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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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Efficiency and fidelity of base incorporation and extension of primers bound to unmodified-G and [AF]G-template by Dpo4. (a) Time course of extension of 32P 5′-endlabeled 13-mer primers bound to 19-mer templates in the presence of all four dNTPs. The 3′-end of the 13-mer primer was paired with the template base on the 3′-side of the unmodified-G, or [AF]G; the 3′-end primer base of the 14-mer is paired with G or [AF]G. In the case of the [AF]G-template, additional 15-, 16-, 17- and 18-mers bands that migrate with different mobilities than the correctly elongated bands arising from the unmodified template strand, are detected, thus indicating mutagenic extension. The green triangles represent the correctly extended products; the magenta triangles represent mutagenic extension. In the case of the [AF]G template, the fully extended 19-mer and the shorter 18-mer products comprise ∼22% and ∼20% of the overall extended and unextended primer strands, respectively, observed after a 20 min incubation time (lane 10). (b) dCTP, dATP, dGTP or dTTP single nucleotide insertion. (c) Nucleotide insertion frequencies normalized relative to the insertion of a C base opposite the unmodified-G. The Michaelis-Menten kinetic data are from the Supplementary Table 1. (d) Efficiency and fidelity of extension from a C base opposite the unmodified-G and [AF]G- by Dpo4. Lanes 1-2 demonstrate extension in the presence of a mixture of dGTP, dATP and dTTP. Lanes 3-6 and 7-10 show extension by a single nucleotide at a time (see labels), and lanes 11-16 show extension in the presence of two nucleotides at a time (see labels). The 15-mer to 18-mer primers containing mutated bases are indicated by the magenta triangles (e) Relative extension frequencies from G•C and [AF]G•C pairs in the presence of dGTP. The Michaelis-Menten kinetic data are from Supplementary Table 1. (f) Profile analysis of 32P-signal intensities shown in lanes 1 and 2 of panel d.
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Figure 6: Efficiency and fidelity of base incorporation and extension of primers bound to unmodified-G and [AF]G-template by Dpo4. (a) Time course of extension of 32P 5′-endlabeled 13-mer primers bound to 19-mer templates in the presence of all four dNTPs. The 3′-end of the 13-mer primer was paired with the template base on the 3′-side of the unmodified-G, or [AF]G; the 3′-end primer base of the 14-mer is paired with G or [AF]G. In the case of the [AF]G-template, additional 15-, 16-, 17- and 18-mers bands that migrate with different mobilities than the correctly elongated bands arising from the unmodified template strand, are detected, thus indicating mutagenic extension. The green triangles represent the correctly extended products; the magenta triangles represent mutagenic extension. In the case of the [AF]G template, the fully extended 19-mer and the shorter 18-mer products comprise ∼22% and ∼20% of the overall extended and unextended primer strands, respectively, observed after a 20 min incubation time (lane 10). (b) dCTP, dATP, dGTP or dTTP single nucleotide insertion. (c) Nucleotide insertion frequencies normalized relative to the insertion of a C base opposite the unmodified-G. The Michaelis-Menten kinetic data are from the Supplementary Table 1. (d) Efficiency and fidelity of extension from a C base opposite the unmodified-G and [AF]G- by Dpo4. Lanes 1-2 demonstrate extension in the presence of a mixture of dGTP, dATP and dTTP. Lanes 3-6 and 7-10 show extension by a single nucleotide at a time (see labels), and lanes 11-16 show extension in the presence of two nucleotides at a time (see labels). The 15-mer to 18-mer primers containing mutated bases are indicated by the magenta triangles (e) Relative extension frequencies from G•C and [AF]G•C pairs in the presence of dGTP. The Michaelis-Menten kinetic data are from Supplementary Table 1. (f) Profile analysis of 32P-signal intensities shown in lanes 1 and 2 of panel d.

Mentions: Dpo4 readily elongates the 13-mer primer strand using the unmodified-G DNA template, to produce predominantly full-length 19-mer extension product in the presence of all four dNTPs; only single bands corresponding to partially extended 14-, 15-, 16-, 17- and 18-mers are evident (Fig. 6a, lanes 1-5 and Supplementary Fig. 5). Dpo4 efficiently inserts a base opposite the [AF]G-adduct forming a 14-mer product, but further extension is inhibited (Fig. 6a, lanes 6-10). Moreover, additional bands for 15-, 16-, 17- and 18-mers are detected, indicating mutagenic extension, while the single band for the 19-mer suggests that the correct fully elongated product was formed (Supplementary Fig. 5). Single base incorporation experiments (Fig. 6b) and Michaelis-Menten kinetic parameters (Fig. 6c, Supplementary Fig. 6 and Supplementary Table 1) indicate that C is preferentially inserted opposite [AF]G with insertion frequency, fins, only 8-fold smaller than it is opposite unmodified G. However, the efficiency of extension from the primer 3′-terminal C•[AF]G base pair via incorporation of the next correct dGTP, is reduced by a factor of ∼150, as compared to extension from the unmodified G•C base pair (Fig. 6d lanes 3 and 7, Fig. 6e, Supplementary Fig. 7, panel a1, Supplementary Table 1).


