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Replication bypass of the trans-4-Hydroxynonenal-derived (6S,8R,11S)-1,N(2)-deoxyguanosine DNA adduct by the sulfolobus solfataricus DNA polymerase IV.

Banerjee S, Christov PP, Kozekova A, Rizzo CJ, Egli M, Stone MP - Chem. Res. Toxicol. (2012)

Bottom Line: The incoming dNTP, either dGTP or dATP, was positioned with Watson-Crick pairing opposite the template 5'-neighbor base, dCyt or dThy, respectively.In contrast, for the 18-mer:14-mer template-primers with a primed (6S,8R,11S)-HNE-1,N(2)-dGuo:dCyd pair, ring opening of the adduct to the corresponding N(2)-dGuo aldehyde species occurred.This allowed Watson-Crick base pairing at the (6S,8R,11S)-HNE-1,N(2)-dGuo:dCyd pair.

View Article: PubMed Central - PubMed

Affiliation: Departments of Chemistry and Biochemistry, Center in Molecular Toxicology, Vanderbilt Institute of Chemical Biology and the Vanderbilt-Ingram Cancer Center, Vanderbilt University , Nashville, Tennessee 37235, United States.

ABSTRACT
trans-4-Hydroxynonenal (HNE) is the major peroxidation product of ω-6 polyunsaturated fatty acids in vivo. Michael addition of the N(2)-amino group of dGuo to HNE followed by ring closure of N1 onto the aldehyde results in four diastereomeric 1,N(2)-dGuo (1,N(2)-HNE-dGuo) adducts. The (6S,8R,11S)-HNE-1,N(2)-dGuo adduct was incorporated into the 18-mer templates 5'-d(TCATXGAATCCTTCCCCC)-3' and d(TCACXGAATCCTTCCCCC)-3', where X = (6S,8R,11S)-HNE-1,N(2)-dGuo adduct. These differed in the identity of the template 5'-neighbor base, which was either Thy or Cyt, respectively. Each of these templates was annealed with either a 13-mer primer 5'-d(GGGGGAAGGATTC)-3' or a 14-mer primer 5'-d(GGGGGAAGGATTCC)-3'. The addition of dNTPs to the 13-mer primer allowed analysis of dNTP insertion opposite to the (6S,8R,11S)-HNE-1,N(2)-dGuo adduct, whereas the 14-mer primer allowed analysis of dNTP extension past a primed (6S,8R,11S)-HNE-1,N(2)-dGuo:dCyd pair. The Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) belongs to the Y-family of error-prone polymerases. Replication bypass studies in vitro reveal that this polymerase inserted dNTPs opposite the (6S,8R,11S)-HNE-1,N(2)-dGuo adduct in a sequence-specific manner. If the template 5'-neighbor base was dCyt, the polymerase inserted primarily dGTP, whereas if the template 5'-neighbor base was dThy, the polymerase inserted primarily dATP. The latter event would predict low levels of Gua → Thy mutations during replication bypass when the template 5'-neighbor base is dThy. When presented with a primed (6S,8R,11S)-HNE-1,N(2)-dGuo:dCyd pair, the polymerase conducted full-length primer extension. Structures for ternary (Dpo4-DNA-dNTP) complexes with all four template-primers were obtained. For the 18-mer:13-mer template-primers in which the polymerase was confronted with the (6S,8R,11S)-HNE-1,N(2)-dGuo adduct, the (6S,8R,11S)-1,N(2)-dGuo lesion remained in the ring-closed conformation at the active site. The incoming dNTP, either dGTP or dATP, was positioned with Watson-Crick pairing opposite the template 5'-neighbor base, dCyt or dThy, respectively. In contrast, for the 18-mer:14-mer template-primers with a primed (6S,8R,11S)-HNE-1,N(2)-dGuo:dCyd pair, ring opening of the adduct to the corresponding N(2)-dGuo aldehyde species occurred. This allowed Watson-Crick base pairing at the (6S,8R,11S)-HNE-1,N(2)-dGuo:dCyd pair.

