SEQUENCE REQUIREMENTS FOR THE TERMINATION OF ROLLING-CIRCLE REPLICATION OF PLASMID PT181

Most small multicopy plasmids of Gram-positive bacteria and many in Gr am-negative bacteria replicate by a rolling-circle (RC) mechanism. The replication initiator proteins encoded by the RC plasmids and single- stranded bacteriophages of Escherichia coil have origin-specific nicki ng-closing activities that are required for the initiation and termina tion of RC replication. We have investigated the sequence requirements for termination of RC replication of plasmid pT181. The initiator nic k site is located in the loop of a hairpin region (IRII) within the pT 181 origin of replication. By mutational analysis, we have found that several nucleotides within the stem of IRII which are critical for the initiation activity are dispensable for termination of replication. W e also demonstrate that nucleotides in the right arm of IRII, but not the left arm, are absolutely required for termination of RC replicatio n. We have also identified specific nucleotides in IRII that are criti cal for its termination activity. The sequence of the right arm of the hairpin must be located downstream of the initiator nick site for ter mination, suggesting that termination requires a specific orientation of the initiator protein at the origin.