Abstract
The Escherichia coli initiator protein, DnaA binds to the replicational origin of the chromosome (oriC) and initiates the replication in a manner depending on ATP binding. Replication of mini-chromosome (a plasmid bearing oriC) has been reconstituted in vitro using crude extracts or purified proteins. In these in vitro systems, only ATP-bound form of DnaA is active to initiate a series of initiation reactions which leads to loading of the DNA polymerase III holoenzyme (pol III HE) onto DNA for DNA synthesis. In a manner dependent on Hda protein and DNA loading of pol III HE, ATP bound on DnaA is hydrolyzed. The resultant ADP-bound form of DnaA is inactive for the initiation. This inactivation system called RIDA (regulatory inactivation of DnaA) ensures the replication initiation only once per cell cycle. We here describe recent progress on our system to reconstitute the inactivation of DnaA by RIDA and a novel system to regenerate active DnaA. The reconstitution of the Hda-dependent DnaA-ATP hydrolysis requires the DNA-loaded form of the sliding clamp. This complex is formed by loading of the β subunit of pol III HE onto DNA during replication process. Hda protein forms a stable complex with the sliding clamp, and most likely has a direct contact to ATP bound on DnaA. The cellular ATP-DnaA level fluctuates during the cell cycle. Regeneration from ADP-DnaA to ATP-DnaA is observed in vivo before the next round of the replication. Recently we have found a specific DNA segment DARS (DnaA-reactivating sequences) that has an activity to release ADP bound on DnaA. ATP-DnaA is regenerated by nucleotide-exchange in the presence of DARS and the elevated concentrations of ATP. The resultant ATP-DnaA was active in the mini-chromosome replication reaction in vitro. Thus, DARS can functionally reactivate ADP-DnaA. The inactivation-reactivation cycle of DnaA might be reconstituted in vitro in a manner controled by RIDA and DARS.
| Original language | English |
|---|---|
| Title of host publication | 2006 IEEE International Symposium on Micro-Nano Mechanical and Human Science, MHS |
| DOIs | |
| Publication status | Published - Dec 1 2006 |
| Event | 2006 IEEE International Symposium on Micro-Nano Mechanical and Human Science, MHS - Nagoya, Japan Duration: Nov 5 2006 → Nov 8 2006 |
Other
| Other | 2006 IEEE International Symposium on Micro-Nano Mechanical and Human Science, MHS |
|---|---|
| Country | Japan |
| City | Nagoya |
| Period | 11/5/06 → 11/8/06 |
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All Science Journal Classification (ASJC) codes
- Computational Mechanics
- Control and Systems Engineering
- Human Factors and Ergonomics
Cite this
Reconstitution of in vitro inactivation and reactivation systems of DnaA protein for the control of chromosomal replication initiation in Escherichia coli. / Su'etsugu, Masayuki; Fujimitsu, Kazuyuki; Katayama, Tsutomu.
2006 IEEE International Symposium on Micro-Nano Mechanical and Human Science, MHS. 2006. 4110408.Research output: Chapter in Book/Report/Conference proceeding › Conference contribution
}
TY - GEN
T1 - Reconstitution of in vitro inactivation and reactivation systems of DnaA protein for the control of chromosomal replication initiation in Escherichia coli
AU - Su'etsugu, Masayuki
AU - Fujimitsu, Kazuyuki
AU - Katayama, Tsutomu
PY - 2006/12/1
Y1 - 2006/12/1
N2 - The Escherichia coli initiator protein, DnaA binds to the replicational origin of the chromosome (oriC) and initiates the replication in a manner depending on ATP binding. Replication of mini-chromosome (a plasmid bearing oriC) has been reconstituted in vitro using crude extracts or purified proteins. In these in vitro systems, only ATP-bound form of DnaA is active to initiate a series of initiation reactions which leads to loading of the DNA polymerase III holoenzyme (pol III HE) onto DNA for DNA synthesis. In a manner dependent on Hda protein and DNA loading of pol III HE, ATP bound on DnaA is hydrolyzed. The resultant ADP-bound form of DnaA is inactive for the initiation. This inactivation system called RIDA (regulatory inactivation of DnaA) ensures the replication initiation only once per cell cycle. We here describe recent progress on our system to reconstitute the inactivation of DnaA by RIDA and a novel system to regenerate active DnaA. The reconstitution of the Hda-dependent DnaA-ATP hydrolysis requires the DNA-loaded form of the sliding clamp. This complex is formed by loading of the β subunit of pol III HE onto DNA during replication process. Hda protein forms a stable complex with the sliding clamp, and most likely has a direct contact to ATP bound on DnaA. The cellular ATP-DnaA level fluctuates during the cell cycle. Regeneration from ADP-DnaA to ATP-DnaA is observed in vivo before the next round of the replication. Recently we have found a specific DNA segment DARS (DnaA-reactivating sequences) that has an activity to release ADP bound on DnaA. ATP-DnaA is regenerated by nucleotide-exchange in the presence of DARS and the elevated concentrations of ATP. The resultant ATP-DnaA was active in the mini-chromosome replication reaction in vitro. Thus, DARS can functionally reactivate ADP-DnaA. The inactivation-reactivation cycle of DnaA might be reconstituted in vitro in a manner controled by RIDA and DARS.
AB - The Escherichia coli initiator protein, DnaA binds to the replicational origin of the chromosome (oriC) and initiates the replication in a manner depending on ATP binding. Replication of mini-chromosome (a plasmid bearing oriC) has been reconstituted in vitro using crude extracts or purified proteins. In these in vitro systems, only ATP-bound form of DnaA is active to initiate a series of initiation reactions which leads to loading of the DNA polymerase III holoenzyme (pol III HE) onto DNA for DNA synthesis. In a manner dependent on Hda protein and DNA loading of pol III HE, ATP bound on DnaA is hydrolyzed. The resultant ADP-bound form of DnaA is inactive for the initiation. This inactivation system called RIDA (regulatory inactivation of DnaA) ensures the replication initiation only once per cell cycle. We here describe recent progress on our system to reconstitute the inactivation of DnaA by RIDA and a novel system to regenerate active DnaA. The reconstitution of the Hda-dependent DnaA-ATP hydrolysis requires the DNA-loaded form of the sliding clamp. This complex is formed by loading of the β subunit of pol III HE onto DNA during replication process. Hda protein forms a stable complex with the sliding clamp, and most likely has a direct contact to ATP bound on DnaA. The cellular ATP-DnaA level fluctuates during the cell cycle. Regeneration from ADP-DnaA to ATP-DnaA is observed in vivo before the next round of the replication. Recently we have found a specific DNA segment DARS (DnaA-reactivating sequences) that has an activity to release ADP bound on DnaA. ATP-DnaA is regenerated by nucleotide-exchange in the presence of DARS and the elevated concentrations of ATP. The resultant ATP-DnaA was active in the mini-chromosome replication reaction in vitro. Thus, DARS can functionally reactivate ADP-DnaA. The inactivation-reactivation cycle of DnaA might be reconstituted in vitro in a manner controled by RIDA and DARS.
UR - http://www.scopus.com/inward/record.url?scp=50449099427&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=50449099427&partnerID=8YFLogxK
U2 - 10.1109/MHS.2006.320253
DO - 10.1109/MHS.2006.320253
M3 - Conference contribution
AN - SCOPUS:50449099427
SN - 1424407176
SN - 9781424407170
BT - 2006 IEEE International Symposium on Micro-Nano Mechanical and Human Science, MHS
ER -