Biochemistry Workgroup, Leibniz Institute for Age Research-Fritz Lipmann Institute, D-07745 Jena, Germany.
Anal Biochem. 2010 Oct 1;405(1):11-8. doi: 10.1016/j.ab.2010.05.019. Epub 2010 Jun 1.
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.
具有 3'-5'外切核酸酶活性的酶在通过校对促进 DNA 复制和 DNA 修复的准确性方面非常重要。因此，这些酶的功能被内源性或外源性效应物改变，可能对 DNA 复制产生重大影响，并最终影响基因组完整性。我们开发了一种无标记的高通量筛选方法，用于定量不同试剂对核酸外切酶活性的影响。该测定基于形成发夹的生物素化寡核苷酸底物，该底物含有一个或多个外切核酸酶抗性硫代磷酸酯核苷酸。使用灵敏的焦磷酸测序反应在样品净化后，间接测定所选 3'-5'外切核酸酶的活性和特异性。在这个焦磷酸测序步骤中，可以定量记录每个位置填充的核苷酸数量，这些核苷酸是通过外切核酸酶从 3'末端外切降解的底物中去除的，这与外切核酸酶的量相等。该系统允许估计外切核酸酶活性的连续性和效率。我们使用文献中报道的化合物来抑制外切核酸酶 III 或聚合酶 I 的大片段（Klenow 片段）的外切核酸酶活性，以评估该测定。
