| Research Abstract |
In the E.coli cell cycle, function of the chromosomal replication initiator DnaA protein is tightly regulated. After the ATP-bound DnaA initiates replication, DnaA-ATP is hydrolyzed by the DNA-loaded clamp subunit of DNA polymerase III holoenzyme and Hda protein, yielding ADP-bound DnaA, the inactivated form. In this study, we analyzed structure-function relationship of Hda protein, and found that the camp-binding site on Hda. Moreover, we identified important amino acid residues in AAA+ motifs of this protein. In the cell cycle, ADP-DnaA is probably reactivated for the next round of initiation of chromosomal replication. We hypothesized that an unknown factor releases ADP from DnaA. As concentration of ATP is 10-fold higher than ADP in the cell, ATP can bind to DnaA, reactivating this protein. As a result of comprehensive biochemical search, we identified a candidate of such DnaA-reactivating factor.
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