A label-free assay of exonuclease activity using a pyrosequencing technique.
Enzymes with 3'-5' exonuclease activities are important in promoting the accuracy of DNA replication and DNA repair by proofreading. The alteration of the function of these enzymes by endogenous or exogenous effectors could, therefore, have a considerable impact on DNA replication and ultimately on genome integrity. We have developed a label-free high-throughput screening method for quantifying the effects of different reagents on exonuclease activity. The assay is based on a hairpin-forming biotinylated oligonucleotide substrate that contains one or more exonuclease-resistant phosphorothioate nucleotides. The activity and specificity of the selected 3'-5' exonuclease is determined indirectly using a sensitive pyrosequencing reaction after cleanup of the samples. In this pyrosequencing step, the amount of nucleotides filled into each position of the exonucleolytically degraded 3' end of the substrate can be recorded quantitatively and equals the amount of the nucleotides removed by the exonuclease. This system allows the estimation of both processivity and efficiency of the exonuclease activity. We have employed compounds reported in the literature to inhibit the exonuclease activities of either exonuclease III or the large fragment of polymerase I (Klenow fragment) to evaluate the assay.
Biochemistry Workgroup, Leibniz Institute for Age Research-Fritz Lipmann Institute, D-07745 Jena, Germany. kguehrs@fli-leibniz.de
Karl-Heinz
KH
Gührs
Marco
M
Groth
Frank
F
Grosse
D004256:DNA Polymerase I / Q000378:metabolism*; D004260:DNA Repair; D004261:DNA Replication; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D057166:High-Throughput Screening Assays; D055029:Inverted Repeat Sequences; D009841:Oligonucleotides / Q000378:metabolism; D010710:Phosphates / Q000737:chemistry; D017422:Sequence Analysis, DNA / Q000379:methods*
D009841:Oligonucleotides; D010710:Phosphates; C083120:phosphorodithioic acid; D004256:DNA Polymerase I; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III