Pidugu Lakshmi S, Gustafson Erin E, Servius Hardler W, Cook Mary E, Lee Nina C, Varney Kristen M, Drohat Alexander C
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, United States.
Molecular and Structural Biology Program, University of Maryland Marlene and Stewart Greenebaum Comprehensive Cancer Center, Baltimore, MD 21201, United States.
Nucleic Acids Res. 2025 Jun 6;53(11). doi: 10.1093/nar/gkaf492.
Many proteins that bind specifically to DNA and RNA employ a mechanism known as nucleotide flipping, whereby a nucleotide rotates out of the helical stack and into an active site. This conformational change can be monitored using spectroscopic methods, including fluorescence and nuclear magnetic resonance (NMR). We previously showed that flipping of a 2'-fluoroarabino-substituted nucleotide by thymine DNA glycosylase (TDG) can be directly monitored using 19F NMR. However, 2'-F-substituted phosphoramidites are typically readily available only for canonical nucleotides, posing an experimental limitation. Given the large conformational change associated with nucleotide flipping, we reasoned that it could be indirectly monitored by 2'-F nucleotides located at proximal sites in the target or complementary DNA strand. Indeed, we find that TDG flipping of deoxythymidine from a G⋅T mispair is robustly monitored by 2'-F probes at six of nine sites examined. Moreover, flipping of cadC (5-carboxyl-2'-deoxycytidine), paired with dG or dA, can be monitored with 2'-F probes in the complementary strand, revealing highly efficient cadC flipping by TDG. Notably, kinetic assays show that the 2'-F probes have a small or negligible effect on TDG activity. The results point to a general and experimentally accessible approach for studying nucleotide flipping and other conformational changes in nucleic acids.
许多与DNA和RNA特异性结合的蛋白质采用一种称为核苷酸翻转的机制，即一个核苷酸从螺旋堆积中旋转出来并进入活性位点。这种构象变化可以使用光谱方法进行监测，包括荧光和核磁共振（NMR）。我们之前表明，胸腺嘧啶DNA糖基化酶（TDG）对2'-氟阿拉伯糖取代核苷酸的翻转可以使用19F NMR直接监测。然而，2'-F取代的亚磷酰胺通常仅对标准核苷酸容易获得，这构成了一个实验限制。鉴于与核苷酸翻转相关的巨大构象变化，我们推断它可以通过位于靶标或互补DNA链近端位点的2'-F核苷酸间接监测。事实上，我们发现，在所检测的九个位点中的六个位点，2'-F探针能够可靠地监测TDG对G·T错配中脱氧胸苷的翻转。此外，与dG或dA配对的cadC（5-羧基-2'-脱氧胞苷）的翻转可以用互补链中的2'-F探针监测，揭示了TDG对cadC的高效翻转。值得注意的是，动力学分析表明，2'-F探针对TDG活性的影响很小或可忽略不计。这些结果指出了一种研究核酸中核苷酸翻转和其他构象变化的通用且实验上可行的方法。
