pi protein- and ATP-dependent transitions from 'closed' to 'open' complexes at the gamma ori of plasmid R6K.
R6K-encoded pi protein can bind to the seven, 22 bp tandem iterons of the gamma origin. In this work, we use a variant of pi, His-pi.F107S, that is hyperactive in replication. In vitro, His-pi.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 gamma origin can occur in the presence of ATP as well as AMP-PCP (a non-hydrolyzable ATP analog). This suggests that, for gamma origin, ATP hydrolysis may be unnecessary for open complex formation facilitated by His-pi.F107S. In the absence of ATP or Mg2+, His-pi.F107S yielded data suggestive of distortions in the iteron attributable to DNA bending rather than DNA melting. Our findings also demonstrate that ATP and pi stimulate open complex formation over a wide range of temperatures, but not at 0 degrees C. These and other results indicate that ATP and/or Mg2+ are not needed for His-pi.F107S binding to iterons and that ATP effects an allosteric change in the protein bound to gamma origin.
Krüger
Ricardo
R
Department of Bacteriology, University of Wisconsin-Madison, 420 Henry Mall, Madison, WI 53706, USA.
Filutowicz
Marcin
M
D020856:AT Rich Sequence / Q000235:genetics; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology*; D004265:DNA Helicases / Q000737:chemistry / Q000378:metabolism*; D004261:DNA Replication / Q000187:drug effects*; D004268:DNA-Binding Proteins; D019281:Dimerization; D024202:Electrophoretic Mobility Shift Assay; D008274:Magnesium / Q000494:pharmacology; D010957:Plasmids / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D011196:Potassium Permanganate / Q000494:pharmacology; D011485:Protein Binding; D018741:Replication Origin / Q000235:genetics; D013696:Temperature; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*
D004268:DNA-Binding Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D011196:Potassium Permanganate; C005147:5'-adenylyl (beta,gamma-methylene)diphosphonate; D000255:Adenosine Triphosphate; D004265:DNA Helicases; D008274:Magnesium