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Structure of the ternaryHNE-dGuo modified template-primer II complexwith the S. solfataricus P2 DNA polymerase Dpo4 andincoming dATP. (A) Electron density at the active site. (B) Watson–Crickbase pair between the 5′-template neighbor T and incoming dATP.(C) Active site with the modified template:primer and the dATP alongwith the polymerase. The Dpo4 polymerase is colored gray and shownin cartoon form. All electron densities are from (2Fo – Fc) maps at the1σ level. The HNE alkyl chain is disordered, resulting in someuncertainty in its position.
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fig4: Structure of the ternaryHNE-dGuo modified template-primer II complexwith the S. solfataricus P2 DNA polymerase Dpo4 andincoming dATP. (A) Electron density at the active site. (B) Watson–Crickbase pair between the 5′-template neighbor T and incoming dATP.(C) Active site with the modified template:primer and the dATP alongwith the polymerase. The Dpo4 polymerase is colored gray and shownin cartoon form. All electron densities are from (2Fo – Fc) maps at the1σ level. The HNE alkyl chain is disordered, resulting in someuncertainty in its position.

Mentions: The structure of the ternary Dpo4-DNA-dATP complexwith 5′-TXG-3′ template pairedwith a −1 primer(II) was determined at 2.4 Å resolution. The catalytic core isshown with electron density for the adducted and neighboring region(Figure 4). Again, the polymerase active siteresembled the “type II” structure with native DNA.47 It accommodated the (6S,8R,11S)-HNE-1,N2-dGuo adduct and its 5′-template dThd neighbor. The 1,N2-dGuo exocyclic ring was inserted into theduplex such that the incoming dATP could not orient in the Watson–Crickplane. The 5′-dThd was positioned to form Watson–Crickhydrogen bonds with the incoming dATP, which resulted in a gap of7.4 Å between the 3′-hydroxyl of the primer dCyd and theα-phosphate of the dATP. This is unlikely to represent a catalyticallycompetent conformation. Of three bound Ca2+ ions, one wasat the active site and 3.5 Å distant from the 3′-terminalhydroxyl of the primer, suggesting that it was positioned to catalyzethe reaction. Again, the electron density for the alkyl side chainof the (6S,8R,11S)-HNE-1,N2-dGuo adduct was weak, butthe side chain was presumably directed into minor groove. The 5′-endof the template was also disordered, and three bases on that sideof the adduct were seen with higher thermal parameters.


Replication bypass of the trans-4-Hydroxynonenal-derived (6S,8R,11S)-1,N(2)-deoxyguanosine DNA adduct by the sulfolobus solfataricus DNA polymerase IV.

Banerjee S, Christov PP, Kozekova A, Rizzo CJ, Egli M, Stone MP - Chem. Res. Toxicol. (2012)

Structure of the ternaryHNE-dGuo modified template-primer II complexwith the S. solfataricus P2 DNA polymerase Dpo4 andincoming dATP. (A) Electron density at the active site. (B) Watson–Crickbase pair between the 5′-template neighbor T and incoming dATP.(C) Active site with the modified template:primer and the dATP alongwith the polymerase. The Dpo4 polymerase is colored gray and shownin cartoon form. All electron densities are from (2Fo – Fc) maps at the1σ level. The HNE alkyl chain is disordered, resulting in someuncertainty in its position.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3285121&req=5

fig4: Structure of the ternaryHNE-dGuo modified template-primer II complexwith the S. solfataricus P2 DNA polymerase Dpo4 andincoming dATP. (A) Electron density at the active site. (B) Watson–Crickbase pair between the 5′-template neighbor T and incoming dATP.(C) Active site with the modified template:primer and the dATP alongwith the polymerase. The Dpo4 polymerase is colored gray and shownin cartoon form. All electron densities are from (2Fo – Fc) maps at the1σ level. The HNE alkyl chain is disordered, resulting in someuncertainty in its position.
Mentions: The structure of the ternary Dpo4-DNA-dATP complexwith 5′-TXG-3′ template pairedwith a −1 primer(II) was determined at 2.4 Å resolution. The catalytic core isshown with electron density for the adducted and neighboring region(Figure 4). Again, the polymerase active siteresembled the “type II” structure with native DNA.47 It accommodated the (6S,8R,11S)-HNE-1,N2-dGuo adduct and its 5′-template dThd neighbor. The 1,N2-dGuo exocyclic ring was inserted into theduplex such that the incoming dATP could not orient in the Watson–Crickplane. The 5′-dThd was positioned to form Watson–Crickhydrogen bonds with the incoming dATP, which resulted in a gap of7.4 Å between the 3′-hydroxyl of the primer dCyd and theα-phosphate of the dATP. This is unlikely to represent a catalyticallycompetent conformation. Of three bound Ca2+ ions, one wasat the active site and 3.5 Å distant from the 3′-terminalhydroxyl of the primer, suggesting that it was positioned to catalyzethe reaction. Again, the electron density for the alkyl side chainof the (6S,8R,11S)-HNE-1,N2-dGuo adduct was weak, butthe side chain was presumably directed into minor groove. The 5′-endof the template was also disordered, and three bases on that sideof the adduct were seen with higher thermal parameters.

