PURIFICATION AND BIOCHEMICAL-CHARACTERIZATION OF TRWC, THE HELICASE INVOLVED IN PLASMID R388 CONJUGAL DNA TRANSFER
PURIFICATION AND BIOCHEMICAL-CHARACTERIZATION OF TRWC, THE HELICASE INVOLVED IN PLASMID R388 CONJUGAL DNA TRANSFER
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DOI：
10.1111/j.1432-1033.1994.tb20065.x
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发表时间：
1994-12-01
期刊：
影响因子：
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通讯作者：
DELACRUZ, F
中科院分区：
文献类型：
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作者：
GRANDOSO, G;LLOSA, M;DELACRUZ, F
TrwC是广宿主范围质粒R388的接合DNA转移中的必需蛋白。在两个色谱步骤中从TrwC过量生产细菌中纯化TrwC。纯化程序产生>90%纯的TrwC蛋白，其不含污染性核酸酶活性。TrwC在550 mM NaCl存在下在凝胶过滤色谱中表现为二聚体，并且具有10.1的pI。纯化的蛋白在体外显示ssDNA依赖的核苷-5 '-三磷酸酶和DNA解旋酶活性。ATP是NTP水解反应的优选底物，其需要Mg 2+。解旋酶活性依赖于ATP和Mg ~（2+）。由TrwC催化的解旋反应的效率范围从对于93个核苷酸序列>90%的片段被置换到
TrwC is an essential protein in conjugative DNA transfer of the broad-host-range plasmid R388. TrwC was purified in two chromatographic steps from TrwC-overproducing bacteria. The purification procedure resulted in >90% pure TrwC protein, which was free of contaminating nuclease activities. TrwC behaved as a dimer in gel-filtration chromatography in the presence of 550 mM NaCl, and had a pI of 10.1. The purified protein showed in-vitro ssDNA-dependent nucleoside-5'-triphosphatase and DNA helicase activities. ATP was the preferred substrate for the NTP hydrolysis reaction, which required Mg2+. The helicase activity was dependent on ATP and Mg2+. The efficiency of the unwinding reaction catalyzed by TrwC ranged from >90% of fragment displaced for a 93-nucleotide sequence to