The DnaA N-terminal domain interacts with Hda to facilitate replicase clamp-mediated inactivation of DnaA.
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| Title: | The DnaA N-terminal domain interacts with Hda to facilitate replicase clamp-mediated inactivation of DnaA. |
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| Authors: | Su'etsugu, Masayuki1, Harada, Yuji1, Keyamura, Kenji1, Matsunaga, Chika1, Kasho, Kazutoshi1, Abe, Yoshito2, Ueda, Tadashi2, Katayama, Tsutomu1 |
| Source: | Environmental Microbiology. Dec2013, Vol. 15 Issue 12, p3183-3195. 13p. |
| Subjects: | DNA, DNA replication, Escherichia coli, Adenosine triphosphate, Solvolysis, Hydrolysis, Molecular biology |
| Abstract: | DnaA activity for replication initiation of the Escherichia coli chromosome is negatively regulated by feedback from the DNA-loaded form of the replicase clamp. In this process, called RIDA (regulatory inactivation of DnaA), ATP-bound DnaA transiently assembles into a complex consisting of Hda and the DNA-clamp, which promotes inter- AAA+ domain association between Hda and DnaA and stimulates hydrolysis of DnaA-bound ATP, producing inactive ADP- DnaA. Using a truncated DnaA mutant, we previously demonstrated that the DnaA N-terminal domain is involved in RIDA. However, the precise role of the N-terminal domain in RIDA has remained largely unclear. Here, we used an in vitro reconstituted system to demonstrate that the Asn-44 residue in the N-terminal domain of DnaA is crucial for RIDA but not for replication initiation. Moreover, an assay termed PDAX (pull-down after cross-linking) revealed an unstable interaction between a DnaA- N44 A mutant and Hda. In vivo, this mutant exhibited an increase in the cellular level of ATP-bound DnaA. These results establish a model in which interaction between DnaA Asn-44 and Hda stabilizes the association between the AAA+ domains of DnaA and Hda to facilitate DnaA- ATP hydrolysis during RIDA. [ABSTRACT FROM AUTHOR] |
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| Database: | Engineering Source |
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