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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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Misalignment-mediated replication errors and proposed mechanism for the [AF]G-induced semi-targeted mutagenesis. (a) Misalignment-mediated DNA synthesis errors on lesion-modified DNA. The intermediates are stabilized by Watson-Crick base-pairing. The schematics are adapted from37,39. The template bases colored in dark red are distinct from the DNA sequence in the present study. (b) Proposed mechanism for [AF]G-induced semi-targeted mutagenesis in the Dpo4 active site. The misaligned intermediates are stabilized by interactions with the DNA polymerase. Left panel: Schematics of base pairing alignment and the Dpo4 little finger (purple) and thumb domain (red) contacts with the ‘top’ portion of the template/primer DNA in an unmodified ternary complex. State 1, molecule 1 of [AF]G•C-1: C5 stacks above the AF-moiety, the base of dGTP is shifted toward the middle of the template/primer helix and the phosphate groups adopt a ‘goat-tail’ conformation50. In this state the thumb domain of Dpo4 is translocated relative to the template/primer DNA compared to the unmodified ternary complex, denoted by a red arrow. The State 1 complex has a ‘binary complex-like’ DNA/Dpo4 interaction pattern that reduces Dpo4 elongation efficiency and allows time for further realignment. State 2, molecule 2 of [AF]G•C-1: The little finger, palm and finger domains are translocated so the [AF]G(anti)•C base pair is now at the (–2) position, the AF-moiety is in a pocket on the surface of the Dpo4 little finger domain. The complex has a ‘ternary complex-like’ interaction pattern. Translocation of the little finger domain is denoted by a purple arrow, and of the finger and palm domains by a blue arrow. The unpaired dGTP can be interchanged with another dNTP. The equilibria between State 2 and State 1 would allow bringing the C5 back into the double helix for correct extension. State 3 (hypothesized): Covalent bond formation following State 2 would result in C5 base deletion, and misinsertion of G opposite template base A4. Binding of the next correct dTTP opposite A3 does not require a change in Dpo4/DNA contacts. The complex now resembles the catalytically competent [AF]G•C-2 post-extension complex.
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Figure 7: Misalignment-mediated replication errors and proposed mechanism for the [AF]G-induced semi-targeted mutagenesis. (a) Misalignment-mediated DNA synthesis errors on lesion-modified DNA. The intermediates are stabilized by Watson-Crick base-pairing. The schematics are adapted from37,39. The template bases colored in dark red are distinct from the DNA sequence in the present study. (b) Proposed mechanism for [AF]G-induced semi-targeted mutagenesis in the Dpo4 active site. The misaligned intermediates are stabilized by interactions with the DNA polymerase. Left panel: Schematics of base pairing alignment and the Dpo4 little finger (purple) and thumb domain (red) contacts with the ‘top’ portion of the template/primer DNA in an unmodified ternary complex. State 1, molecule 1 of [AF]G•C-1: C5 stacks above the AF-moiety, the base of dGTP is shifted toward the middle of the template/primer helix and the phosphate groups adopt a ‘goat-tail’ conformation50. In this state the thumb domain of Dpo4 is translocated relative to the template/primer DNA compared to the unmodified ternary complex, denoted by a red arrow. The State 1 complex has a ‘binary complex-like’ DNA/Dpo4 interaction pattern that reduces Dpo4 elongation efficiency and allows time for further realignment. State 2, molecule 2 of [AF]G•C-1: The little finger, palm and finger domains are translocated so the [AF]G(anti)•C base pair is now at the (–2) position, the AF-moiety is in a pocket on the surface of the Dpo4 little finger domain. The complex has a ‘ternary complex-like’ interaction pattern. Translocation of the little finger domain is denoted by a purple arrow, and of the finger and palm domains by a blue arrow. The unpaired dGTP can be interchanged with another dNTP. The equilibria between State 2 and State 1 would allow bringing the C5 back into the double helix for correct extension. State 3 (hypothesized): Covalent bond formation following State 2 would result in C5 base deletion, and misinsertion of G opposite template base A4. Binding of the next correct dTTP opposite A3 does not require a change in Dpo4/DNA contacts. The complex now resembles the catalytically competent [AF]G•C-2 post-extension complex.

Mentions: Primer extension via the correct dGTP from the mismatched 3′-A, G, and T opposite the [AF]G adduct is inefficient (Supplementary Table 1). Surprisingly, Dpo4 is robust in extending from an [AF]G•G terminus via addition of dTTP (Supplementary Fig. 7a, panel a4 and Supplementary Fig. 7b,c). This intermediate is stabilized by an incoming dTTP opposite template A4 and a correct terminal base pair between primer base G14 and template C5 (Supplementary Fig. 7b). The dNTP-stabilized misinsertion misalignment mechanism (Fig. 7a) has been demonstrated previously for aromatic amine adducts and other lesions21,40-42.


