Ingenierie de l'ADN pour l'étude des lésions
Didier Gasparutto, Jean Breton

DNA is the target of several endogenous and exogenous processes that give rise to a large set of DNA lesions. These include base modifications, base losses, cross-links and strand-breaks. Such genetic alterations if not repaired are able to evolve in lethal and mutagenic events during DNA replication and transcription steps. To determine the toxicity and the reparability of the different DNA lesions, we have developed a wide panel of tools consisting in DNA fragments (oligonucleotides) that contain a selective lesion at a defined place. Then such modified DNA probes are used in several in vitro repair, replication and ligand-fishing assays to evaluate the biological consequences of the targeted DNA lesions.

 

Modified oligodeoxyribonucleotides (ODNs) are powerful tools to assess the biological significance of lesions to DNA. For this purpose, we have developed synthetic pathways for the site-specific insertion of several modified bases into DNA fragments. Thus, the chemical solid-phase synthesis of ODNs using original strategies of protection and mild conditions of deprotection, as well as a specific post-modification approach of an unique nucleoside residue within the sequence have been applied. These two approaches of preparation allowed us to have access to a set of modified DNA probes that contain a single modified nucleoside. The resulting DNA substrates are used to investigate recognition and removal of the lesions by DNA repair enzymes. In addition the DNA polymerase-mediated nucleotide incorporation opposite the damage was determined using such modified ODNs as templates.

 

 

A significant example is shown in Figure 1. An intrastrand cross-link lesion, in which two neighbouring nucleobases are covalently tethered, has been site-specifically synthesized into defined sequences in order to perform in vitro replication studies using either 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 ODNs. The lesion-containing modified ODNs were used as DNA templates in primer extension reactions catalyzed by several DNA polymerases. 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-CH2thymine lesion. These results are strongly suggestive that the present intrastrand cross-link lesion, if not repaired, would constitute a blocking lesion for DNA polymerases, being likely lethal for the cell.

 

More recently, to investigate the reparability of DNA alterations we set up new devices based on bio-activable Fluorescence substrates (FRET probes) or surface plasmon resonance imaging (SPRi). In the SPRi-based approach, ODNs bearing a base lesion were grafted by a pyrrole electro-copolymerization process on a glass prism coated with a gold layer. The latter label-free DNA sensor chip permits the detection of binding as well as cleavage activities of proteins to DNA damage. Using the current SPR imaging-based DNA array we observed original binding and excision activities of several DNA repair proteins towards a set of oxidized or alkylated nucleobases. These results show that SPR imaging constitutes a powerful technique to screen modulators of DNA repair and to identify new cellular DNA lesion sensors. This latter concept has been validated by detecting interactions between HMGB proteins with DNA damage generated by platined anticancer drugs.

 

Selected publication(s)  S. Bellon et al. , Org. Biomol. Chem. 4 (2006) 3831-3837.   M. D'Abbadie et al. Nature Biotechnology  25 (2007) 939-943.  C. Corne et al.  The Analyst 113 (2008) 1036-1045.  D. Gasparutto et al.  BBA 1790 (2009) 16-24.

 

Maj : 20/07/2012 (555)

 

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