Journal of Bacteriology, June 2005, p. 3913-3920, Vol. 187, No. 12
0021-9193/05/$08.00+0 doi:10.1128/JB.187.12.3913-3920.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Deletion of the datA Site Does Not Affect Once-per-Cell-Cycle Timing but Induces Rifampin-Resistant Replication
Morigen,
Felipe Molina, and
Kirsten Skarstad*
Department of Cell Biology, Institute for Cancer Research, The Norwegian Radium Hospital, 0310 Oslo, Norway
Received 10 December 2004/
Accepted 11 March 2005
In Escherichia coli, three mechanisms have been proposed to maintain proper regulation of replication so that initiation occurs once, and only once, per cell cycle. First, newly formed origins are inactivated by sequestration; second, the initiator, DnaA, is inactivated by the Hda protein at active replication forks; and third, the level of free DnaA protein is reduced by replication of the datA site. The datA site titrates unusually large amounts of DnaA and it has been reported that reinitiation, and thus asynchrony of replication, occurs in cells lacking this site. Here, we show that reinitiation in
datA cells does not occur during exponential growth and that an apparent asynchrony phenotype results from the occurrence of rifampin-resistant initiations. This shows that the datA site is not required to prevent reinitiation and limit initiation of replication to once per generation. The datA site may, however, play a role in timing of initiation relative to cell growth. Inactivation of active ATP-DnaA by the Hda protein and the sliding clamp of the polymerase was found to be required to prevent reinitiation and asynchrony of replication.
* Corresponding author. Mailing address: Department of Cell Biology, Institute for Cancer Research, The Norwegian Radium Hospital, 0310 Oslo, Norway. Phone: 47-22934255. Fax: 47-22934580. E-mail: kirsten.skarstad{at}labmed.uio.no.
Journal of Bacteriology, June 2005, p. 3913-3920, Vol. 187, No. 12
0021-9193/05/$08.00+0 doi:10.1128/JB.187.12.3913-3920.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
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Copyright © 2005 by the American Society for Microbiology. All rights reserved.