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Mechanism of error-free and semitargeted mutagenic bypass of an aromatic amine lesion by Y-family polymerase Dpo4.

Rechkoblit O, Kolbanovskiy A, Malinina L, Geacintov NE, Broyde S, Patel DJ - Nat. Struct. Mol. Biol. (2010)

Bottom Line: This extension leads to cognate full-length product, as well as mis-elongated products containing base mutations and deletions.The mutagenic template-primer-dNTP arrangement is promoted by interactions between the polymerase and the bulky lesion rather than by a base pair-stabilized misaligment.Further extension leads to semitargeted mutations via this proposed polymerase-guided mechanism.

View Article: PubMed Central - PubMed

Affiliation: Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.

ABSTRACT
The aromatic amine carcinogen 2-aminofluorene (AF) forms covalent adducts with DNA, predominantly with guanine at the C8 position. Such lesions are bypassed by Y-family polymerases such as Dpo4 via error-free and error-prone mechanisms. We show that Dpo4 catalyzes elongation from a correct 3'-terminal cytosine opposite [AF]G in a nonrepetitive template sequence with low efficiency. This extension leads to cognate full-length product, as well as mis-elongated products containing base mutations and deletions. Crystal structures of the Dpo4 ternary complex, with the 3'-terminal primer cytosine base opposite [AF]G in the anti conformation and with the AF moiety positioned in the major groove, reveal both accurate and misalignment-mediated mutagenic extension pathways. The mutagenic template-primer-dNTP arrangement is promoted by interactions between the polymerase and the bulky lesion rather than by a base pair-stabilized misaligment. Further extension leads to semitargeted mutations via this proposed polymerase-guided mechanism.

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Structure of the [AF]G•A-2 Dpo4 post-extension ternary complex containing two molecules per AU with different positions of the partner A14 base. (a) Schematic of the expected template/primer-dTTP pairing. (b) Schematic of the observed base pairing arrangement within the Dpo4 active site of molecule 1, with the A14 apparently oriented outside the helix. (c) The active site of molecule 1. [AF]G(anti) at the (–2) position opposite primer A14 base. Simulated annealing Fo-Fc omit map contoured at 3σ level is colored in blue and at 2σ level is colored in gray (2.10 Å resolution). (d) The A14 base positioned opposite [AF]G(anti), appears to be oriented outside the helix on the minor groove side. (e) Schematic of the observed base pairing arrangement within the Dpo4 active site of molecule 2 with A14 apparently oriented inside the helix. (f) The active site of molecule 2. Simulated annealing Fo-Fc omit map contoured at 3σ level is colored in blue and at 2σ level it is colored in gray. (g) The A14 base positioned opposite [AF]G(anti), appears to be oriented inside the helix.
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Figure 5: Structure of the [AF]G•A-2 Dpo4 post-extension ternary complex containing two molecules per AU with different positions of the partner A14 base. (a) Schematic of the expected template/primer-dTTP pairing. (b) Schematic of the observed base pairing arrangement within the Dpo4 active site of molecule 1, with the A14 apparently oriented outside the helix. (c) The active site of molecule 1. [AF]G(anti) at the (–2) position opposite primer A14 base. Simulated annealing Fo-Fc omit map contoured at 3σ level is colored in blue and at 2σ level is colored in gray (2.10 Å resolution). (d) The A14 base positioned opposite [AF]G(anti), appears to be oriented outside the helix on the minor groove side. (e) Schematic of the observed base pairing arrangement within the Dpo4 active site of molecule 2 with A14 apparently oriented inside the helix. (f) The active site of molecule 2. Simulated annealing Fo-Fc omit map contoured at 3σ level is colored in blue and at 2σ level it is colored in gray. (g) The A14 base positioned opposite [AF]G(anti), appears to be oriented inside the helix.

Mentions: The two molecules in the AU of the [AF]G•A-2 complex (Fig. 5a-g) are similar to the [AF]G•C-2 complex, with the AF-moiety of the [AF]G(anti) residing in the little finger domain pocket (Figs. 5c and 5f). However, the partner A14 base appears to adopt multiple conformations (Figs. 5d and 5g). Despite the high quality of the overall 2Fo-Fc electron density map at 2.1 Å for both molecules, the density for the nucleobase of A14 is poor, while the A14 phosphate and sugar moieties are well-defined (molecules 1 and 2 in Supplementary Figs. 4a and 4b, respectively). While the exact orientation of the A14 base is not clear, we are confident of A14 placement in the minor groove in molecule 1 (Fig. 5d). In molecule 2, the electron density between the phosphate group and the sugar ring of A14 is visible on a lower σ-level than it is in molecule 1; nevertheless, an inside-the-helix position of the A14 sugar is indicated (Fig. 5g). The C1′-C1′ distance between the modified-[AF]G and A14 is ∼10.6 Å, which would be appropriate for involvement of some variation of the [AF]G(anti)•A(syn) alignment51.


