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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-1 Dpo4 extension ternary complex containing two distinct molecules per asymmetric unit (AU), with ‘correct’ and ‘mutagenic’ alignment for extension from the [AF]G(anti)•C base pair. (a) Schematic of the expected pairing of the [AF]G-template with the 13-mer primer, ending with a 2′,3′-dideoxy-C, and dGTP. (b) Schematic of the observed base pairing arrangement within the Dpo4 active site of molecule 1. (c) Structure of the active site of molecule 1. [AF]G(anti) at the (–1) position is opposite the primer C14 base. The simulated annealing Fo-Fc omit map contoured at 3σ level is colored in blue (2.70 Å resolution). (d) Watson-Crick base pair between the [AF]G(anti) and C14(anti) at the (–1) position. (e) Base stacking arrangement of the [AF]G(anti) opposite C14 and neighboring base pairs. The little finger domain residues Leu293, Arg331 and Arg 332 contact the AF-moiety. (f) Schematic of the observed base pairing arrangement within the Dpo4 active site of molecule 2. (g) Structure of the active site of molecule 2. The [AF]G(anti) opposite the primer C14 base are shifted to the (–2) position. Arg336 has repositioned to stack with the ‘bottom’ face of the AF-moiety. Simulated annealing Fo-Fc omit map contoured at 3σ level is colored in blue (2.70 Å resolution). The loop connecting β2 and β3 of the Dpo4 finger domain (residues Phe33 to Ala42) is highlighted by the shaded area. (h) Accommodation of the AF-moiety in a pocket on the surface of the little finger domain. (i) Watson-Crick base pair between the [AF]G(anti) and C14(anti) at the (–2) position.
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Figure 2: Structure of the [AF]G•C-1 Dpo4 extension ternary complex containing two distinct molecules per asymmetric unit (AU), with ‘correct’ and ‘mutagenic’ alignment for extension from the [AF]G(anti)•C base pair. (a) Schematic of the expected pairing of the [AF]G-template with the 13-mer primer, ending with a 2′,3′-dideoxy-C, and dGTP. (b) Schematic of the observed base pairing arrangement within the Dpo4 active site of molecule 1. (c) Structure of the active site of molecule 1. [AF]G(anti) at the (–1) position is opposite the primer C14 base. The simulated annealing Fo-Fc omit map contoured at 3σ level is colored in blue (2.70 Å resolution). (d) Watson-Crick base pair between the [AF]G(anti) and C14(anti) at the (–1) position. (e) Base stacking arrangement of the [AF]G(anti) opposite C14 and neighboring base pairs. The little finger domain residues Leu293, Arg331 and Arg 332 contact the AF-moiety. (f) Schematic of the observed base pairing arrangement within the Dpo4 active site of molecule 2. (g) Structure of the active site of molecule 2. The [AF]G(anti) opposite the primer C14 base are shifted to the (–2) position. Arg336 has repositioned to stack with the ‘bottom’ face of the AF-moiety. Simulated annealing Fo-Fc omit map contoured at 3σ level is colored in blue (2.70 Å resolution). The loop connecting β2 and β3 of the Dpo4 finger domain (residues Phe33 to Ala42) is highlighted by the shaded area. (h) Accommodation of the AF-moiety in a pocket on the surface of the little finger domain. (i) Watson-Crick base pair between the [AF]G(anti) and C14(anti) at the (–2) position.

Mentions: The [AF]G•C-1 extension complex (Fig. 2a) has two distinct molecules in the asymmetric unit (AU), labeled 1 and 2. These molecules reflect ‘correct’ (molecule 1) and ‘mutagenic’ (molecule 2) extension from a C base opposite the [AF]G lesion (see Discussion section). In molecule 1, the incoming dGTP forms a nascent base pair with template C5 (Fig. 2b,c), as expected. The AF-moiety is positioned outside the template/primer helix on the major groove side of the nascent duplex; the modified G is in the anti conformation and forms a Watson-Crick base pair with 3′-terminal primer base C14 (Fig. 2c,d). The dGTP is positioned in a lower alignment relative to the active site, with the sugar ring of dGTP no longer in a stacking range with Tyr12, in contrast to the alignment observed in [AF]G•A-1 (Fig. 1e). The phosphate groups of the dGTP are in the unusual ‘goat-tail’ conformation similar to that observed previously in the Dpo4 complex containing a mismatch50. Moreover, the nascent C5•dGTP base pair is shifted relative to the adjacent [AF]G(anti)•C pair, so that the base of dGTP is above the center of the modified-G•C base pair (Fig. 2e), and C5 is above the AF-moiety. Interestingly, the base of A4, 5′ to C5, resides in the same plane as the base of C5, thus forming an A4-C5 platform that stacks against the long hydrophobic face of the AF. The side chains of the Dpo4 little finger domain residues Arg331, Arg332 and Leu293 partially enclose and shield the AF-moiety from the solvent (Fig. 2e).


