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Site-specific incorporation of N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF) into oligonucleotides using modified 'ultra-mild' DNA synthesis.

Gillet LC, Alzeer J, Schärer OD - Nucleic Acids Res. (2005)

Bottom Line: Key to this endeavor was the development of improved deprotection conditions (10% diisopropylamine in methanol supplemented with 0.25 M of beta-mercaptoethanol) designed to remove protecting groups of commercially available 'ultra-mild' phosphoramidite building blocks without compromising the integrity of the exquisitely base-labile acetyl group at N8 of dG-AAF.We demonstrate the suitability of these oligonucleotides in the nucleotide excision repair reaction.Our synthetic approach should facilitate comprehensive studies of the mechanisms of repair and mutagenesis induced by dG-AAF adducts in DNA and should be of general use for the incorporation of base-labile functionalities into DNA.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Cancer Research, University of Zürich August Forel Strasse 7, 8008 Zürich, Switzerland.

ABSTRACT
Aromatic amino and nitro compounds are potent carcinogens found in the environment that exert their toxic effects by reacting with DNA following metabolic activation. One important adduct is N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF), which has been extensively used in studies of the mechanisms of DNA repair and mutagenesis. Despite the importance of dG-AAF adducts in DNA, an efficient method for its incorporation into DNA using solid-phase synthesis is still missing. We report the development of a modified 'ultra-mild' DNA synthesis protocol that allows the incorporation of dG-AAF into oligonucleotides of any length accessible by solid-phase DNA synthesis with high efficiency and independent of sequence context. Key to this endeavor was the development of improved deprotection conditions (10% diisopropylamine in methanol supplemented with 0.25 M of beta-mercaptoethanol) designed to remove protecting groups of commercially available 'ultra-mild' phosphoramidite building blocks without compromising the integrity of the exquisitely base-labile acetyl group at N8 of dG-AAF. We demonstrate the suitability of these oligonucleotides in the nucleotide excision repair reaction. Our synthetic approach should facilitate comprehensive studies of the mechanisms of repair and mutagenesis induced by dG-AAF adducts in DNA and should be of general use for the incorporation of base-labile functionalities into DNA.

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NER dual incision activity on dG-AAF and dG-AF-containing plasmids. Plasmids containing site-specific dG-AAF or dG-AF residues were incubated with cell extracts prepared from HeLa cells. The 24mer to 32mer excision products containing dG-AAF or dG-AF were detected by annealing to a complementary oligonucleotide with a 5′-GpGpGpG overhang, which served as a template for end-labeling with [α-32P]dCTP with sequenase. The reaction products were resolved on a 14% denaturing polyacrylamide gel. An MspI digest of pBR322 was used as a size marker and the position of the 27 and 35 nt bands are indicated.
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fig4: NER dual incision activity on dG-AAF and dG-AF-containing plasmids. Plasmids containing site-specific dG-AAF or dG-AF residues were incubated with cell extracts prepared from HeLa cells. The 24mer to 32mer excision products containing dG-AAF or dG-AF were detected by annealing to a complementary oligonucleotide with a 5′-GpGpGpG overhang, which served as a template for end-labeling with [α-32P]dCTP with sequenase. The reaction products were resolved on a 14% denaturing polyacrylamide gel. An MspI digest of pBR322 was used as a size marker and the position of the 27 and 35 nt bands are indicated.

Mentions: To demonstrate the usefulness of the dG-AAF- and dG-AF-modified oligonucleotides, we used them as substrates for in vitro NER assays. This assay consists in mixing a plasmid or an oligonucleotide containing a single lesion, with a NER proficient cell extract. The excision reaction (36–38) results in the release in solution of short oligonucleotides (24mer to 32mer) containing the lesion, which can be indirectly detected with an appropriate ‘fill-in’ labeling (see Figure 4 for details). We wished to use, directly from the DNA synthesizer, the 120mer containing the dG-AAF modification as substrate for the NER reaction. Although 120-AAF was devised to meet the size and damage-position requirements to allow the assembly of the NER machinery (51), we were unable to detect any significant signal of repair activity after incubation with HeLa cell extract (data not shown). We do not presently know whether the 120mer was too short or whether an unrelated activity in the cell extract such as a non-specific nuclease or a protein that binds to DNA ends (52–54) prevented the NER reaction from occurring.