Mechanism of error-free and semitargeted mutagenic bypass of an aromatic amine lesion by Y-family polymerase Dpo4.

Rechkoblit O, Kolbanovskiy A, Malinina L, Geacintov NE, Broyde S, Patel DJ - Nat. Struct. Mol. Biol. (2010)

Structure of the [AF]G•A-2 Dpo4 post-extension ternary complex containing two molecules per AU with different positions of the partner A14 base. (a) Schematic of the expected template/primer-dTTP pairing. (b) Schematic of the observed base pairing arrangement within the Dpo4 active site of molecule 1, with the A14 apparently oriented outside the helix. (c) The active site of molecule 1. [AF]G(anti) at the (–2) position opposite primer A14 base. Simulated annealing Fo-Fc omit map contoured at 3σ level is colored in blue and at 2σ level is colored in gray (2.10 Å resolution). (d) The A14 base positioned opposite [AF]G(anti), appears to be oriented outside the helix on the minor groove side. (e) Schematic of the observed base pairing arrangement within the Dpo4 active site of molecule 2 with A14 apparently oriented inside the helix. (f) The active site of molecule 2. Simulated annealing Fo-Fc omit map contoured at 3σ level is colored in blue and at 2σ level it is colored in gray. (g) The A14 base positioned opposite [AF]G(anti), appears to be oriented inside the helix.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4215948&req=5

Figure 5: Structure of the [AF]G•A-2 Dpo4 post-extension ternary complex containing two molecules per AU with different positions of the partner A14 base. (a) Schematic of the expected template/primer-dTTP pairing. (b) Schematic of the observed base pairing arrangement within the Dpo4 active site of molecule 1, with the A14 apparently oriented outside the helix. (c) The active site of molecule 1. [AF]G(anti) at the (–2) position opposite primer A14 base. Simulated annealing Fo-Fc omit map contoured at 3σ level is colored in blue and at 2σ level is colored in gray (2.10 Å resolution). (d) The A14 base positioned opposite [AF]G(anti), appears to be oriented outside the helix on the minor groove side. (e) Schematic of the observed base pairing arrangement within the Dpo4 active site of molecule 2 with A14 apparently oriented inside the helix. (f) The active site of molecule 2. Simulated annealing Fo-Fc omit map contoured at 3σ level is colored in blue and at 2σ level it is colored in gray. (g) The A14 base positioned opposite [AF]G(anti), appears to be oriented inside the helix.
Mentions: The two molecules in the AU of the [AF]G•A-2 complex (Fig. 5a-g) are similar to the [AF]G•C-2 complex, with the AF-moiety of the [AF]G(anti) residing in the little finger domain pocket (Figs. 5c and 5f). However, the partner A14 base appears to adopt multiple conformations (Figs. 5d and 5g). Despite the high quality of the overall 2Fo-Fc electron density map at 2.1 Å for both molecules, the density for the nucleobase of A14 is poor, while the A14 phosphate and sugar moieties are well-defined (molecules 1 and 2 in Supplementary Figs. 4a and 4b, respectively). While the exact orientation of the A14 base is not clear, we are confident of A14 placement in the minor groove in molecule 1 (Fig. 5d). In molecule 2, the electron density between the phosphate group and the sugar ring of A14 is visible on a lower σ-level than it is in molecule 1; nevertheless, an inside-the-helix position of the A14 sugar is indicated (Fig. 5g). The C1′-C1′ distance between the modified-[AF]G and A14 is ∼10.6 Å, which would be appropriate for involvement of some variation of the [AF]G(anti)•A(syn) alignment51.

Bottom Line: This extension leads to cognate full-length product, as well as mis-elongated products containing base mutations and deletions.The mutagenic template-primer-dNTP arrangement is promoted by interactions between the polymerase and the bulky lesion rather than by a base pair-stabilized misaligment.Further extension leads to semitargeted mutations via this proposed polymerase-guided mechanism.

View Article: PubMed Central - PubMed

Affiliation: Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.

ABSTRACT
The aromatic amine carcinogen 2-aminofluorene (AF) forms covalent adducts with DNA, predominantly with guanine at the C8 position. Such lesions are bypassed by Y-family polymerases such as Dpo4 via error-free and error-prone mechanisms. We show that Dpo4 catalyzes elongation from a correct 3'-terminal cytosine opposite [AF]G in a nonrepetitive template sequence with low efficiency. This extension leads to cognate full-length product, as well as mis-elongated products containing base mutations and deletions. Crystal structures of the Dpo4 ternary complex, with the 3'-terminal primer cytosine base opposite [AF]G in the anti conformation and with the AF moiety positioned in the major groove, reveal both accurate and misalignment-mediated mutagenic extension pathways. The mutagenic template-primer-dNTP arrangement is promoted by interactions between the polymerase and the bulky lesion rather than by a base pair-stabilized misaligment. Further extension leads to semitargeted mutations via this proposed polymerase-guided mechanism.

Show MeSH
Related in: MedlinePlus