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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-1 Dpo4 extension ternary complex. (a) Chemical formula of 2-aminofluorene-C8-guanine, [AF]G, and 2-acetylaminofluorene-C8-guanine, [AAF]G, adducts. (b) Schematic of the expected pairing of the [AF]G-modified 19-mer template with the 13-mer primer, ending with a 2′,3′-dideoxy-A, and dGTP in the extension ternary complex with Dpo4. The insertion position at the Dpo4 active site is denoted by (0), and the post-insertion position is denoted by (–1). (c) Schematic of the observed base pairing arrangement within the Dpo4 active site. (d) Overall structure of the [AF]G•A-1 complex. (e) Structure of the active site of the [AF]G•A-1 complex. [AF]G(syn) at the (–1) position is opposite disordered 3′-terminal A14 base of the primer strand. The next template base C5 is paired with an incoming dGTP at the (0) position of the active site. The first Ca2+, cation A, is coordinated by invariant D7, D105, and E106 residues. The second Ca2+, cation B, is chelated by the phosphate groups of the incoming dGTP. Simulated annealing Fo-Fc omit map contoured at 3σ level and colored in blue (2.96 Å resolution) is shown for [AF]G, A14 and Arg336 residues. (f) [AF]G(syn) opposite A14 of the primer strand. [AF]G in the anti conformation (black lines) does not fit the map. Only the phosphate group of A14 has well-ordered density. (g) The N2 group of modified-G(syn) forms hydrogen bonds with the phosphate oxygens of C5. (h) Base stacking arrangement of the [AF]G(syn) and neighboring base pairs. The intercalated AF-moiety leaves no room for the disordered base of A14 within the template/primer helix.
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Figure 1: Structure of the [AF]G•A-1 Dpo4 extension ternary complex. (a) Chemical formula of 2-aminofluorene-C8-guanine, [AF]G, and 2-acetylaminofluorene-C8-guanine, [AAF]G, adducts. (b) Schematic of the expected pairing of the [AF]G-modified 19-mer template with the 13-mer primer, ending with a 2′,3′-dideoxy-A, and dGTP in the extension ternary complex with Dpo4. The insertion position at the Dpo4 active site is denoted by (0), and the post-insertion position is denoted by (–1). (c) Schematic of the observed base pairing arrangement within the Dpo4 active site. (d) Overall structure of the [AF]G•A-1 complex. (e) Structure of the active site of the [AF]G•A-1 complex. [AF]G(syn) at the (–1) position is opposite disordered 3′-terminal A14 base of the primer strand. The next template base C5 is paired with an incoming dGTP at the (0) position of the active site. The first Ca2+, cation A, is coordinated by invariant D7, D105, and E106 residues. The second Ca2+, cation B, is chelated by the phosphate groups of the incoming dGTP. Simulated annealing Fo-Fc omit map contoured at 3σ level and colored in blue (2.96 Å resolution) is shown for [AF]G, A14 and Arg336 residues. (f) [AF]G(syn) opposite A14 of the primer strand. [AF]G in the anti conformation (black lines) does not fit the map. Only the phosphate group of A14 has well-ordered density. (g) The N2 group of modified-G(syn) forms hydrogen bonds with the phosphate oxygens of C5. (h) Base stacking arrangement of the [AF]G(syn) and neighboring base pairs. The intercalated AF-moiety leaves no room for the disordered base of A14 within the template/primer helix.

Mentions: Aromatic amines are potent carcinogens, present in cigarette smoke and coal-derived synthetic fuels1, and formed during the cooking of protein-rich foods such as meat and fish2. Dye industry workers have been susceptible to bladder cancer, now known to be preventable by eliminating exposure to specific aromatic amines3. 2-acetylaminofluorene, AAF, and its deacetylated derivative aminofluorene, AF, have been intensively studied as model chemical mutagens and carcinogens4. Metabolic processing of AAF by cytochrome P450 enzymes creates reactive intermediates that predominantly bind to the C8 position of guanine, forming covalent [AF]G and [AAF]G adducts (Fig. 1a)4.


