AUTHOR=Katayama Tsutomu , Kasho Kazutoshi , Kawakami Hironori 

TITLE=The DnaA Cycle in Escherichia coli: Activation, Function and Inactivation of the Initiator Protein

JOURNAL=Frontiers in Microbiology

VOLUME=8

YEAR=2017

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2017.02496

DOI=10.3389/fmicb.2017.02496

ISSN=1664-302X

ABSTRACT=<p>This review summarizes the mechanisms of the initiator protein DnaA in replication initiation and its regulation in <italic>Escherichia coli</italic>. The chromosomal origin (<italic>oriC</italic>) DNA is unwound by the replication initiation complex to allow loading of DnaB helicases and replisome formation. The initiation complex consists of the DnaA protein, DnaA-initiator-associating protein DiaA, integration host factor (IHF), and <italic>oriC</italic>, which contains a duplex-unwinding element (DUE) and a DnaA-oligomerization region (DOR) containing DnaA-binding sites (DnaA boxes) and a single IHF-binding site that induces sharp DNA bending. DiaA binds to DnaA and stimulates DnaA assembly at the DOR. DnaA binds tightly to ATP and ADP. ATP-DnaA constructs functionally different sub-complexes at DOR, and the DUE-proximal DnaA sub-complex contains IHF and promotes DUE unwinding. The first part of this review presents the structures and mechanisms of <italic>oriC</italic>-DnaA complexes involved in the regulation of replication initiation. During the cell cycle, the level of ATP-DnaA level, the active form for initiation, is strictly regulated by multiple systems, resulting in timely replication initiation. After initiation, regulatory inactivation of DnaA (RIDA) intervenes to reduce ATP-DnaA level by hydrolyzing the DnaA-bound ATP to ADP to yield ADP-DnaA, the inactive form. RIDA involves the binding of the DNA polymerase clamp on newly synthesized DNA to the DnaA-inactivator Hda protein. In <italic>datA</italic>-dependent DnaA-ATP hydrolysis (DDAH), binding of IHF at the chromosomal locus <italic>datA</italic>, which contains a cluster of DnaA boxes, results in further hydrolysis of DnaA-bound ATP. SeqA protein inhibits untimely initiation at <italic>oriC</italic> by binding to newly synthesized <italic>oriC</italic> DNA and represses <italic>dnaA</italic> transcription in a cell cycle dependent manner. To reinitiate DNA replication, ADP-DnaA forms oligomers at DnaA-reactivating sequences (<italic>DARS1</italic> and <italic>DARS2</italic>), resulting in the dissociation of ADP and the release of nucleotide-free apo-DnaA, which then binds ATP to regenerate ATP-DnaA. <italic>In vivo, DARS2</italic> plays an important role in this process and its activation is regulated by timely binding of IHF to <italic>DARS2</italic> in the cell cycle. Chromosomal locations of <italic>DARS</italic> sites are optimized for the strict regulation for timely replication initiation. The last part of this review describes how DDAH and DARS regulate DnaA activity.</p>