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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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Interactions of the Dpo4 little finger and thumb domains with template/primer DNA in extension and post-extension [AF]G-modified Dpo4 ternary complexes. The backbone DNA phosphate groups in contact with Dpo4 are labeled in color and are shown by CPK spheres. The template strand is colored in cyan, the primer strand is in green. (a) The [AF]G•A-1 extension complex has a ‘ternary complex-like’ Dpo4/DNA interaction pattern. The little finger domain contacts the phosphate groups of template C5-A9 and of primer A7-G9 positions (top panel). The thumb domain contacts the phosphates of the template A12 and primer A12-G13 positions (bottom panel). The DNA segment, containing template C5-A16, primer T4-A14 bases and dGTP, is shown. (b) Molecule 1 of the [AF]G•C-1 complex with ‘correct’ template/primer-dNTP pairing alignment has a ‘binary complex-like’ Dpo4/DNA contact pattern. The little finger domain contacts the phosphate groups of template C5-A9 and primer A7-G9 positions (top panel), the thumb domain is translocated compared to the position observed in the [AF]G•A-1 complex and contacts the phosphate groups of the template C11 and primer G13-C14 positions (bottom panel). The DNA segment, containing template C5-A16, primer T4-C14 bases and dGTP is shown. (c) Molecule 2 of the [AF]G•C-1 complex with ‘mutagenic’ template/primer-dNTP pairing alignment has a ‘ternary complex-like’ Dpo4/DNA contact pattern. The little finger domain is translocated relative to the position observed in molecule 1; it contacts the phosphate groups of template [AF]G6-T8 (the C5 base is looped out and the phosphate of A4 is disordered) and primer T8-G10 positions (top panel), and the thumb domain interacts with the phosphate groups of the template C11 and primer G13-C14 positions (bottom panel). The DNA segment containing template A4-A16, primer T4-C14 bases and dGTP is shown. (d) The [AF]G•C-2 post-extension complex has a ‘ternary complex-like’ Dpo4/DNA contact pattern. The little finger domain contacts the phosphate groups of template A4-T8 and primer T8-G10 positions (top panel), and the thumb domain interacts with the phosphate groups of the template C11 and primer G13-C14 positions (bottom panel). The DNA segment containing template A4-A16, primer T4-C14 bases and dGTP is shown. The [AF]G•A-2 post-extension complex has a similar Dpo4/DNA interaction pattern to that of [AF]G•C-2.
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Figure 3: Interactions of the Dpo4 little finger and thumb domains with template/primer DNA in extension and post-extension [AF]G-modified Dpo4 ternary complexes. The backbone DNA phosphate groups in contact with Dpo4 are labeled in color and are shown by CPK spheres. The template strand is colored in cyan, the primer strand is in green. (a) The [AF]G•A-1 extension complex has a ‘ternary complex-like’ Dpo4/DNA interaction pattern. The little finger domain contacts the phosphate groups of template C5-A9 and of primer A7-G9 positions (top panel). The thumb domain contacts the phosphates of the template A12 and primer A12-G13 positions (bottom panel). The DNA segment, containing template C5-A16, primer T4-A14 bases and dGTP, is shown. (b) Molecule 1 of the [AF]G•C-1 complex with ‘correct’ template/primer-dNTP pairing alignment has a ‘binary complex-like’ Dpo4/DNA contact pattern. The little finger domain contacts the phosphate groups of template C5-A9 and primer A7-G9 positions (top panel), the thumb domain is translocated compared to the position observed in the [AF]G•A-1 complex and contacts the phosphate groups of the template C11 and primer G13-C14 positions (bottom panel). The DNA segment, containing template C5-A16, primer T4-C14 bases and dGTP is shown. (c) Molecule 2 of the [AF]G•C-1 complex with ‘mutagenic’ template/primer-dNTP pairing alignment has a ‘ternary complex-like’ Dpo4/DNA contact pattern. The little finger domain is translocated relative to the position observed in molecule 1; it contacts the phosphate groups of template [AF]G6-T8 (the C5 base is looped out and the phosphate of A4 is disordered) and primer T8-G10 positions (top panel), and the thumb domain interacts with the phosphate groups of the template C11 and primer G13-C14 positions (bottom panel). The DNA segment containing template A4-A16, primer T4-C14 bases and dGTP is shown. (d) The [AF]G•C-2 post-extension complex has a ‘ternary complex-like’ Dpo4/DNA contact pattern. The little finger domain contacts the phosphate groups of template A4-T8 and primer T8-G10 positions (top panel), and the thumb domain interacts with the phosphate groups of the template C11 and primer G13-C14 positions (bottom panel). The DNA segment containing template A4-A16, primer T4-C14 bases and dGTP is shown. The [AF]G•A-2 post-extension complex has a similar Dpo4/DNA interaction pattern to that of [AF]G•C-2.

