PLASMID R6K CONTAINS 2 FUNCTIONAL ORITS WHICH CAN ASSEMBLE SIMULTANEOUSLY IN RELAXOSOMES IN-VIVO

Plasmid R6K contains two functional origins or transfer (oriT), in con trast to previously characterized conjugative plasmids. The oriTs are formed by 98 bp palindromic sequences invertedly orientated with respe ct to each other and located in the immediate vicinity of the alpha an d beta origins of replication. The gene for R6K oriT-nickase, taxC, wa s identified by transposon mutagenesis and sequenced, revealing that T axC belongs to the VirD2 nickase family. The protein was overproduced and purified. It catalysed a cleaving-joining reaction on single-stran ded DNA containing its target sequence. Identification of the nic site s suggested that the R6K oriTs belong to the RP4/VirD2 oriT family. Cl eavage was highly specific and did not occur with oligonucleotides cle aved by related nickases like TraI of RP4 or VirD2 of the Ti plasmid. Mic cleavage of in vivo preassembled relaxation complexes was induced by incubation of plasmid cleared lysates with ethidium bromide. Nicked molecules obtained in this way were treated with snake venom phosphod iesterase to produce double strand cleavages at the nic sites. 35% of the molecules were cleaved simultaneously at both nic sites, both in t he case of R6K and of R6Kdrd1, a derepressed mutant whose frequency of transfer is 1000-fold higher. This figure represents the minimum perc entage of individual R6K molecules containing two pre-assembled relaxa tion complexes. (C) 1996 Academic Press Limited