Ac. Zhao et Sa. Khan, SEQUENCE REQUIREMENTS FOR THE TERMINATION OF ROLLING-CIRCLE REPLICATION OF PLASMID PT181, Molecular microbiology, 24(3), 1997, pp. 535-544
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.