Dorjsuren Dorjbal, Wilson David M, Beard William A, McDonald John P, Austin Christopher P, Woodgate Roger, Wilson Samuel H, Simeonov Anton
NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892-3370, USA.
Nucleic Acids Res. 2009 Oct;37(19):e128. doi: 10.1093/nar/gkp641. Epub 2009 Aug 14.
Specialized DNA polymerases are involved in DNA synthesis during base-excision repair and translesion synthesis across a wide range of chemically modified DNA templates. Notable features of these enzymes include low catalytic efficiency, low processivity and low fidelity. Traditionally, in vitro studies of these enzymes have utilized radiolabeled substrates and gel electrophoretic separation of products. We have developed a simple homogeneous fluorescence-based method to study the enzymology of specialized DNA polymerases in real time. The method is based on fluorescent reporter strand displacement from a tripartite substrate containing a quencher-labeled template strand, an unlabeled primer and a fluorophore-labeled reporter. With this method, we could follow the activity of human DNA polymerases beta, eta, iota and kappa under different reaction conditions, and we investigated incorporation of the aberrant nucleotide, 8-oxodGTP, as well as bypass of an abasic site or 8-oxoG DNA template lesion in different configurations. Lastly, we demonstrate that the method can be used for small molecule inhibitor discovery and characterization in highly miniaturized settings, and we report the first nanomolar inhibitors of Y-family DNA polymerases iota and eta. The fluorogenic method presented here should facilitate mechanistic and inhibitor investigations of these polymerases and is also applicable to the study of highly processive replicative polymerases.
特殊的DNA聚合酶参与碱基切除修复过程中的DNA合成以及跨多种化学修饰DNA模板的跨损伤合成。这些酶的显著特点包括催化效率低、持续合成能力低和保真度低。传统上，对这些酶的体外研究利用放射性标记的底物和产物的凝胶电泳分离。我们开发了一种简单的基于均相荧光的方法来实时研究特殊DNA聚合酶的酶学。该方法基于从包含淬灭剂标记的模板链、未标记的引物和荧光团标记的报告链的三方底物中进行荧光报告链置换。通过这种方法，我们可以追踪人DNA聚合酶β、η、ι和κ在不同反应条件下的活性，并研究异常核苷酸8-氧代鸟嘌呤三磷酸（8-oxodGTP）的掺入，以及不同构型下无碱基位点或8-氧代鸟嘌呤（8-oxoG）DNA模板损伤的跨越。最后，我们证明该方法可用于在高度微型化的环境中发现和表征小分子抑制剂，并且我们报告了Y家族DNA聚合酶ι和η的首个纳摩尔级抑制剂。本文介绍的荧光方法应有助于对这些聚合酶进行机制和抑制剂研究，并且也适用于研究高度持续合成的复制性聚合酶。
