Abstract—

Noncovalent interactions between DNA and proteins are vital for cells. DNA-protein crosslinking may occur due to various endogenous and exogenous factors, as well as chemotherapeutic agents, and often affects the performance of DNA-binding proteins. Oligodeoxyribonucleotides with a reactive group can serve as a tool to investigate the structure and action of DNA-binding proteins. Here, we report the crosslinking of MutS, which is a sensor protein of DNA mismatch repair (MMR) in Escherichia coli, via the protein’s cysteine. This was realized via DNA that harboured a 2′-deoxy-2′-[3-(2-pyridyldithio)propionamide] group as part of a nucleoside at a given position, and two fluorophores. The proposed DNA duplex consisted of complementary oligonucleotides with breaks in the top and bottom strands. This feature allowed us to introduce the reactive group at different positions and made it possible to synthesize only the central fragment of the DNA with a minimal length. The MutS-DNA conjugate was obtained by thiol-disulfide exchange with quantitative yield and separated from the unreacted DNA by size-exclusion chromatography. Fluorescence resonance energy transfer was used to show that MutS remained functionally active within the conjugate, since the protein was able to change its conformation and DNA conformation as well.

中文翻译：

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具有C2'原子的2-吡啶基二硫基的DNA：保留其功能活性的MutS蛋白交联的有前途的工具

摘要—

DNA和蛋白质之间的非共价相互作用对细胞至关重要。DNA-蛋白质交联可能由于各种内源性和外源性因素以及化学治疗剂而发生，并经常影响DNA结合蛋白的性能。具有反应性基团的寡脱氧核糖核苷酸可以用作研究DNA结合蛋白的结构和作用的工具。在这里，我们报告了MutS的交联，它是大肠杆菌中DNA错配修复（MMR）的传感蛋白。，通过蛋白质的半胱氨酸。这是通过在给定位置带有2'-脱氧-2'-[3-（2-吡啶基二硫代）丙酰胺]基团作为核苷的一部分的DNA和两个荧光团实现的。提出的DNA双链体由互补的寡核苷酸组成，其顶部和底部链断裂。此功能使我们可以在不同位置引入反应基团，从而可以仅合成最短长度的DNA中心片段。MutS-DNA共轭物是通过硫醇-二硫键交换以定量收率获得的，并通过尺寸排阻色谱法与未反应的DNA分离。荧光共振能量转移用于显示MutS在缀合物内仍具有功能活性，因为该蛋白质也能够改变其构象和DNA构象。