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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 ofthe ternary HNE-dGuo-modified template-primer IV complexwith the S. solfataricus P2 DNA polymerase Dpo4 andincoming dATP. (A) Electron density at the active site. (B) Watson–Crickbase pair between HNE-dGuo and 3′-primer terminus dCyd. (C)Watson–Crick base pair between the 5′-template neighborT and the incoming dATP. (D) Active site with the modified template:primerand the dATP along with the polymerase. The Dpo4 polymerase is coloredgray and shown in cartoon form. All electron densities are from (2Fo – Fc) mapsat the 1σ level.
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fig6: Structure ofthe ternary HNE-dGuo-modified template-primer IV complexwith the S. solfataricus P2 DNA polymerase Dpo4 andincoming dATP. (A) Electron density at the active site. (B) Watson–Crickbase pair between HNE-dGuo and 3′-primer terminus dCyd. (C)Watson–Crick base pair between the 5′-template neighborT and the incoming dATP. (D) Active site with the modified template:primerand the dATP along with the polymerase. The Dpo4 polymerase is coloredgray and shown in cartoon form. All electron densities are from (2Fo – Fc) mapsat the 1σ level.

Mentions: The structure with incoming dATP for the ternarycomplex with the5′-TXG-3′ template in which theadduct was paired with dCyd (IV) was determined at 2.9 Å resolution(Table 2). The catalytic core is shown withthe electron density map for the adducted and neighboring region (Figure 6). The Fo – Fc omit map calculated at 2σ is providedin Figure S3 in the Supporting Information. In this case, the HNE moiety also underwent rearrangement to aring open conformation. The ring-opening exposed the Watson–Crickface of the adducted base toward the dCyd of the primer (Scheme 2B). Watson–Crick hydrogen bonding was conservedfor both neighboring base pairs (Figure 6C).The distance between the primer 3-OH and the incoming α- phosphateof the dATP was 5.3 Å. The Ca2+ binding sites weresimilar to the corresponding structure of template-primer complexIII with incoming dGTP.


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 ofthe ternary HNE-dGuo-modified template-primer IV complexwith the S. solfataricus P2 DNA polymerase Dpo4 andincoming dATP. (A) Electron density at the active site. (B) Watson–Crickbase pair between HNE-dGuo and 3′-primer terminus dCyd. (C)Watson–Crick base pair between the 5′-template neighborT and the incoming dATP. (D) Active site with the modified template:primerand the dATP along with the polymerase. The Dpo4 polymerase is coloredgray and shown in cartoon form. All electron densities are from (2Fo – Fc) mapsat the 1σ level.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig6: Structure ofthe ternary HNE-dGuo-modified template-primer IV complexwith the S. solfataricus P2 DNA polymerase Dpo4 andincoming dATP. (A) Electron density at the active site. (B) Watson–Crickbase pair between HNE-dGuo and 3′-primer terminus dCyd. (C)Watson–Crick base pair between the 5′-template neighborT and the incoming dATP. (D) Active site with the modified template:primerand the dATP along with the polymerase. The Dpo4 polymerase is coloredgray and shown in cartoon form. All electron densities are from (2Fo – Fc) mapsat the 1σ level.
Mentions: The structure with incoming dATP for the ternarycomplex with the5′-TXG-3′ template in which theadduct was paired with dCyd (IV) was determined at 2.9 Å resolution(Table 2). The catalytic core is shown withthe electron density map for the adducted and neighboring region (Figure 6). The Fo – Fc omit map calculated at 2σ is providedin Figure S3 in the Supporting Information. In this case, the HNE moiety also underwent rearrangement to aring open conformation. The ring-opening exposed the Watson–Crickface of the adducted base toward the dCyd of the primer (Scheme 2B). Watson–Crick hydrogen bonding was conservedfor both neighboring base pairs (Figure 6C).The distance between the primer 3-OH and the incoming α- phosphateof the dATP was 5.3 Å. The Ca2+ binding sites weresimilar to the corresponding structure of template-primer complexIII with incoming dGTP.

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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