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Determination of DNA guanine sites forming photo-adducts with Ru(II)-labeled oligonucleotides; DNA polymerase inhibition by the resulting photo-crosslinking

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Abstract

The influence of the distance between the anchoring site of the tethered [Ru(TAP)2dip]2+ complex (TAP=1,4,5,8-tetraazaphenanthrene; dip=4,7-diphenyl-1,10-phenanthroline) on a probe sequence and the guanines of the complementary target strand was studied by (1) the luminescence quenching of the complex (by electron transfer) and (2) the oligodeoxyribonucleotide adduct (ODN adduct) formation which results in photo-crosslinking of the two strands. Moving the guanine moieties away from the complex induces an important decrease of the efficiency of both processes, but clearly affects the ODN adduct formation more specifically than the quenching process. From these results, we determined the positions of the guanine bases in the duplex ODN that are able to form a photo-adduct with the tethered complex. We also examined the possible competition between a long-range hole migration in the duplex ODN and the formation of a photo-adduct by using a sequence labeled with the complex at the 5′-phosphate end. Such a hole migration appears to be inefficient as compared to the ODN adduct formation. Finally, we studied the influence of the photo-crosslinking on the function of two different DNA polymerases. A 17-mer Ru(II)-labeled ODN was hybridized to its complementary sequence located on the 5′-side of a 40-mer matrix. After illumination, the elongation of a 13-mer DNA primer hybridized to the 3′-extremity of the same matrix was stopped at a position corresponding to the formation of the ODN adduct.

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Acknowledgements

The authors wish to thank the Centre National de la Recherche Scientifique (CNRS, France), the Fonds National de la Recherche Scientifique (FNRS, Belgium), the E.U. program (TMR) and the Communauté française de Belgique “Action de recherche concertée” (ARC) for financial support. O.L. thanks the “Fonds pour la formation à la recherche dans l’industrie et dans l’agriculture” (FRIA) for a fellowship. D.G.-F. is grateful to the Complutense University of Madrid (Spain) for a post-doctoral grant. The authors wish to thank E. Loire for technical support. The COST D14 is gratefully acknowledged.

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Author notes

  1. Stephan Schumm

    Present address: Unilever R&D Colworth, Colworth House, Sharnbrook, Bedford, MK44 1LQ, UK

  2. David García-Fresnadillo

    Present address: Organic Chemistry, Universidad Complutense de Madrid, Avenida Complutense s/n, 28040 , Madrid, Spain

Authors and Affiliations

  1. Organic Chemistry and Photochemistry, Laboratoire Européen Associé Ingénierie Biomoléculaire, Université Libre de Bruxelles, 50 Avenue F.D. Roosevelt, CP 160/08, 1050, Brussels, Belgium

    Olivier Lentzen, Stephan Schumm, David García-Fresnadillo, Cécile Moucheron & Andrée Kirsch-De Mesmaeker

  2. Laboratoire Européen Associé Ingénierie Biomoléculaire, LEDSS, UMR CNRS 5616, Université Joseph Fourier, BP 53, 38041 Cedex 9, Grenoble, France

    Eric Defrancq, Jean-François Constant & Pascal Dumy

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  1. Olivier Lentzen
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  2. Eric Defrancq
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Corresponding authors

Correspondence to Pascal Dumy or Andrée Kirsch-De Mesmaeker.

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Lentzen, O., Defrancq, E., Constant, JF. et al. Determination of DNA guanine sites forming photo-adducts with Ru(II)-labeled oligonucleotides; DNA polymerase inhibition by the resulting photo-crosslinking. J Biol Inorg Chem 9, 100–108 (2004). https://doi.org/10.1007/s00775-003-0502-3

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  • DOI: https://doi.org/10.1007/s00775-003-0502-3

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