A microarray to measure repair of damaged plasmids by cell lysates

Abstract

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.

PARP inhibition and the radiosensitizing effects of the PARP inhibitor ABT-888 in in vitrohepatocellular carcinoma models

“…Cell nuclear extracts were prepared as previously described [23]. Protein content was in the 1–3 mg/mL range.…”

Effects of micronutrients on DNA repair

“…The alternative to the challenge assay, that is, the in vitro repair assay, takes various forms; the substrate can be an oligonucleotide or plasmid engineered with a specific altered base in the sequence, repair activity being indicated by either nicking of the DNA or incorporation of labelled nucleotide into a repair patch [12][13][14][15]. Alternatively, the comet assay can be used, in which case the substrate is in the form of agarose-embedded nucleoids derived by lysis of cells treated with a specific DNA-damaging agent [16][17][18][19].…”

Preparation of bacteria microarray using selective patterning of polyelectrolyte multilayer and poly(ethylene glycol)-poly(lactide) diblock copolymer

“…This procedure was repeated until the desired number of polyelectrolyte layers was assembled with a positively charged PAH on the outermost layer because negatively charged proteins bind well on positively charged polymeric surface owing to the strong electrostatic interaction force. [5][6][7][8][9][10][11][12][13][14][15][16][17] The Preparation of Functional Surface Using the MIMIC Method. PDMS (Sylgard 184, Dow Corning, USA) molds having micro or nanostructures were fabricated against a complementary relief structure that was prepared by conventional photolithography.…”

“…[3][4][5][6] In contrast to conventional DNA, peptide and protein microarrys based on molecular binding event, cell based microarrays provides essential information of the biological activities of samples rather than the identity of them because global cellular responses including expression of gene and protein, viability, bioavailability, and toxicity can be assessed. [7][8][9] Recently, bacterial microarrays have been used in microbial genetics, microbial ecology, biomedical study, and environmental monitoring because of their low cost, fast growth, rich genetic information and alteration, easy handling, and reliable sensitivity to a wide variety of environmental stimuli. [10][11][12][13] Thus, the technique for efficient immobilization of bacteria is essential for the achievement of specific bacterial microarrays.…”