title: A fluorescence-based exonuclease assay to characterize DmWRNexo, orthologue of human progeroid WRN exonuclease, and its application to other nucleases.
abstract: WRN exonuclease is involved in resolving DNA damage that occurs either during DNA replication or following exposure to endogenous or exogenous genotoxins. It is likely to play a role in preventing accumulation of recombinogenic intermediates that would otherwise accumulate at transiently stalled replication forks, consistent with a hyper-recombinant phenotype of cells lacking WRN. In humans, the exonuclease domain comprises an N-terminal portion of a much larger protein that also possesses helicase activity, together with additional sites important for DNA and protein interaction. By contrast, in Drosophila, the exonuclease activity of WRN (DmWRNexo) is encoded by a distinct genetic locus from the presumptive helicase, allowing biochemical (and genetic) dissection of the role of the exonuclease activity in genome stability mechanisms. Here, we demonstrate a fluorescent method to determine WRN exonuclease activity using purified recombinant DmWRNexo and end-labeled fluorescent oligonucleotides. This system allows greater reproducibility than radioactive assays as the substrate oligonucleotides remain stable for months, and provides a safer and relatively rapid method for detailed analysis of nuclease activity, permitting determination of nuclease polarity, processivity, and substrate preferences.
authors.affiliation: Department of Biochemistry, University of Oxford.
authors.forename: Penelope A
authors.initials: PA
authors.lastname: Mason
authors.forename: Ivan
authors.initials: I
authors.lastname: Boubriak
authors.forename: Lynne S
authors.initials: LS
authors.lastname: Cox
mesh_terms: D000180:Acrylic Resins / Q000737:chemistry; D000818:Animals; D004330:Drosophila / Q000201:enzymology; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005092:Exonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019793:Fluorescein / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D061848:Optical Imaging / Q000379:methods*; D053484:RecQ Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D000071657:Werner Syndrome Helicase; D000099166:Polyacrylamides
chemical_list: D000180:Acrylic Resins; D029721:Drosophila Proteins; D005090:Exodeoxyribonucleases; D005092:Exonucleases; D019793:Fluorescein; D005456:Fluorescent Dyes; D009841:Oligonucleotides; D053484:RecQ Helicases; D011994:Recombinant Proteins; D000071657:Werner Syndrome Helicase; D000099166:Polyacrylamides; C542170:WRNexo protein, Drosophila; C098785:WRN protein, human