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.
Millau
J-F
JF
Laboratoire des Lésions des Acides Nucléiques, LCIB (UMR-E 3 CEA-UJF), INAC, CEA Grenoble, 38054, Grenoble cedex 9, France.
Raffin
A-L
AL
Caillat
S
S
Claudet
C
C
Arras
G
G
Ugolin
N
N
Douki
T
T
Ravanat
J-L
JL
Breton
J
J
Oddos
T
T
Dumontet
C
C
Sarasin
A
A
Chevillard
S
S
Favier
A
A
Sauvaigo
S
S
D002461:Cell Line, Transformed; D004260:DNA Repair; D006367:HeLa Cells; D006801:Humans; D010957:Plasmids; D013050:Spectrometry, Fluorescence