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-1 Dpo4 extension ternary complex. (a) Chemical formula of 2-aminofluorene-C8-guanine, [AF]G, and 2-acetylaminofluorene-C8-guanine, [AAF]G, adducts. (b) Schematic of the expected pairing of the [AF]G-modified 19-mer template with the 13-mer primer, ending with a 2′,3′-dideoxy-A, and dGTP in the extension ternary complex with Dpo4. The insertion position at the Dpo4 active site is denoted by (0), and the post-insertion position is denoted by (–1). (c) Schematic of the observed base pairing arrangement within the Dpo4 active site. (d) Overall structure of the [AF]G•A-1 complex. (e) Structure of the active site of the [AF]G•A-1 complex. [AF]G(syn) at the (–1) position is opposite disordered 3′-terminal A14 base of the primer strand. The next template base C5 is paired with an incoming dGTP at the (0) position of the active site. The first Ca2+, cation A, is coordinated by invariant D7, D105, and E106 residues. The second Ca2+, cation B, is chelated by the phosphate groups of the incoming dGTP. Simulated annealing Fo-Fc omit map contoured at 3σ level and colored in blue (2.96 Å resolution) is shown for [AF]G, A14 and Arg336 residues. (f) [AF]G(syn) opposite A14 of the primer strand. [AF]G in the anti conformation (black lines) does not fit the map. Only the phosphate group of A14 has well-ordered density. (g) The N2 group of modified-G(syn) forms hydrogen bonds with the phosphate oxygens of C5. (h) Base stacking arrangement of the [AF]G(syn) and neighboring base pairs. The intercalated AF-moiety leaves no room for the disordered base of A14 within the template/primer helix.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Structure of the [AF]G•A-1 Dpo4 extension ternary complex. (a) Chemical formula of 2-aminofluorene-C8-guanine, [AF]G, and 2-acetylaminofluorene-C8-guanine, [AAF]G, adducts. (b) Schematic of the expected pairing of the [AF]G-modified 19-mer template with the 13-mer primer, ending with a 2′,3′-dideoxy-A, and dGTP in the extension ternary complex with Dpo4. The insertion position at the Dpo4 active site is denoted by (0), and the post-insertion position is denoted by (–1). (c) Schematic of the observed base pairing arrangement within the Dpo4 active site. (d) Overall structure of the [AF]G•A-1 complex. (e) Structure of the active site of the [AF]G•A-1 complex. [AF]G(syn) at the (–1) position is opposite disordered 3′-terminal A14 base of the primer strand. The next template base C5 is paired with an incoming dGTP at the (0) position of the active site. The first Ca2+, cation A, is coordinated by invariant D7, D105, and E106 residues. The second Ca2+, cation B, is chelated by the phosphate groups of the incoming dGTP. Simulated annealing Fo-Fc omit map contoured at 3σ level and colored in blue (2.96 Å resolution) is shown for [AF]G, A14 and Arg336 residues. (f) [AF]G(syn) opposite A14 of the primer strand. [AF]G in the anti conformation (black lines) does not fit the map. Only the phosphate group of A14 has well-ordered density. (g) The N2 group of modified-G(syn) forms hydrogen bonds with the phosphate oxygens of C5. (h) Base stacking arrangement of the [AF]G(syn) and neighboring base pairs. The intercalated AF-moiety leaves no room for the disordered base of A14 within the template/primer helix.
Mentions: Aromatic amines are potent carcinogens, present in cigarette smoke and coal-derived synthetic fuels1, and formed during the cooking of protein-rich foods such as meat and fish2. Dye industry workers have been susceptible to bladder cancer, now known to be preventable by eliminating exposure to specific aromatic amines3. 2-acetylaminofluorene, AAF, and its deacetylated derivative aminofluorene, AF, have been intensively studied as model chemical mutagens and carcinogens4. Metabolic processing of AAF by cytochrome P450 enzymes creates reactive intermediates that predominantly bind to the C8 position of guanine, forming covalent [AF]G and [AAF]G adducts (Fig. 1a)4.

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