Structural and functional analysis of a bipolar replication terminus - Implications for the origin of polarity of fork arrest

We have delineated the amino acid to nucleotide contacts made by two intera cting dimers of the replication terminator protein (RTP) of Bacillus subtil is with a novel naturally occurring bipolar replication terminus by convert ing RTP to a site-directed chemical nuclease and mapping its cleavage sites on the terminus. The data show a relatively symmetrical arrangement of the amino acid to base contacts, and a comparison of the bipolar contacts with that of a normal unipolar terminus suggests that the DNA-protein contacts play an important determinative role in generating polarity from structural ly symmetrical RTP dimers, The amino acid to nucleotide contacts provided d istance constraints that enabled us to build a three-dimensional model of t he protein-DNA complex. The model is consistent with features of the bipola r Ter RTP complex derived from mutational and cross-linking data. The bipol ar terminus arrested Escherichia coil DNA replication and DnaB helicase and T7 RNA polymerase in vitro in both orientations. RTP arrested the unwindin g of duplex DNA on the bipolar Ter DNA substrate regardless of the length o f the duplex DNA The latter result suggested further that the terminus arre sted authentic DNA unwinding by the helicase rather than just translocation of helicase on DNA.