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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•C-2 Dpo4 post-extension ternary complex. (a) Schematic of the expected pairing of the [AF]G-template with the 13-mer primer, ending with a 2′,3′-dideoxy-G, and added dTTP. (b) Schematic of the observed base pairing arrangement within the Dpo4 active site. (c) Structure of the active site. Simulated annealing Fo-Fc omit map contoured at 3σ level is colored in blue (2.0 Å resolution). (d) Watson-Crick base pair between the [AF]G(anti) and C14(anti) at the (–2) position. (e) Accommodation of the AF-moiety in a pocket on the surface of the little finger domain.
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Figure 4: Structure of the [AF]G•C-2 Dpo4 post-extension ternary complex. (a) Schematic of the expected pairing of the [AF]G-template with the 13-mer primer, ending with a 2′,3′-dideoxy-G, and added dTTP. (b) Schematic of the observed base pairing arrangement within the Dpo4 active site. (c) Structure of the active site. Simulated annealing Fo-Fc omit map contoured at 3σ level is colored in blue (2.0 Å resolution). (d) Watson-Crick base pair between the [AF]G(anti) and C14(anti) at the (–2) position. (e) Accommodation of the AF-moiety in a pocket on the surface of the little finger domain.

Mentions: The [AF]G•C-2 and [AF]G•A-2 post-extension complexes with either C or A base opposite the [AF]G at the (–2) position represent a further primer extension step. The two molecules in the AU of [AF]G•C-2 (Fig. 4a) are similar to each other and have the expected ‘ternary complex-like’ Dpo4/DNA contact patterns (Fig. 3d, top and bottom). The nascent base pair is formed by the A4 template base and incoming dTTP, with its sugar stacked normally against the aromatic ring of Tyr12 (Fig. 4b,c). The AF-moiety of the [AF]G(anti), Watson-Crick paired with C14 (Fig. 4c,d), is placed in the same pocket on the surface of the little finger domain with the relocated side chain of Arg336 (Fig. 4e), as in the molecule 2 of [AF]G•C-1 (Fig. 2h). The modified-[AF]G•C base pair maintains the normal base stacking alignment with the adjacent C7•G13 and C5•G15 base pairs (Fig. 4c). The distance between the α-phosphate of dTTP and the 3′-C of the terminal G15 primer base is ∼3.9 Å and the normal alignment of the Dpo4 active site is maintained.


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•C-2 Dpo4 post-extension ternary complex. (a) Schematic of the expected pairing of the [AF]G-template with the 13-mer primer, ending with a 2′,3′-dideoxy-G, and added dTTP. (b) Schematic of the observed base pairing arrangement within the Dpo4 active site. (c) Structure of the active site. Simulated annealing Fo-Fc omit map contoured at 3σ level is colored in blue (2.0 Å resolution). (d) Watson-Crick base pair between the [AF]G(anti) and C14(anti) at the (–2) position. (e) Accommodation of the AF-moiety in a pocket on the surface of the little finger domain.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Structure of the [AF]G•C-2 Dpo4 post-extension ternary complex. (a) Schematic of the expected pairing of the [AF]G-template with the 13-mer primer, ending with a 2′,3′-dideoxy-G, and added dTTP. (b) Schematic of the observed base pairing arrangement within the Dpo4 active site. (c) Structure of the active site. Simulated annealing Fo-Fc omit map contoured at 3σ level is colored in blue (2.0 Å resolution). (d) Watson-Crick base pair between the [AF]G(anti) and C14(anti) at the (–2) position. (e) Accommodation of the AF-moiety in a pocket on the surface of the little finger domain.
Mentions: The [AF]G•C-2 and [AF]G•A-2 post-extension complexes with either C or A base opposite the [AF]G at the (–2) position represent a further primer extension step. The two molecules in the AU of [AF]G•C-2 (Fig. 4a) are similar to each other and have the expected ‘ternary complex-like’ Dpo4/DNA contact patterns (Fig. 3d, top and bottom). The nascent base pair is formed by the A4 template base and incoming dTTP, with its sugar stacked normally against the aromatic ring of Tyr12 (Fig. 4b,c). The AF-moiety of the [AF]G(anti), Watson-Crick paired with C14 (Fig. 4c,d), is placed in the same pocket on the surface of the little finger domain with the relocated side chain of Arg336 (Fig. 4e), as in the molecule 2 of [AF]G•C-1 (Fig. 2h). The modified-[AF]G•C base pair maintains the normal base stacking alignment with the adjacent C7•G13 and C5•G15 base pairs (Fig. 4c). The distance between the α-phosphate of dTTP and the 3′-C of the terminal G15 primer base is ∼3.9 Å and the normal alignment of the Dpo4 active site is maintained.

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