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

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.