SPR imaging for label-free multiplexed analyses of DNA N-glycosylase interactions with damaged DNA duplexes.
Base excision repair (BER) is the major mechanism for the correction of damaged nucleobases resulting from the alkylation and oxidation of DNA. The first step in the BER pathway consists of excision of the abnormal base by several specific DNA N-glycosylases. A decrease in BER activity was found to be related to an increased risk of carcinogenesis and aging. To investigate BER activities we set up a new device for DNA repair analysis based on surface plasmon resonance imaging (SPRi). Oligonucleotides bearing an abnormal nucleoside, namely 8-oxo-7,8-dihydro-2'-deoxyguanosine and (5'S)-5',8-cyclopurine-2'-deoxynucleoside, 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 N-glycosylase/AP-lyase activity as well as the binding of repair proteins to DNA damage without cleavage activity. Thus, the Fapy DNA N-glycosylase (Fpg) protein is shown as expected to bind and then cleave its natural substrate, namely 8-oxo-7,8-dihydro-guanine, together with the resulting abasic site. Using the current SPR imaging-based DNA array we observed an original binding activity of Fpg towards the (5'S)-5',8-cyclodAdenosine residue. These results altogether show that SPR imaging may be used to simultaneously and specifically detect recognition and excision of several damaged DNA nucleobases, and constitutes an interesting technique to screen inhibitors of DNA repair proteins.
Corne
Christelle
C
Laboratoire des Lésions des Acides Nucléiques, SCIB/UMR E3 CEA-UJF, INAC, CEA-Grenoble, F-38054 Grenoble Cedex 9, France.
Fiche
Jean-Bernard
JB
Gasparutto
Didier
D
Cunin
Valérie
V
Suraniti
Emmanuel
E
Buhot
Arnaud
A
Fuchs
Julia
J
Calemczuk
Roberto
R
Livache
Thierry
T
Favier
Alain
A
D000818:Animals; D020137:Base Pair Mismatch; D004249:DNA Damage; D045647:DNA Glycosylases / Q000378:metabolism*; D004260:DNA Repair; D045643:DNA Repair Enzymes; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D020349:Surface Plasmon Resonance
D045647:DNA Glycosylases; D045643:DNA Repair Enzymes