Nucleic Acids Research, 2003, Vol. 31, No. 20 5993-6003
© 2003 Oxford University Press
Protein- and ATP-dependent transitions from ‘closed’ to ‘open’ complexes at the 
ori of plasmid R6K
Department of Bacteriology, University of Wisconsin-Madison, 420 Henry Mall, Madison, WI 53706, USA
*To whom correspondence should be addressed. Tel: +1 608 262 6947; Fax: +1 608 262 9865; Email: msfiluto{at}facstaff.wisc.edu
Present address:
Ricardo Krüger, Universidade Católica de Brasília, Campus II, SGAN 916, Módulo B,W5 Norte, Brasília, DF, Brazil
R6K-encoded 
protein can bind to the seven, 22 bp tandem iterons of the 
origin. In this work, we use a variant of , His-·F107S, that is hyperactive in replication. In vitro, His-·F107S-dependent local DNA melting (open complex formation) occurs in the absence of host proteins (IHF/HU or DnaA) and it is positioned in the A + T-rich region adjacent to iterons. Experiments described here examine the effects of ATP, Mg2+ and temperature on the opening reaction. We show that the opening of the origin can occur in the presence of ATP as well as AMP-PCP (a non-hydrolyzable ATP analog). This suggests that, for origin, ATP hydrolysis may be unnecessary for open complex formation facilitated by His-·F107S. In the absence of ATP or Mg2+, His-·F107S yielded data suggestive of distortions in the iteron attributable to DNA bending rather than DNA melting. Our findings also demonstrate that ATP and stimulate open complex formation over a wide range of temperatures, but not at 0°C. These and other results indicate that ATP and/or Mg2+ are not needed for His-·F107S binding to iterons and that ATP effects an allosteric change in the protein bound to origin.