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)

Misalignment-mediated replication errors and proposed mechanism for the [AF]G-induced semi-targeted mutagenesis. (a) Misalignment-mediated DNA synthesis errors on lesion-modified DNA. The intermediates are stabilized by Watson-Crick base-pairing. The schematics are adapted from37,39. The template bases colored in dark red are distinct from the DNA sequence in the present study. (b) Proposed mechanism for [AF]G-induced semi-targeted mutagenesis in the Dpo4 active site. The misaligned intermediates are stabilized by interactions with the DNA polymerase. Left panel: Schematics of base pairing alignment and the Dpo4 little finger (purple) and thumb domain (red) contacts with the ‘top’ portion of the template/primer DNA in an unmodified ternary complex. State 1, molecule 1 of [AF]G•C-1: C5 stacks above the AF-moiety, the base of dGTP is shifted toward the middle of the template/primer helix and the phosphate groups adopt a ‘goat-tail’ conformation50. In this state the thumb domain of Dpo4 is translocated relative to the template/primer DNA compared to the unmodified ternary complex, denoted by a red arrow. The State 1 complex has a ‘binary complex-like’ DNA/Dpo4 interaction pattern that reduces Dpo4 elongation efficiency and allows time for further realignment. State 2, molecule 2 of [AF]G•C-1: The little finger, palm and finger domains are translocated so the [AF]G(anti)•C base pair is now at the (–2) position, the AF-moiety is in a pocket on the surface of the Dpo4 little finger domain. The complex has a ‘ternary complex-like’ interaction pattern. Translocation of the little finger domain is denoted by a purple arrow, and of the finger and palm domains by a blue arrow. The unpaired dGTP can be interchanged with another dNTP. The equilibria between State 2 and State 1 would allow bringing the C5 back into the double helix for correct extension. State 3 (hypothesized): Covalent bond formation following State 2 would result in C5 base deletion, and misinsertion of G opposite template base A4. Binding of the next correct dTTP opposite A3 does not require a change in Dpo4/DNA contacts. The complex now resembles the catalytically competent [AF]G•C-2 post-extension complex.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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Figure 7: Misalignment-mediated replication errors and proposed mechanism for the [AF]G-induced semi-targeted mutagenesis. (a) Misalignment-mediated DNA synthesis errors on lesion-modified DNA. The intermediates are stabilized by Watson-Crick base-pairing. The schematics are adapted from37,39. The template bases colored in dark red are distinct from the DNA sequence in the present study. (b) Proposed mechanism for [AF]G-induced semi-targeted mutagenesis in the Dpo4 active site. The misaligned intermediates are stabilized by interactions with the DNA polymerase. Left panel: Schematics of base pairing alignment and the Dpo4 little finger (purple) and thumb domain (red) contacts with the ‘top’ portion of the template/primer DNA in an unmodified ternary complex. State 1, molecule 1 of [AF]G•C-1: C5 stacks above the AF-moiety, the base of dGTP is shifted toward the middle of the template/primer helix and the phosphate groups adopt a ‘goat-tail’ conformation50. In this state the thumb domain of Dpo4 is translocated relative to the template/primer DNA compared to the unmodified ternary complex, denoted by a red arrow. The State 1 complex has a ‘binary complex-like’ DNA/Dpo4 interaction pattern that reduces Dpo4 elongation efficiency and allows time for further realignment. State 2, molecule 2 of [AF]G•C-1: The little finger, palm and finger domains are translocated so the [AF]G(anti)•C base pair is now at the (–2) position, the AF-moiety is in a pocket on the surface of the Dpo4 little finger domain. The complex has a ‘ternary complex-like’ interaction pattern. Translocation of the little finger domain is denoted by a purple arrow, and of the finger and palm domains by a blue arrow. The unpaired dGTP can be interchanged with another dNTP. The equilibria between State 2 and State 1 would allow bringing the C5 back into the double helix for correct extension. State 3 (hypothesized): Covalent bond formation following State 2 would result in C5 base deletion, and misinsertion of G opposite template base A4. Binding of the next correct dTTP opposite A3 does not require a change in Dpo4/DNA contacts. The complex now resembles the catalytically competent [AF]G•C-2 post-extension complex.
Mentions: Primer extension via the correct dGTP from the mismatched 3′-A, G, and T opposite the [AF]G adduct is inefficient (Supplementary Table 1). Surprisingly, Dpo4 is robust in extending from an [AF]G•G terminus via addition of dTTP (Supplementary Fig. 7a, panel a4 and Supplementary Fig. 7b,c). This intermediate is stabilized by an incoming dTTP opposite template A4 and a correct terminal base pair between primer base G14 and template C5 (Supplementary Fig. 7b). The dNTP-stabilized misinsertion misalignment mechanism (Fig. 7a) has been demonstrated previously for aromatic amine adducts and other lesions21,40-42.

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