A microarray to measure repair of damaged plasmids by cell lysates.

DNA repair mechanisms constitute major defences against agents that cause cancer, degenerative disease and aging. Different repair systems cooperate to maintain the integrity of genetic information. Investigations of DNA repair involvement in human pathology require an efficient tool that takes into account the variety and complexity of repair systems. We have developed a highly sensitive damaged plasmid microarray to quantify cell lysate excision/synthesis (ES) capacities using small amounts of proteins. This microsystem is based on efficient immobilization and conservation on hydrogel coated glass slides of plasmid DNA damaged with a panel of genotoxic agents. Fluorescent signals are generated from incorporation of labelled dNTPs by DNA excision-repair synthesis mechanisms at plasmid sites. Highly precise DNA repair phenotypes i.e. simultaneous quantitative measures of ES capacities toward seven lesions repaired by distinct repair pathways, are obtained. Applied to the characterization of xeroderma pigmentosum (XP) cells at basal level and in response to a low dose of UVB irradiation, the assay showed the multifunctional role of different XP proteins in cell protection against all types of damage. On the other hand, measurement of the ES of peripheral blood mononuclear cells from six donors revealed significant diversity between individuals. Our results illustrate the power of such a parallelized approach with high potential for several applications including the discovery of new cancer biomarkers and the screening of chemical agents modulating DNA repair systems.

Pulling the chromatin.

Nucleosome is the basic subunit of the chromatin, which organizes the genomic DNA within the cell nucleus. It was understood in the last decade that beside the DNA compaction it plays an important role in the regulation of the gene expression. In its intact form, the nucleosome represents an important mechanical barrier and, among others, it prevents access to the DNA and blocks the transcription elongation. Therefore, it has become important to know the forces and energies necessary to destabilize the nucleosome in order to understand the DNA-related processes. Stretching the chromatin fibre using micromanipulation techniques (e.g. optical tweezers) is an ideal approach to study the nucleosomal stability and the parameters that can modify it. In this short review we will discuss the existing data and potential difficulties that this state-of-the-art technique still has to overcome.