title: Measurement of translesion synthesis by fluorescent capillary electrophoresis: 7,8-dihydro-8-oxodeoxyguanosine bypass modulation by natural products.
abstract: Translesion synthesis (TLS) relies on a series of specialized DNA polymerases able to insert a base either correctly or incorrectly opposite a lesion on a DNA template strand during replication or post-repair synthesis. To measure the correct or mutagenic outcome of 7,8-dihydro-8-oxodeoxyguanosine (8-oxodG) bypass by TLS DNA polymerases, a capillary electrophoresis (CE) method with fluorescent label has been developed. Two oligonucleotides were designed and hybridized: (i) a 72-mer oligonucleotide framing one 8-oxodG at position 40 and (ii) the 39-mer oligonucleotide complementary to the first strand from the 3' end to the lesion and labeled at the 5' end with a fluorochrome. After incubation with FHs 74 Int human intestinal epithelial cell nuclear proteins, in the presence of either deoxyadenosine triphosphate (dATP) or deoxycytidine triphosphate (dCTP), and denaturation, the resulting elongated oligomers were analyzed by fluorescent capillary electrophoresis. This primer extension assay was then validated in terms of linearity (linear range=0.5-2.5 nM), detectability (limits of detection and quantification=0.023 and 0.091 nM, respectively), and precision (total precisions=8.1% and 3.7% for dATP and dCTP, respectively, n=9). The addition of some natural phytochemicals to the reaction mix significantly influences the outcome of TLS either in an error-free way or in a mutagenic way.
authors.affiliation: Laboratory of Therapeutic Chemistry and Pharmacognosy, Research Institute for Health Sciences and Technology, Université de Mons (UMONS), 7000 Mons, Belgium. amandine.nachtergael@umons.ac.be
authors.forename: Amandine
authors.initials: A
authors.lastname: Nachtergael
authors.forename: Catherine
authors.initials: C
authors.lastname: Charles
authors.forename: Martin
authors.initials: M
authors.lastname: Spanoghe
authors.forename: Martine
authors.initials: M
authors.lastname: Gadenne
authors.forename: Alexandra
authors.initials: A
authors.lastname: Belayew
authors.forename: Pierre
authors.initials: P
authors.lastname: Duez
mesh_terms: D000080242:8-Hydroxy-2'-Deoxyguanosine; D001688:Biological Products / Q000032:analysis*; D018929:Cell Culture Techniques; D017931:DNA Primers / Q000032:analysis*; D004261:DNA Replication / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000032:analysis; D019075:Electrophoresis, Capillary / Q000379:methods*; D005453:Fluorescence; D006801:Humans; D009153:Mutagens
chemical_list: D001688:Biological Products; D017931:DNA Primers; D009153:Mutagens; D000080242:8-Hydroxy-2'-Deoxyguanosine; D004259:DNA-Directed DNA Polymerase; D003849:Deoxyguanosine
keywords: 8-oxodG; Capillary electrophoresis; Genistein; Indole-3-carbinol; Myricetin; Translesion synthesis