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)

Efficiency and fidelity of base incorporation and extension of primers bound to unmodified-G and [AF]G-template by Dpo4. (a) Time course of extension of 32P 5′-endlabeled 13-mer primers bound to 19-mer templates in the presence of all four dNTPs. The 3′-end of the 13-mer primer was paired with the template base on the 3′-side of the unmodified-G, or [AF]G; the 3′-end primer base of the 14-mer is paired with G or [AF]G. In the case of the [AF]G-template, additional 15-, 16-, 17- and 18-mers bands that migrate with different mobilities than the correctly elongated bands arising from the unmodified template strand, are detected, thus indicating mutagenic extension. The green triangles represent the correctly extended products; the magenta triangles represent mutagenic extension. In the case of the [AF]G template, the fully extended 19-mer and the shorter 18-mer products comprise ∼22% and ∼20% of the overall extended and unextended primer strands, respectively, observed after a 20 min incubation time (lane 10). (b) dCTP, dATP, dGTP or dTTP single nucleotide insertion. (c) Nucleotide insertion frequencies normalized relative to the insertion of a C base opposite the unmodified-G. The Michaelis-Menten kinetic data are from the Supplementary Table 1. (d) Efficiency and fidelity of extension from a C base opposite the unmodified-G and [AF]G- by Dpo4. Lanes 1-2 demonstrate extension in the presence of a mixture of dGTP, dATP and dTTP. Lanes 3-6 and 7-10 show extension by a single nucleotide at a time (see labels), and lanes 11-16 show extension in the presence of two nucleotides at a time (see labels). The 15-mer to 18-mer primers containing mutated bases are indicated by the magenta triangles (e) Relative extension frequencies from G•C and [AF]G•C pairs in the presence of dGTP. The Michaelis-Menten kinetic data are from Supplementary Table 1. (f) Profile analysis of 32P-signal intensities shown in lanes 1 and 2 of panel d.
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Figure 6: Efficiency and fidelity of base incorporation and extension of primers bound to unmodified-G and [AF]G-template by Dpo4. (a) Time course of extension of 32P 5′-endlabeled 13-mer primers bound to 19-mer templates in the presence of all four dNTPs. The 3′-end of the 13-mer primer was paired with the template base on the 3′-side of the unmodified-G, or [AF]G; the 3′-end primer base of the 14-mer is paired with G or [AF]G. In the case of the [AF]G-template, additional 15-, 16-, 17- and 18-mers bands that migrate with different mobilities than the correctly elongated bands arising from the unmodified template strand, are detected, thus indicating mutagenic extension. The green triangles represent the correctly extended products; the magenta triangles represent mutagenic extension. In the case of the [AF]G template, the fully extended 19-mer and the shorter 18-mer products comprise ∼22% and ∼20% of the overall extended and unextended primer strands, respectively, observed after a 20 min incubation time (lane 10). (b) dCTP, dATP, dGTP or dTTP single nucleotide insertion. (c) Nucleotide insertion frequencies normalized relative to the insertion of a C base opposite the unmodified-G. The Michaelis-Menten kinetic data are from the Supplementary Table 1. (d) Efficiency and fidelity of extension from a C base opposite the unmodified-G and [AF]G- by Dpo4. Lanes 1-2 demonstrate extension in the presence of a mixture of dGTP, dATP and dTTP. Lanes 3-6 and 7-10 show extension by a single nucleotide at a time (see labels), and lanes 11-16 show extension in the presence of two nucleotides at a time (see labels). The 15-mer to 18-mer primers containing mutated bases are indicated by the magenta triangles (e) Relative extension frequencies from G•C and [AF]G•C pairs in the presence of dGTP. The Michaelis-Menten kinetic data are from Supplementary Table 1. (f) Profile analysis of 32P-signal intensities shown in lanes 1 and 2 of panel d.
Mentions: Dpo4 readily elongates the 13-mer primer strand using the unmodified-G DNA template, to produce predominantly full-length 19-mer extension product in the presence of all four dNTPs; only single bands corresponding to partially extended 14-, 15-, 16-, 17- and 18-mers are evident (Fig. 6a, lanes 1-5 and Supplementary Fig. 5). Dpo4 efficiently inserts a base opposite the [AF]G-adduct forming a 14-mer product, but further extension is inhibited (Fig. 6a, lanes 6-10). Moreover, additional bands for 15-, 16-, 17- and 18-mers are detected, indicating mutagenic extension, while the single band for the 19-mer suggests that the correct fully elongated product was formed (Supplementary Fig. 5). Single base incorporation experiments (Fig. 6b) and Michaelis-Menten kinetic parameters (Fig. 6c, Supplementary Fig. 6 and Supplementary Table 1) indicate that C is preferentially inserted opposite [AF]G with insertion frequency, fins, only 8-fold smaller than it is opposite unmodified G. However, the efficiency of extension from the primer 3′-terminal C•[AF]G base pair via incorporation of the next correct dGTP, is reduced by a factor of ∼150, as compared to extension from the unmodified G•C base pair (Fig. 6d lanes 3 and 7, Fig. 6e, Supplementary Fig. 7, panel a1, Supplementary Table 1).

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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