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Conformational changes of the phenyl and naphthyl isocyanate-DNA adducts during DNA replication and by minor groove binding molecules.

Nakano S, Uotani Y, Sato Y, Oka H, Fujii M, Sugimoto N - Nucleic Acids Res. (2013)

Bottom Line: Nevertheless, the kinetic analysis shows that these DNA lesions are compatible with DNA ligase and DNA polymerase reactions, as much as natural DNA bases.We suggest that the adduct lesions have a capability of adopting dual conformations, depending on the difference in their interaction energies between stacking of the attached aromatic group and base pairing through hydrogen bonds.The nucleotide derivatives would be useful for enhancing the phenotypic diversity of DNA molecules and for exploring new non-natural nucleotides.

View Article: PubMed Central - PubMed

Affiliation: Department of Nanobiochemistry, Faculty of Frontiers of Innovative Research in Science and Technology (FIRST), Konan University, 7-1-20, Minatojima-minamimachi, Chuo-ku, Kobe, 650-0047, Japan, Frontier Institute for Biomolecular Engineering Research (FIBER), Konan University, 7-1-20, Minatojima-minamimachi, Chuo-ku, Kobe, 650-0047, Japan, Department of Chemistry, Faculty of Science and Engineering, Konan University, 8-9-1, Okamoto, Higashinada-ku, Kobe, 658-8501, Japan, Molecular Engineering Institute (MEI), Kinki University, 11-6 Kayanomori, Iizuka, Fukuoka, 820-8555, Japan and Department of Environmental and Biological Chemistry, Kinki University, 11-6 Kayanomori, Iizuka, Fukuoka, 820-8555, Japan.

ABSTRACT
DNA lesions produced by aromatic isocyanates have an extra bulky group on the nucleotide bases, with the capability of forming stacking interaction within a DNA helix. In this work, we investigated the conformation of the 2'-deoxyadenosine and 2'-deoxycytidine derivatives tethering a phenyl or naphthyl group, introduced in a DNA duplex. The chemical modification experiments using KMnO4 and 1-cyclohexyl-3 -(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate have shown that the 2'-deoxycytidine lesions form the base pair with guanine while the 2'-deoxyadenosine lesions have less ability of forming the base pair with thymine in solution. Nevertheless, the kinetic analysis shows that these DNA lesions are compatible with DNA ligase and DNA polymerase reactions, as much as natural DNA bases. We suggest that the adduct lesions have a capability of adopting dual conformations, depending on the difference in their interaction energies between stacking of the attached aromatic group and base pairing through hydrogen bonds. It is also presented that the attached aromatic groups change their orientation by interacting with the minor groove binding netropsin, distamycin and synthetic polyamide. The nucleotide derivatives would be useful for enhancing the phenotypic diversity of DNA molecules and for exploring new non-natural nucleotides.

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Related in: MedlinePlus

(A) DNA sequences and PAGE for the incorporation of dApheTP opposite N (dA, dG, dC, dT or dF) in the DNA template by the Klenow fragment (0.45 units). The reaction was conducted with 500 µM dApheTP for 2 min. The arrow indicates the extended DNA primer. (B) Kinetic traces for the primer extension incorporating dApheTP (circles) and dATP (triangles) opposite dT (closed symbols) or dF (open symbols) by the Klenow fragment.
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gkt608-F5: (A) DNA sequences and PAGE for the incorporation of dApheTP opposite N (dA, dG, dC, dT or dF) in the DNA template by the Klenow fragment (0.45 units). The reaction was conducted with 500 µM dApheTP for 2 min. The arrow indicates the extended DNA primer. (B) Kinetic traces for the primer extension incorporating dApheTP (circles) and dATP (triangles) opposite dT (closed symbols) or dF (open symbols) by the Klenow fragment.

Mentions: We also investigated the polymerase reactions. It is known that the shapes and sizes are important for the polymerization with non-natural nucleotides (13). We prepared the 5′-O triphosphate of dAphe (dApheTP) by oxidative treatment of its H-phosphonate monoester through silyl phosphite (14–16) (Supplementary Figure S4) and incorporated it into a DNA primer using the Klenow fragment of E. coli DNA polymerase I without 3′→5′ exonuclease activity. This polymerase strictly recognizes a dNTP according to the rules of Watson–Crick base pairing or selects a large hydrophobic base analog opposite an abasic site in the DNA template (17–20). Incorporation of dApheTP opposite an abasic site is expected if the phenyl group of dAphe preferentially adopts the stacking conformation. However, the Klenow fragment incorporated dApheTP opposite thymine, but not opposite the abasic site (Figure 5A). The rate constant for the dApheTP incorporation opposite thymine was lower, but significant, than that for the reaction with dATP (0.77 and 1.9 min−1, respectively). The result suggests that the incoming dApheTP binds to the polymerase and forms the base pair with thymine. As shown in Figure 5B, the incorporation of dApheTP opposite the abasic site was markedly inhibited (<0.001 min−1), whereas the incorporation of dATP was substantial (0.32 min−1); referred to as the A-rule (21,22).Figure 5.


