Characterization of purine-motif triplex DNA-binding proteins in HeLa extracts.
Characterization of purine-motif triplex DNA-binding proteins in HeLa extracts.
复制标题
HeLa 提取物中嘌呤基序三链体 DNA 结合蛋白的表征。
作者：
Marco Musso;Laura D. Nelson;M. V. Van Dyke
表明体内存在三螺旋 DNA 的证据之一是识别这种结构的细胞蛋白。使用含有富含 GT 的嘌呤基序三链体的寡核苷酸探针、来自 HeLa 核或细胞质提取物的蛋白质以及电泳迁移率变动分析，我们鉴定了四种特定的人类蛋白质三链体复合物。这些复合物中的蛋白质不识别类似的高嘌呤/高嘧啶双链体DNA或嘧啶基序三链体，但确实识别嘌呤基序三链体，无论它们在第三链中是否具有磷酸二酯或硫代磷酸酯主链或涉及A*AT而不是T*AT碱基三联体。对于这些蛋白质中的每一种，结合亲和力随着三链体长度的增加而增加。对于一些三链体结合蛋白，注意到对单个富含G的寡核苷酸的亲和力较弱，尽管这实际上可能反映了对四链体结构的亲和力，而这些寡核苷酸易于采用这种结构。 HeLa 核提取物的离子交换色谱分级表明，至少三种不同的蛋白质导致了观察到的电泳迁移率变化。西南印迹方法鉴定了三种主要多肽，表观分子质量分别为 100、60 和 15 kDa，它们优先识别嘌呤基序三链体。这些数据证明存在特异性识别一个三螺旋基序的真核蛋白质，并支持三螺旋 DNA 的生物学作用的想法。
One piece of evidence indicating that triple-helical DNAs exist in vivo would be the demonstration of cellular proteins that recognize such structures. Using oligonucleotide probes containing a GT-rich purine-motif triplex, proteins from either HeLa nuclear or cytoplasmic extracts and electrophoretic mobility shift assays, we identified four specific human protein-triplex complexes. Proteins in these complexes did not recognize an analogous homopurine/homopyrimidine duplex DNA or a pyrimidine-motif triplex but did recognize purine-motif triplexes regardless of whether they possessed a phosphodiester or phosphorothioate backbone in the third strand or involved A*AT instead of T*AT base triplets. For each of these proteins, binding affinity increased with increasing triplex length. For some triplex-binding proteins, a weak affinity was noted for individual G-rich oligonucleotides, though this may actually reflect an affinity for quadruplex structures, which these oligonucleotides are prone to adopt. Ion exchange chromatographic fractionation of HeLa nuclear extracts indicated that at least three different proteins were responsible for the observed electrophoretic mobility shifts. Southwestern blotting methods identified three major polypeptides, with apparent molecular masses of 100, 60, and 15 kDa, that preferentially recognized purine-motif triplexes. These data demonstrate the existence of eukaryotic proteins that specifically recognize one triplex motif and support the idea of a biological role for triple helical DNA.