TERMINATION OF DNA-REPLICATION IN-VITRO - REQUIREMENT FOR STEREOSPECIFIC INTERACTION BETWEEN 2 DIMERS OF THE REPLICATION TERMINATOR PROTEINOF BACILLUS-SUBTILIS AND WITH THE TERMINATOR SITE TO ELICIT POLAR CONTRAHELICASE AND FORK IMPEDANCE

The termination of DNA replication at a sequence-specific replication terminus in Bacillus subtilis is catalyzed by a dimeric replication te rminator protein (RTP) of subunit mol. wt 14 500. RTP has become an at tractive protein with which to study the molecular mechanism of termin ation because its crystal structure has now been solved and the previo us lack of an in vitro replication system has been largely overcome by our discovery that the protein terminates replication in vivo and in vitro in the well-studied Gram-negative Escherichia coil system. We ha ve exploited the surrogate in vitro system to show that RTP acts as a polar contrahelicase to DnaB helicase of E.coli only when two RTP dime rs are bound co-operatively to overlapping core and auxiliary sequence s comprising the terminus. A core sequence by itself binds one dimer o f RTP, but elicits no contrahelicase activity. Binding of two RTP dime rs to a tandem head-to-tail core repeat also elicits no contrahelicase activity, thus suggesting that a specific stereochemical interaction between two RTP dimers and with the terminator site is essential for t ermination. RTP blocks unwinding of DNA substrates containing heterodu plex regions that include the terminus and are in the size range of si milar to 50 to >1000 bp in length. Thus, the protein blocks authentic helicase-catalyzed unwinding rather than just the translocation of the helicase on DNA.