Róna Gergely, Scheer Ildikó, Nagy Kinga, Pálinkás Hajnalka L, Tihanyi Gergely, Borsos Máté, Békési Angéla, Vértessy Beáta G
Institute of Enzymology, RCNS, Hungarian Academy of Sciences, Magyar Tudósok Str. 2, H-1117 Budapest, Hungary Department of Applied Biotechnology and Food Sciences, Budapest University of Technology and Economics, Szt Gellért Square 4, H-1111 Budapest, Hungary
Institute of Enzymology, RCNS, Hungarian Academy of Sciences, Magyar Tudósok Str. 2, H-1117 Budapest, Hungary Department of Applied Biotechnology and Food Sciences, Budapest University of Technology and Economics, Szt Gellért Square 4, H-1111 Budapest, Hungary.
Nucleic Acids Res. 2016 Feb 18;44(3):e28. doi: 10.1093/nar/gkv977. Epub 2015 Oct 1.
The role of uracil in genomic DNA has been recently re-evaluated. It is now widely accepted to be a physiologically important DNA element in diverse systems from specific phages to antibody maturation and Drosophila development. Further relevant investigations would largely benefit from a novel reliable and fast method to gain quantitative and qualitative information on uracil levels in DNA both in vitro and in situ, especially since current techniques does not allow in situ cellular detection. Here, starting from a catalytically inactive uracil-DNA glycosylase protein, we have designed several uracil sensor fusion proteins. The designed constructs can be applied as molecular recognition tools that can be detected with conventional antibodies in dot-blot applications and may also serve as in situ uracil-DNA sensors in cellular techniques. Our method is verified on numerous prokaryotic and eukaryotic cellular systems. The method is easy to use and can be applied in a high-throughput manner. It does not require expensive equipment or complex know-how, facilitating its easy implementation in any basic molecular biology laboratory. Elevated genomic uracil levels from cells of diverse genetic backgrounds and/or treated with different drugs can be demonstrated also in situ, within the cell.
尿嘧啶在基因组DNA中的作用最近得到了重新评估。现在人们普遍认为，在从特定噬菌体到抗体成熟和果蝇发育的各种系统中，尿嘧啶是一种具有重要生理意义的DNA元素。进一步的相关研究将极大地受益于一种新颖、可靠且快速的方法，以获取有关体外和原位DNA中尿嘧啶水平的定量和定性信息，特别是因为目前的技术无法进行原位细胞检测。在此，我们从一种催化失活的尿嘧啶-DNA糖基化酶蛋白出发，设计了几种尿嘧啶传感器融合蛋白。所设计的构建体可作为分子识别工具，在斑点印迹应用中可用常规抗体进行检测，也可在细胞技术中用作原位尿嘧啶-DNA传感器。我们的方法在众多原核和真核细胞系统中得到了验证。该方法易于使用，且可高通量应用。它不需要昂贵的设备或复杂的专业知识，便于在任何基础分子生物学实验室轻松实施。在细胞内原位也可证明，来自不同遗传背景和/或用不同药物处理的细胞中基因组尿嘧啶水平升高。