Bottom Line: The incoming dNTP, either dGTP or dATP, was positioned with Watson-Crick pairing opposite the template 5'-neighbor base, dCyt or dThy, respectively.In contrast, for the 18-mer:14-mer template-primers with a primed (6S,8R,11S)-HNE-1,N(2)-dGuo:dCyd pair, ring opening of the adduct to the corresponding N(2)-dGuo aldehyde species occurred.This allowed Watson-Crick base pairing at the (6S,8R,11S)-HNE-1,N(2)-dGuo:dCyd pair.

View Article: PubMed Central - PubMed

Affiliation: Departments of Chemistry and Biochemistry, Center in Molecular Toxicology, Vanderbilt Institute of Chemical Biology and the Vanderbilt-Ingram Cancer Center, Vanderbilt University , Nashville, Tennessee 37235, United States.

ABSTRACT
trans-4-Hydroxynonenal (HNE) is the major peroxidation product of ω-6 polyunsaturated fatty acids in vivo. Michael addition of the N(2)-amino group of dGuo to HNE followed by ring closure of N1 onto the aldehyde results in four diastereomeric 1,N(2)-dGuo (1,N(2)-HNE-dGuo) adducts. The (6S,8R,11S)-HNE-1,N(2)-dGuo adduct was incorporated into the 18-mer templates 5'-d(TCATXGAATCCTTCCCCC)-3' and d(TCACXGAATCCTTCCCCC)-3', where X = (6S,8R,11S)-HNE-1,N(2)-dGuo adduct. These differed in the identity of the template 5'-neighbor base, which was either Thy or Cyt, respectively. Each of these templates was annealed with either a 13-mer primer 5'-d(GGGGGAAGGATTC)-3' or a 14-mer primer 5'-d(GGGGGAAGGATTCC)-3'. The addition of dNTPs to the 13-mer primer allowed analysis of dNTP insertion opposite to the (6S,8R,11S)-HNE-1,N(2)-dGuo adduct, whereas the 14-mer primer allowed analysis of dNTP extension past a primed (6S,8R,11S)-HNE-1,N(2)-dGuo:dCyd pair. The Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) belongs to the Y-family of error-prone polymerases. Replication bypass studies in vitro reveal that this polymerase inserted dNTPs opposite the (6S,8R,11S)-HNE-1,N(2)-dGuo adduct in a sequence-specific manner. If the template 5'-neighbor base was dCyt, the polymerase inserted primarily dGTP, whereas if the template 5'-neighbor base was dThy, the polymerase inserted primarily dATP. The latter event would predict low levels of Gua → Thy mutations during replication bypass when the template 5'-neighbor base is dThy. When presented with a primed (6S,8R,11S)-HNE-1,N(2)-dGuo:dCyd pair, the polymerase conducted full-length primer extension. Structures for ternary (Dpo4-DNA-dNTP) complexes with all four template-primers were obtained. For the 18-mer:13-mer template-primers in which the polymerase was confronted with the (6S,8R,11S)-HNE-1,N(2)-dGuo adduct, the (6S,8R,11S)-1,N(2)-dGuo lesion remained in the ring-closed conformation at the active site. The incoming dNTP, either dGTP or dATP, was positioned with Watson-Crick pairing opposite the template 5'-neighbor base, dCyt or dThy, respectively. In contrast, for the 18-mer:14-mer template-primers with a primed (6S,8R,11S)-HNE-1,N(2)-dGuo:dCyd pair, ring opening of the adduct to the corresponding N(2)-dGuo aldehyde species occurred. This allowed Watson-Crick base pairing at the (6S,8R,11S)-HNE-1,N(2)-dGuo:dCyd pair.

Show MeSH
Related in: MedlinePlus