Living Systems Institute, University of Exeter, Stocker Road, Exeter, EX4 4QD, UK.
Institute of Experimental Immunology, University of Zurich, 8057, Zurich, Switzerland.
Sci Rep. 2019 Jun 20;9(1):8853. doi: 10.1038/s41598-019-45356-z.
DNA and RNA nucleases play a critical role in a growing number of cellular processes ranging from DNA repair to immune surveillance. Nevertheless, many nucleases have unknown or poorly characterized activities. Elucidating nuclease substrate specificities and co-factors can support a more definitive understanding of cellular mechanisms in physiology and disease. Using fluorescence-based methods, we present a quick, safe, cost-effective, and real-time versatile nuclease assay, which uniquely studies nuclease enzyme kinetics. In conjunction with a substrate library we can now analyse nuclease catalytic rates, directionality, and substrate preferences. The assay is sensitive enough to detect kinetics of repair enzymes when confronted with DNA mismatches or DNA methylation sites. We have also extended our analysis to study the kinetics of human single-strand DNA nuclease TREX2, DNA polymerases, RNA, and RNA:DNA nucleases. These nucleases are involved in DNA repair, immune regulation, and have been associated with various diseases, including cancer and immune disorders.
DNA 和 RNA 核酸酶在越来越多的细胞过程中发挥着关键作用，从 DNA 修复到免疫监视。然而，许多核酸酶的活性尚不清楚或特征描述较差。阐明核酸酶的底物特异性和辅助因子可以更明确地理解生理和疾病中的细胞机制。我们使用基于荧光的方法，提出了一种快速、安全、具有成本效益且实时通用的核酸酶测定法，该方法独特地研究了核酸酶的酶动力学。结合底物文库，我们现在可以分析核酸酶的催化速率、方向性和底物偏好。该测定法足够灵敏，可以检测到修复酶在面对 DNA 错配或 DNA 甲基化位点时的动力学。我们还扩展了分析范围，以研究人类单链 DNA 核酸酶 TREX2、DNA 聚合酶、RNA 和 RNA:DNA 核酸酶的动力学。这些核酸酶参与 DNA 修复、免疫调节，并与各种疾病相关，包括癌症和免疫紊乱。
