Purification and Biochemical Characterization of TrwC, the Helicase Involved in Plasmid R388 Conjugal DNA Transfer

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Abstract
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 E. coli.

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