Fluorogenic DNA ligase and base excision repair enzyme assays using substrates labeled with single fluorophores.
Continuing our work on fluorogenic substrates labeled with single fluorophores for nucleic acid modifying enzymes, here we describe the development of such substrates for DNA ligases and some base excision repair enzymes. These substrates are hairpin-type synthetic DNA molecules with a single fluorophore located on a base close to the 3' ends, an arrangement that results in strong fluorescence quenching. When such substrates are subjected to an enzymatic reaction, the position of the dyes relative to that end of the molecules is altered, resulting in significant fluorescence intensity changes. The ligase substrates described here were 5' phosphorylated and either blunt-ended or carrying short, self-complementary single-stranded 5' extensions. The ligation reactions resulted in the covalent joining of the ends of the molecules, decreasing the quenching effect of the terminal bases on the dyes. To generate fluorogenic substrates for the base excision repair enzymes formamido-pyrimidine-DNA glycosylase (FPG), human 8-oxo-G DNA glycosylase/AP lyase (hOGG1), endonuclease IV (EndoIV), and apurinic/apyrimidinic endonuclease (APE1), we introduced abasic sites or a modified nucleotide, 8-oxo-dG, at such positions that their enzymatic excision would result in the release of a short fluorescent fragment. This was also accompanied by strong fluorescence increases. Overall fluorescence changes ranged from approximately 4-fold (ligase reactions) to more than 20-fold (base excision repair reactions).
Thermo Fisher Scientific, Carlsbad, CA 92008, USA. Electronic address: theo.nikiforov@thermofisher.com.
Theo T
TT
Nikiforov
Thermo Fisher Scientific, Carlsbad, CA 92008, USA.
Steven
S
Roman
D000080242:8-Hydroxy-2'-Deoxyguanosine; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011088:DNA Ligases / Q000378:metabolism*; D004260:DNA Repair; D045643:DNA Repair Enzymes / Q000378:metabolism*; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000378:metabolism; D057075:Enzyme Assays / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans
D005456:Fluorescent Dyes; D000080242:8-Hydroxy-2'-Deoxyguanosine; D004247:DNA; D011088:DNA Ligases; D045643:DNA Repair Enzymes; D003849:Deoxyguanosine
Base excision repair enzymes; DNA ligase assay; Fluorescence quenching; Fluorogenic assay