Site-specific incorporation of N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF) into oligonucleotides using modified 'ultra-mild' DNA synthesis.

Gillet LC, Alzeer J, Schärer OD - Nucleic Acids Res. (2005)

NER dual incision activity on dG-AAF and dG-AF-containing plasmids. Plasmids containing site-specific dG-AAF or dG-AF residues were incubated with cell extracts prepared from HeLa cells. The 24mer to 32mer excision products containing dG-AAF or dG-AF were detected by annealing to a complementary oligonucleotide with a 5′-GpGpGpG overhang, which served as a template for end-labeling with [α-32P]dCTP with sequenase. The reaction products were resolved on a 14% denaturing polyacrylamide gel. An MspI digest of pBR322 was used as a size marker and the position of the 27 and 35 nt bands are indicated.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1074722&req=5

fig4: NER dual incision activity on dG-AAF and dG-AF-containing plasmids. Plasmids containing site-specific dG-AAF or dG-AF residues were incubated with cell extracts prepared from HeLa cells. The 24mer to 32mer excision products containing dG-AAF or dG-AF were detected by annealing to a complementary oligonucleotide with a 5′-GpGpGpG overhang, which served as a template for end-labeling with [α-32P]dCTP with sequenase. The reaction products were resolved on a 14% denaturing polyacrylamide gel. An MspI digest of pBR322 was used as a size marker and the position of the 27 and 35 nt bands are indicated.
Mentions: To demonstrate the usefulness of the dG-AAF- and dG-AF-modified oligonucleotides, we used them as substrates for in vitro NER assays. This assay consists in mixing a plasmid or an oligonucleotide containing a single lesion, with a NER proficient cell extract. The excision reaction (36–38) results in the release in solution of short oligonucleotides (24mer to 32mer) containing the lesion, which can be indirectly detected with an appropriate ‘fill-in’ labeling (see Figure 4 for details). We wished to use, directly from the DNA synthesizer, the 120mer containing the dG-AAF modification as substrate for the NER reaction. Although 120-AAF was devised to meet the size and damage-position requirements to allow the assembly of the NER machinery (51), we were unable to detect any significant signal of repair activity after incubation with HeLa cell extract (data not shown). We do not presently know whether the 120mer was too short or whether an unrelated activity in the cell extract such as a non-specific nuclease or a protein that binds to DNA ends (52–54) prevented the NER reaction from occurring.

Bottom Line: Key to this endeavor was the development of improved deprotection conditions (10% diisopropylamine in methanol supplemented with 0.25 M of beta-mercaptoethanol) designed to remove protecting groups of commercially available 'ultra-mild' phosphoramidite building blocks without compromising the integrity of the exquisitely base-labile acetyl group at N8 of dG-AAF.We demonstrate the suitability of these oligonucleotides in the nucleotide excision repair reaction.Our synthetic approach should facilitate comprehensive studies of the mechanisms of repair and mutagenesis induced by dG-AAF adducts in DNA and should be of general use for the incorporation of base-labile functionalities into DNA.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Cancer Research, University of Zürich August Forel Strasse 7, 8008 Zürich, Switzerland.

ABSTRACT
Aromatic amino and nitro compounds are potent carcinogens found in the environment that exert their toxic effects by reacting with DNA following metabolic activation. One important adduct is N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF), which has been extensively used in studies of the mechanisms of DNA repair and mutagenesis. Despite the importance of dG-AAF adducts in DNA, an efficient method for its incorporation into DNA using solid-phase synthesis is still missing. We report the development of a modified 'ultra-mild' DNA synthesis protocol that allows the incorporation of dG-AAF into oligonucleotides of any length accessible by solid-phase DNA synthesis with high efficiency and independent of sequence context. Key to this endeavor was the development of improved deprotection conditions (10% diisopropylamine in methanol supplemented with 0.25 M of beta-mercaptoethanol) designed to remove protecting groups of commercially available 'ultra-mild' phosphoramidite building blocks without compromising the integrity of the exquisitely base-labile acetyl group at N8 of dG-AAF. We demonstrate the suitability of these oligonucleotides in the nucleotide excision repair reaction. Our synthetic approach should facilitate comprehensive studies of the mechanisms of repair and mutagenesis induced by dG-AAF adducts in DNA and should be of general use for the incorporation of base-labile functionalities into DNA.

Show MeSH
Related in: MedlinePlus