PURIFICATION AND BIOCHEMICAL-CHARACTERIZATION OF TRWC, THE HELICASE INVOLVED IN PLASMID R388 CONJUGAL DNA TRANSFER

TrwC is an essential protein in conjugative DNA transfer of the broad- host-range plasmid R388. TrwC was purified in two chromatographic step s from TrwC-overproducing bacteria. The purification procedure resulte d in >90% pure TrwC protein, which was free of contaminating nuclease activities. TrwC behaved as a dimer in gel-filtration chromatography i n the presence of 550 mM NaCl, and had a pI of 10.1. The purified prot ein showed in-vitro ssDNA-dependent nucleoside-5'-triphosphatase and D NA helicase activities. ATP was the preferred substrate for the NTP hy drolysis reaction, which required Mg2+. The helicase activity was depe ndent on ATP and Mg2+. The efficiency of the unwinding reaction cataly zed by TrwC ranged from >90% of fragment displaced for a 93-nucleotide sequence to <5% for a 365-nucleotide sequence. Unwinding was unidirec tional in the 5' to 3' direction. The enzyme turned over very slowly f rom one DNA substrate molecule to another. TrwC is only the second DNA helicase to be described which is involved in conjugative DNA transfe r. The biochemical properties of TrwC described here confirm its funct ional relatedness to helicase I (TraI) encoded by plasmid F of E. coli .