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The DnaA N ‐terminal domain interacts with Hda to facilitate replicase clamp‐mediated inactivation of DnaA
Author(s) -
Su'etsugu Masayuki,
Harada Yuji,
Keyamura Kenji,
Matsunaga Chika,
Kasho Kazutoshi,
Abe Yoshito,
Ueda Tadashi,
Katayama Tsutomu
Publication year - 2013
Publication title -
environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.954
H-Index - 188
eISSN - 1462-2920
pISSN - 1462-2912
DOI - 10.1111/1462-2920.12147
Subject(s) - dnaa , biology , mutant , dnab helicase , microbiology and biotechnology , atp hydrolysis , dna replication , biophysics , biochemistry , dna , origin of replication , enzyme , rna , gene , helicase , atpase
Summary DnaA activity for replication initiation of the E scherichia 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 ‐ N 44 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 .