Conformational changes of the phenyl and naphthyl isocyanate-DNA adducts during DNA replication and by minor groove binding molecules.

Nakano S, Uotani Y, Sato Y, Oka H, Fujii M, Sugimoto N - Nucleic Acids Res. (2013)

(A) DNA sequences and PAGE for the incorporation of dApheTP opposite N (dA, dG, dC, dT or dF) in the DNA template by the Klenow fragment (0.45 units). The reaction was conducted with 500 µM dApheTP for 2 min. The arrow indicates the extended DNA primer. (B) Kinetic traces for the primer extension incorporating dApheTP (circles) and dATP (triangles) opposite dT (closed symbols) or dF (open symbols) by the Klenow fragment.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3794578&req=5

gkt608-F5: (A) DNA sequences and PAGE for the incorporation of dApheTP opposite N (dA, dG, dC, dT or dF) in the DNA template by the Klenow fragment (0.45 units). The reaction was conducted with 500 µM dApheTP for 2 min. The arrow indicates the extended DNA primer. (B) Kinetic traces for the primer extension incorporating dApheTP (circles) and dATP (triangles) opposite dT (closed symbols) or dF (open symbols) by the Klenow fragment.
Mentions: We also investigated the polymerase reactions. It is known that the shapes and sizes are important for the polymerization with non-natural nucleotides (13). We prepared the 5′-O triphosphate of dAphe (dApheTP) by oxidative treatment of its H-phosphonate monoester through silyl phosphite (14–16) (Supplementary Figure S4) and incorporated it into a DNA primer using the Klenow fragment of E. coli DNA polymerase I without 3′→5′ exonuclease activity. This polymerase strictly recognizes a dNTP according to the rules of Watson–Crick base pairing or selects a large hydrophobic base analog opposite an abasic site in the DNA template (17–20). Incorporation of dApheTP opposite an abasic site is expected if the phenyl group of dAphe preferentially adopts the stacking conformation. However, the Klenow fragment incorporated dApheTP opposite thymine, but not opposite the abasic site (Figure 5A). The rate constant for the dApheTP incorporation opposite thymine was lower, but significant, than that for the reaction with dATP (0.77 and 1.9 min−1, respectively). The result suggests that the incoming dApheTP binds to the polymerase and forms the base pair with thymine. As shown in Figure 5B, the incorporation of dApheTP opposite the abasic site was markedly inhibited (<0.001 min−1), whereas the incorporation of dATP was substantial (0.32 min−1); referred to as the A-rule (21,22).Figure 5.

Bottom Line: Nevertheless, the kinetic analysis shows that these DNA lesions are compatible with DNA ligase and DNA polymerase reactions, as much as natural DNA bases.We suggest that the adduct lesions have a capability of adopting dual conformations, depending on the difference in their interaction energies between stacking of the attached aromatic group and base pairing through hydrogen bonds.The nucleotide derivatives would be useful for enhancing the phenotypic diversity of DNA molecules and for exploring new non-natural nucleotides.

View Article: PubMed Central - PubMed

Affiliation: Department of Nanobiochemistry, Faculty of Frontiers of Innovative Research in Science and Technology (FIRST), Konan University, 7-1-20, Minatojima-minamimachi, Chuo-ku, Kobe, 650-0047, Japan, Frontier Institute for Biomolecular Engineering Research (FIBER), Konan University, 7-1-20, Minatojima-minamimachi, Chuo-ku, Kobe, 650-0047, Japan, Department of Chemistry, Faculty of Science and Engineering, Konan University, 8-9-1, Okamoto, Higashinada-ku, Kobe, 658-8501, Japan, Molecular Engineering Institute (MEI), Kinki University, 11-6 Kayanomori, Iizuka, Fukuoka, 820-8555, Japan and Department of Environmental and Biological Chemistry, Kinki University, 11-6 Kayanomori, Iizuka, Fukuoka, 820-8555, Japan.

ABSTRACT
DNA lesions produced by aromatic isocyanates have an extra bulky group on the nucleotide bases, with the capability of forming stacking interaction within a DNA helix. In this work, we investigated the conformation of the 2'-deoxyadenosine and 2'-deoxycytidine derivatives tethering a phenyl or naphthyl group, introduced in a DNA duplex. The chemical modification experiments using KMnO4 and 1-cyclohexyl-3 -(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate have shown that the 2'-deoxycytidine lesions form the base pair with guanine while the 2'-deoxyadenosine lesions have less ability of forming the base pair with thymine in solution. Nevertheless, the kinetic analysis shows that these DNA lesions are compatible with DNA ligase and DNA polymerase reactions, as much as natural DNA bases. We suggest that the adduct lesions have a capability of adopting dual conformations, depending on the difference in their interaction energies between stacking of the attached aromatic group and base pairing through hydrogen bonds. It is also presented that the attached aromatic groups change their orientation by interacting with the minor groove binding netropsin, distamycin and synthetic polyamide. The nucleotide derivatives would be useful for enhancing the phenotypic diversity of DNA molecules and for exploring new non-natural nucleotides.

Show MeSH
Related in: MedlinePlus