Mentions: The [AF]G•A-1 complex has a pattern of Dpo4 interactions with DNA backbone phosphates similar to the one in an unmodified ternary complex48-49 (Fig. 3a, top and bottom). In molecule 1 of [AF]G•C-1, the little finger domain contacts the same residues as in [AF]G•A-1 (Fig. 3b, top), while the thumb domain is shifted towards the active site (see arrow, Fig. 3b, bottom and stereo view of translocation at the full structure level in Supplementary Fig. 2a, as well as thumb domain level in Supplementary Fig. 2b). The pattern of Dpo4 interactions with DNA in molecule 1 of the [AF]G•C-1 ternary complex resembles that of an unmodified binary complex (without dNTP)49. In ‘mutagenic’ molecule 2 of [AF]G•C-1, the little finger domain has translocated from the position observed in molecule 1 (see arrow, Fig. 3c, top), while the thumb domain maintains its contacts as in molecule 1 (Fig. 3c, bottom and Supplementary Fig. 2d,e). The Dpo4-DNA interaction pattern in molecule 2 corresponds to that of a ternary complex.


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)

Interactions of the Dpo4 little finger and thumb domains with template/primer DNA in extension and post-extension [AF]G-modified Dpo4 ternary complexes. The backbone DNA phosphate groups in contact with Dpo4 are labeled in color and are shown by CPK spheres. The template strand is colored in cyan, the primer strand is in green. (a) The [AF]G•A-1 extension complex has a ‘ternary complex-like’ Dpo4/DNA interaction pattern. The little finger domain contacts the phosphate groups of template C5-A9 and of primer A7-G9 positions (top panel). The thumb domain contacts the phosphates of the template A12 and primer A12-G13 positions (bottom panel). The DNA segment, containing template C5-A16, primer T4-A14 bases and dGTP, is shown. (b) Molecule 1 of the [AF]G•C-1 complex with ‘correct’ template/primer-dNTP pairing alignment has a ‘binary complex-like’ Dpo4/DNA contact pattern. The little finger domain contacts the phosphate groups of template C5-A9 and primer A7-G9 positions (top panel), the thumb domain is translocated compared to the position observed in the [AF]G•A-1 complex and contacts the phosphate groups of the template C11 and primer G13-C14 positions (bottom panel). The DNA segment, containing template C5-A16, primer T4-C14 bases and dGTP is shown. (c) Molecule 2 of the [AF]G•C-1 complex with ‘mutagenic’ template/primer-dNTP pairing alignment has a ‘ternary complex-like’ Dpo4/DNA contact pattern. The little finger domain is translocated relative to the position observed in molecule 1; it contacts the phosphate groups of template [AF]G6-T8 (the C5 base is looped out and the phosphate of A4 is disordered) and primer T8-G10 positions (top panel), and the thumb domain interacts with the phosphate groups of the template C11 and primer G13-C14 positions (bottom panel). The DNA segment containing template A4-A16, primer T4-C14 bases and dGTP is shown. (d) The [AF]G•C-2 post-extension complex has a ‘ternary complex-like’ Dpo4/DNA contact pattern. The little finger domain contacts the phosphate groups of template A4-T8 and primer T8-G10 positions (top panel), and the thumb domain interacts with the phosphate groups of the template C11 and primer G13-C14 positions (bottom panel). The DNA segment containing template A4-A16, primer T4-C14 bases and dGTP is shown. The [AF]G•A-2 post-extension complex has a similar Dpo4/DNA interaction pattern to that of [AF]G•C-2.
