Site-directed mutants of RTP of Bacillus subtilis and the mechanism of replication fork arrest

DNA replication fork arrest during the termination phase of chromosome repl ication in Bacillus subtilis is brought about by the replication terminator protein (RTP) bound to specific DNA terminator sequences (Tev sites) distr ibuted throughout the terminus region. An attractive suggestion by others w as that crucial to the functioning of the RTP-Ter complex is a specific int eraction between RTP positioned on the DNA and the helicase associated with the approaching replication fork. Ln support of this was the behaviour of two site-directed mutants of RTP. They appeared to bind Ter DNA normally bu t were ineffective in fork arrest as ascertained by in vitro Escherichia co li DnaB helicase and replication assays. We describe here a system for asse ssing the fork-arrest behaviour of RTP mutants in a bona fide ill vivo assa y in B. subtilis. One of the previously studied mutants, RTP.Y33N, was non- functional in fork arrest in vivo, as predicted. But through extensive anal yses, this RTP mutant was shown to be severely defective in binding to Ter DNA, contrary to expectation. Taken in conjunction with recent findings on the other mutant (RTP.E30K), it is concluded that there is as yet no substa ntive evidence from the behaviour of RTP mutants to support the Rm-helicase interaction model for fork arrest. In an extension of the present work on RTP.Y33N, we determined the dissociation rates of complexes formed by wild- type (wt) RTP and another RTP mutant with various terminator sequences. The functional wtRTP-TerI complex was quite stable (half-life of 182 minutes), reminiscent of the great stability of the E. coli Tus-Ter complex. More si gnificant were the exceptional stabilities of complexes comprising wtRTP an d an RTP double-mutant (E39K.R42Q) bound to some particular terminator sequ ences. From the measurement of in vivo fork-arrest activities of the variou s complexes, it is concluded that the stability (half-life) of the whole RT P-Ter complex is not the overriding determinant of arrest, and that the RTP -Ter complex must be actively disrupted, or RTP removed, by the action of t he approaching replication fork. (C) 1999 Academic Press.