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-1 Dpo4 extension ternary complex containing two distinct molecules per asymmetric unit (AU), with ‘correct’ and ‘mutagenic’ alignment for extension from the [AF]G(anti)•C base pair. (a) Schematic of the expected pairing of the [AF]G-template with the 13-mer primer, ending with a 2′,3′-dideoxy-C, and dGTP. (b) Schematic of the observed base pairing arrangement within the Dpo4 active site of molecule 1. (c) Structure of the active site of molecule 1. [AF]G(anti) at the (–1) position is opposite the primer C14 base. The simulated annealing Fo-Fc omit map contoured at 3σ level is colored in blue (2.70 Å resolution). (d) Watson-Crick base pair between the [AF]G(anti) and C14(anti) at the (–1) position. (e) Base stacking arrangement of the [AF]G(anti) opposite C14 and neighboring base pairs. The little finger domain residues Leu293, Arg331 and Arg 332 contact the AF-moiety. (f) Schematic of the observed base pairing arrangement within the Dpo4 active site of molecule 2. (g) Structure of the active site of molecule 2. The [AF]G(anti) opposite the primer C14 base are shifted to the (–2) position. Arg336 has repositioned to stack with the ‘bottom’ face of the AF-moiety. Simulated annealing Fo-Fc omit map contoured at 3σ level is colored in blue (2.70 Å resolution). The loop connecting β2 and β3 of the Dpo4 finger domain (residues Phe33 to Ala42) is highlighted by the shaded area. (h) Accommodation of the AF-moiety in a pocket on the surface of the little finger domain. (i) Watson-Crick base pair between the [AF]G(anti) and C14(anti) at the (–2) position.
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Figure 2: Structure of the [AF]G•C-1 Dpo4 extension ternary complex containing two distinct molecules per asymmetric unit (AU), with ‘correct’ and ‘mutagenic’ alignment for extension from the [AF]G(anti)•C base pair. (a) Schematic of the expected pairing of the [AF]G-template with the 13-mer primer, ending with a 2′,3′-dideoxy-C, and dGTP. (b) Schematic of the observed base pairing arrangement within the Dpo4 active site of molecule 1. (c) Structure of the active site of molecule 1. [AF]G(anti) at the (–1) position is opposite the primer C14 base. The simulated annealing Fo-Fc omit map contoured at 3σ level is colored in blue (2.70 Å resolution). (d) Watson-Crick base pair between the [AF]G(anti) and C14(anti) at the (–1) position. (e) Base stacking arrangement of the [AF]G(anti) opposite C14 and neighboring base pairs. The little finger domain residues Leu293, Arg331 and Arg 332 contact the AF-moiety. (f) Schematic of the observed base pairing arrangement within the Dpo4 active site of molecule 2. (g) Structure of the active site of molecule 2. The [AF]G(anti) opposite the primer C14 base are shifted to the (–2) position. Arg336 has repositioned to stack with the ‘bottom’ face of the AF-moiety. Simulated annealing Fo-Fc omit map contoured at 3σ level is colored in blue (2.70 Å resolution). The loop connecting β2 and β3 of the Dpo4 finger domain (residues Phe33 to Ala42) is highlighted by the shaded area. (h) Accommodation of the AF-moiety in a pocket on the surface of the little finger domain. (i) Watson-Crick base pair between the [AF]G(anti) and C14(anti) at the (–2) position.
Mentions: The [AF]G•C-1 extension complex (Fig. 2a) has two distinct molecules in the asymmetric unit (AU), labeled 1 and 2. These molecules reflect ‘correct’ (molecule 1) and ‘mutagenic’ (molecule 2) extension from a C base opposite the [AF]G lesion (see Discussion section). In molecule 1, the incoming dGTP forms a nascent base pair with template C5 (Fig. 2b,c), as expected. The AF-moiety is positioned outside the template/primer helix on the major groove side of the nascent duplex; the modified G is in the anti conformation and forms a Watson-Crick base pair with 3′-terminal primer base C14 (Fig. 2c,d). The dGTP is positioned in a lower alignment relative to the active site, with the sugar ring of dGTP no longer in a stacking range with Tyr12, in contrast to the alignment observed in [AF]G•A-1 (Fig. 1e). The phosphate groups of the dGTP are in the unusual ‘goat-tail’ conformation similar to that observed previously in the Dpo4 complex containing a mismatch50. Moreover, the nascent C5•dGTP base pair is shifted relative to the adjacent [AF]G(anti)•C pair, so that the base of dGTP is above the center of the modified-G•C base pair (Fig. 2e), and C5 is above the AF-moiety. Interestingly, the base of A4, 5′ to C5, resides in the same plane as the base of C5, thus forming an A4-C5 platform that stacks against the long hydrophobic face of the AF. The side chains of the Dpo4 little finger domain residues Arg331, Arg332 and Leu293 partially enclose and shield the AF-moiety from the solvent (Fig. 2e).

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