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Figure 3: Interactions of the Dpo4 little finger and thumb domains with template/primer DNA in extension and post-extension [AF]G-modified Dpo4 ternary complexes. The backbone DNA phosphate groups in contact with Dpo4 are labeled in color and are shown by CPK spheres. The template strand is colored in cyan, the primer strand is in green. (a) The [AF]G•A-1 extension complex has a ‘ternary complex-like’ Dpo4/DNA interaction pattern. The little finger domain contacts the phosphate groups of template C5-A9 and of primer A7-G9 positions (top panel). The thumb domain contacts the phosphates of the template A12 and primer A12-G13 positions (bottom panel). The DNA segment, containing template C5-A16, primer T4-A14 bases and dGTP, is shown. (b) Molecule 1 of the [AF]G•C-1 complex with ‘correct’ template/primer-dNTP pairing alignment has a ‘binary complex-like’ Dpo4/DNA contact pattern. The little finger domain contacts the phosphate groups of template C5-A9 and primer A7-G9 positions (top panel), the thumb domain is translocated compared to the position observed in the [AF]G•A-1 complex and contacts the phosphate groups of the template C11 and primer G13-C14 positions (bottom panel). The DNA segment, containing template C5-A16, primer T4-C14 bases and dGTP is shown. (c) Molecule 2 of the [AF]G•C-1 complex with ‘mutagenic’ template/primer-dNTP pairing alignment has a ‘ternary complex-like’ Dpo4/DNA contact pattern. The little finger domain is translocated relative to the position observed in molecule 1; it contacts the phosphate groups of template [AF]G6-T8 (the C5 base is looped out and the phosphate of A4 is disordered) and primer T8-G10 positions (top panel), and the thumb domain interacts with the phosphate groups of the template C11 and primer G13-C14 positions (bottom panel). The DNA segment containing template A4-A16, primer T4-C14 bases and dGTP is shown. (d) The [AF]G•C-2 post-extension complex has a ‘ternary complex-like’ Dpo4/DNA contact pattern. The little finger domain contacts the phosphate groups of template A4-T8 and primer T8-G10 positions (top panel), and the thumb domain interacts with the phosphate groups of the template C11 and primer G13-C14 positions (bottom panel). The DNA segment containing template A4-A16, primer T4-C14 bases and dGTP is shown. The [AF]G•A-2 post-extension complex has a similar Dpo4/DNA interaction pattern to that of [AF]G•C-2.
Mentions: The [AF]G•A-1 complex has a pattern of Dpo4 interactions with DNA backbone phosphates similar to the one in an unmodified ternary complex48-49 (Fig. 3a, top and bottom). In molecule 1 of [AF]G•C-1, the little finger domain contacts the same residues as in [AF]G•A-1 (Fig. 3b, top), while the thumb domain is shifted towards the active site (see arrow, Fig. 3b, bottom and stereo view of translocation at the full structure level in Supplementary Fig. 2a, as well as thumb domain level in Supplementary Fig. 2b). The pattern of Dpo4 interactions with DNA in molecule 1 of the [AF]G•C-1 ternary complex resembles that of an unmodified binary complex (without dNTP)49. In ‘mutagenic’ molecule 2 of [AF]G•C-1, the little finger domain has translocated from the position observed in molecule 1 (see arrow, Fig. 3c, top), while the thumb domain maintains its contacts as in molecule 1 (Fig. 3c, bottom and Supplementary Fig. 2d,e). The Dpo4-DNA interaction pattern in molecule 2 corresponds to that of a ternary complex.

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