【摘要】 【目的】以嗜酸嗜热硫化叶菌Sulfolobus acidocaldarius的DNA聚合酶Ⅳ （Saci₀554）为例,表征其跨越模板上损伤碱基的DNA合成效果。【方法】将DNA聚合酶Ⅳ （SacpolⅣ）在大肠杆菌中进行重组表达,经亲和层析纯化得到SacpolⅣ蛋白;利用人工合成的带有不同损伤的寡核苷酸片段作为模板DNA,用尿素变性聚丙烯酰胺凝胶电泳技术,鉴定SacpolⅣ在体外跨越各种损伤碱基进行跨损伤合成的催化能力。【结果】SacpolⅣ重组蛋白能够不同程度地跨越嘌呤和嘧啶损伤,跨越能力的高低取决于损伤碱基与正常碱基形成氢键的能力。本研究还发现,SacpolⅣ能够在DNA链中掺入核糖核苷酸,但掺入核糖核苷酸的效率低于脱氧核糖核苷酸。【结论】本研究证实SacpolⅣ具有很强的跨越损伤合成能力,能够跨越多种氢键配对能力减弱的损伤碱基,为其在细胞内的跨越损伤合成功能提供了生化证据。更多还原

【Abstract】 [Objective] To characterize the translesion synthesis of different damages by DNA polymerase Ⅳ from thermophilic archaea Sulfolobus acidocaldarius. [Methods] We detected the translesion synthesis ability of Sulfolobus acidocaldarius polymerase Ⅳ(SacpolⅣ) using fluorescence labeled oligonucleotides as substrates through denaturing polyacrylamide gel electrophoresis. [Results] We successfully purified SacpolⅣ from induced E. coli and confirmed SacpolⅣ possessed strong translesion synthesis ability to DNA damages on purines and pyrimidines. The TLS ability depends on whether damaged bases can pair normal bases perfectly. Besides, we found Sacpol Ⅳ can incorporate rNMP into DNA strand. [Conclusion] We confirmed that SacpolⅣ is a typical TLS DNA polymerase with the abilities of bypassing damaged purines and pyrimidines on DNA, and incorporating rNMP into DNA.更多还原