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Issue 20, 2006
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Guanine–thymine intrastrand cross-linked lesion containing oligonucleotides: from chemical synthesis to in vitro enzymatic replication

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Abstract

An intrastrand cross-link lesion, in which two neighboring nucleobases are covalently tethered, has been site-specifically synthesized into defined sequence oligonucleotides in order to perform in vitro replication studies using either bacterial replicative or translesional synthesis polymerases. The investigated tandem base lesion that involves a cross-link between the methylene group of thymine and the C8 of an adjacent guanine residue has been prepared by UV-photolysis under anaerobic condition of the photolabile precursor 5-(phenylthiomethyl)-2′-deoxyuridine that has been site-specifically incorporated into a 9-mer oligonucleotide. After ligation, the lesion-containing modified oligonucleotide was used as a DNA template in primer extension reactions catalyzed by several DNA polymerases including the fragment Klenow exo-(Kf-) of E. coli polymerase I, the Thermus aquaticus polymerase (Taq pol) and the E. coli translesional DNA polymerase Pol IV (dinB). It was found that the primer extension reaction was stopped after the incorporation of the correct nucleotide dAMP opposite the 3′-thymine residue of guanineC8–CH2 thymine lesion by Kf- and Pol IV; however it was noted that the efficiency of the nucleotide incorporation was reduced. In contrast, the Taq polymerase was totally blocked at the nucleotide preceding the tandem lesion. These results are strongly suggestive that the present intrastrand cross-link lesion, if not repaired, would constitute a blocking lesion for prokaryotic DNA polymerases, being likely lethal for the cell.

Graphical abstract: Guanine–thymine intrastrand cross-linked lesion containing oligonucleotides: from chemical synthesis to in vitro enzymatic replication

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Supplementary files

Article information


Submitted
03 Jul 2006
Accepted
08 Sep 2006
First published
19 Sep 2006

Org. Biomol. Chem., 2006,4, 3831-3837
Article type
Paper

Guaninethymine intrastrand cross-linked lesion containing oligonucleotides: from chemical synthesis to in vitro enzymatic replication

S. Bellon, D. Gasparutto, C. Saint-Pierre and J. Cadet, Org. Biomol. Chem., 2006, 4, 3831
DOI: 10.1039/B609460K

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