one metal, and two metal pol β structures. (D) A mechanistic model inferred from the three pol β structures with possible polymerase checkpoints indicated under each step.

Bret D. Freudenthal, et al. Structure. 2012 November 7;20(11):1829-1837.
3.
Figure 2

Figure 2. Metal-free and one-metal ternary DNA polymerase β structures in the open conformation. From: Structures of dNTP Intermediate States During DNA Polymerase Active Site Assembly.

The metal-free and one-metal structures are tan and cyan, respectively. A 2Fo-Fc electron density map of the incoming nucleotide in the metal-free (A) and one-metal (C) pol β structures contoured at 1.2σ is shown. The protein is omitted for clarity. The base pairing between the templating base and incoming nucleotide is highlighted (dashed lines). A close up of the active site for the metal-free (B) and one-metal (D) pol β structures. Key protein side chains are shown in stick representation while the nucleotide Mg2+ ion and bridging water molecules are shown as red and blue balls, respectively. (See also Figures S1–S3)

Bret D. Freudenthal, et al. Structure. 2012 November 7;20(11):1829-1837.
4.
Figure 5

Figure 5. One metal dG–dAPCPP mismatch open R283K pol β structure. From: Structures of dNTP Intermediate States During DNA Polymerase Active Site Assembly.

(A) The one metal mismatch pol β structure is shown in magenta with the Mg2+ ion and water molecule shown in red and blue, respectively. Key active site residues are shown with important interactions highlighted with dashed lines. A 2Fo-Fc electron density map of the incoming nucleotide contoured at 1.2σ highlights the disordered base and sugar moiety for the incoming nucleotide. (B) Overlay of the pol β dG–dAPCPP one metal open R283K and two metal closed wild-type enzyme (3C2M) structures. The one metal structure is in magenta and the two metal structure is shown in purple. The open and closed position of Helix N, the active site aspartate residues, and the divalent metal ions are indicated. Mn2+ is shown in purple and corresponds to the two metal closed mismatch wild-type pol β structure.

Bret D. Freudenthal, et al. Structure. 2012 November 7;20(11):1829-1837.
5.
Figure 4

Figure 4. From: Structures of dNTP Intermediate States During DNA Polymerase Active Site Assembly.

Active site of each pol β molecule in the two molecules per asymmetric unit structure (4F5R). The active site carbons for the closed two metal (A) and open metal free (B) pol β molecules are shown in green and tan, respectively. The carbons of the primer terminus base pair are gray. The Mn ions are shown as red spheres. A Fo-Fc density map for each incoming nucleotide is shown in green and contoured to 2.7σ. An anomalous map contoured at 5σ over the entire asymmetric unit is shown in red for both active sites, highlighting the presence of Mn2+ in the closed active site (A) and lack of metal in the open active site (B) soaked with 200 mM MnCl2. The base pairing between the complementary bases is highlighted (dashed lines). Key protein side chains are shown in stick representation. (See also Figure S5)

Bret D. Freudenthal, et al. Structure. 2012 November 7;20(11):1829-1837.

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