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CHEMBIORAD Working Groups

Base and sugar radical reactivity in nucleic acids (WG3)

WG Leader: Prof. Thomas Carell; e-mail: Thomas.Carell@cup.uni-muenchen.de

This WG deals with the radical reactivity involving the base and sugar units of nucleic acids. This subject needs a comprehensive and multidisciplinary investigation. The Action will strengthen the collaboration among groups of diverse research areas, in order to realize the optimal competence mix and contribute to solve some key radical initiated DNA damage associated problems. The investigations and the activity of this WG are closely related to other radical pathways studied in the Action, such as the transformation of membrane lipids, proteins and the formation of small radical species.

The groups involved in WG3 are:

  1. Thomas Carell (WG leader) - LMU Munich (Germany)
  2. Jean Cadet - CEA-Grenoble (France)
  3. Luciano Cellai - IC-CNR, Roma (Italy)
  4. Thanasis Gimisis - University of Athens (Greece)
  5. Miguel Miranda - Universidad Politecnica de Valencia (Spain)
  6. Peter O’Neill - University of Oxford (UK)
  7. Tomris Ozben - Akdeniz University, Antalya (Turkey)
  8. Marc Roberts - University of Paris 7 (France)
  9. Maria Davidkova - Nuclear Physics Institute AS (Czech Republic)
  10. Marian Wolszczak - Technical University of Lodz (Poland)
  11. Caroline Stévigny - Université Libre de Bruxelles (Belgium)

The main subjects for collaborative research are:

Base and sugar radical reactivity in nucleic acids:
  • Methods for identification and quantification of damages in vivo and in vitro in cellular DNA and if possible human fluids such as blood.
  • Structural features of nucleic acids (tautomerisation) for the generation of radical centres.
  • Design and study of chemically prepared nucleic acid modifications to decipher radical based damage and repair mechanisms.
  • Synthesis and investigation of tandem damages involving nucleic acids with lipids and proteins.
  • Charge movement through DNA to study the preferred sites for damage formation.

Tasks will investigate how the biomolecule DNA mediates damaging and repair processes (radical reactivity modulation), and the biochemical and biological consequences of radical induced DNA lesions (radical based damages).

Radical Reactivity Modulation:
Effects of the DNA macromolecular structure on electron transfer processes and generation of radical centres. Activities based on the generation of radical species through transition metal complexes, acting as oxidant or photooxidant, depending on the complex composition. Changes of the nucleic acid structures.

Radical Based Damages:
Preparation of DNA substrates with lesion modifications in a defined sequence context and use of such DNA for the investigation of cellular and biochemical aspects of DNA damage. Effect of radical-based DNA damages on mutagenesis, cancer, and aging through investigation of the mechanisms of DNA repair, using enzymatic and chemical systems. Optimisation and unification of analytical methods for lesion detection in vitro and in vivo.

Some details of the WG3 tasks are given below:

Radical Reactivity Modulation:
  • Synthesis of site-specific modified oligonucleotides with electron injectors and acceptors for biophysical studies.
  • Mechanistic investigation of one-electron reduction processes. Phenomena of electron-hole and excess-electron transfer and effects of the macromolecular structure.
  • Tautomerisation of nucleobases and role in the reactivity of nucleic acid structures.
  • Fate of radical species followed by time-resolved spectroscopy techniques (pulse radiolysis, laser flash photolysis).

Radical Based Damages:
  • Synthesis of DNA lesions and precursors (cyclonucleosides, 8-bromopurines, etc.). Insertion of these building blocks into defined DNA sequences for structure-reactivity studies and for NMR and X-ray measurements.
  • Primer extension studies using various high and low fidelity polymerases using the DNA described above.
  • Mechanistic studies on different classes of oxidatively generated nucleic acid damage formed by OH radical, Fenton-type reactions, one-electron oxidation, or peroxynitrite. Computational studies for envisaging radical degradation pathways.
  • Development of assays aimed at monitoring oxidatively generated base and sugar damages.
  • Assessment of the reparability of oxidatively damaged DNA via nucleotide excision repair, base excision repair and single strand break repair using purified proteins, protein complexes or cell extracts.
  • Assessment of mutagenic features of oxidatively damaged DNA. This will be achieved using various biochemical assays such as the shuttle vector methods that are based on the transfection of cells with plasmids in which a dedicated modification has been site-specifically inserted.

Collaboration with other WGs:

There will be activities in collaboration with other WGs in particular for the following subjects:
- experiments in liposomes for the study of nucleoside-lipid cross-linking products in collaboration with WG1;
- interaction with WG2 for the formation of tandem protein-nucleobase damages;
- interaction with the WG4, for the application of DNA assays to the evaluation of the cell damage and repair pathways initiated by biologically relevant small radicals;
- evaluation of apoptosis signalling in collaboration with all WGs and biological research units.