A Nucleotide Switch in the Escherichia coli DnaA Protein Initiates Chromosomal Replication

Abstract

The ATP-bound DnaA protein opens duplex DNA at the Escherichia coli origin of replication, leading to a series of initiation reactions in vitro. When loaded on DNA, the DNA polymerase III sliding clamp stimulates hydrolysis of DnaA-bound ATP in the presence of the IdaB/Hda protein, thereby yielding ADP-DnaA, which is inactive for initiation in vitro. This negative feedback regulation of DnaA activity is proposed to play a crucial role in the replication cycle. We here report that the mutant protein DnaA R334A is inert to hydrolysis of bound ATP, although its affinities for ATP and ADP remain unaffected. The ATP-bound DnaA R334A protein, but not the ADP form, initiates minichromosomal replication in vitro at a level similar to that seen for wild-type DnaA. When expressed at moderate levels in vivo, DnaA R334A is predominantly in the ATP-bound form, unlike the wild-type and DnaA E204Q proteins, which in vitro hydrolyze ATP in a sliding clamp-and IdaB/Hda-dependent manner. Furthermore, DnaA R334A, but not the wild-type or the DnaA E204Q proteins, promotes overinitiation of chromosomal replication. These in vivo data support a crucial role for bound nucleotides in regulating the activity of DnaA during replication. Based on a homology modeling analysis, we suggest that the Arg-334 residue closely interacts with bound nucleotides.Chromosomal duplication occurs only once during the cell cycle and is regulated mainly by ingenious controls that act during the initiation of replication (1-3). The Escherichia coli initiator protein, DnaA, binds to the chromosomal origin of replication (oriC) and promotes a series of reactions leading to the formation of a replication fork (4, 5). This protein has high affinity for ATP and ADP, but only the ATP-bound form (ATPDnaA) can initiate replication at oriC. ATP-DnaA (but not ADP-DnaA) causes local unwinding of the oriC DNA duplex, which creates a site of entry for the DnaB helicase. DnaB helicase loaded on the unwound site forms a complex with DnaG primase and expands the single-stranded region. DNA polymerase (pol) 1 III holoenzyme is then loaded on the primed DNA and begins synthesis (6, 7). Several features of both DnaA and the oriC locus are important for the regulation of initiation.Immediately after initiation, re-replication of oriC is restrained temporarily (8). E. coli DNA is modified at the adenine residue of the palindromic sequence GATC by DNA-adenine methyltransferase, but newly replicated DNA remains hemimethylated until acted upon by DNA-adenine methyltransferase (9). The hemimethylated oriC locus is bound by the protein SeqA, which likely inhibits the initiation of replication until full methylation is re-established (10 -12). This eclipse time is speculated to persist for ϳ10 min under conditions in which the cellular doubling time is 30 min (9, 10).Also after initiation, DnaA is likely inactivated by the hydrolysis of bound ATP to yield ADP-DnaA (3). In vitro, ATP hydrolysis is promoted by the pol III ␤ subunit that is loaded onto DNA (the so-called sliding clamp)...

Functional dissection of YabA, a negative regulator of DNA replication initiation in Bacillus subtilis

“…In different organisms, such as E. coli, B. subtilis and C. crescentus, the control of DNA replication at the initiation step is mediated by different regulatory proteins. In E. coli, the well characterized DnaA-related Hda protein regulates initiation by promoting the hydrolysis of DnaA-ATP to produce the inactive DnaA-ADP (3,29,30). In vitro, this hydrolysis requires Hda interaction with DnaA and with DnaN loaded onto the DNA and involves a conserved arginine-containing motif in the AAAϩ proteins (21,31).…”

“…In Escherichia coli, considered as a paradigm for initiation studies in eubacteria, the regulatory inactivation of DnaA (RIDA) acts after initiation to promote the switch from the active ATP-DnaA to the inactive ADP-DnaA form. RIDA is mediated by the Hda protein and requires the slidingclamp DnaN (2)(3)(4)(5). A second level of regulation is mediated by the sequestration of the oriC region by the SeqA protein, which binds with a high affinity to hemimethylated GATC sequences within oriC, thus temporarily restraining reintitiation (6)(7)(8).…”

Formation of an ATP-DnaA-specific Initiation Complex Requires DnaA Arginine 285, a Conserved Motif in the AAA+ Protein Family

“…Strains, Nucleic Acids, and Purified Proteins-E. coli K12 strains KA413 (dnaA46), KA450 (⌬oriC1071::Tn10 rnhA199(Am) dnaA17(Am)), and KA451 (dnaA850::Tn10 rnhA::cat) are derivatives of KH5402-1 (wild-type) (22). WM433 (dnaA204) was previously described (22).…”

“…Mutagenic primers used for construction of pKW23-2 (bearing dnaA R281A) are 5Ј-GAGAT-CAACGGCGTTGAGGACGCGTTGAAATCCCGCTTCGGTTG and its complementary strand, and those for pKW24-1 (dnaA R285A) are 5Ј-GTTGAGGATCGTTTGAAATCCGCATTCGGTTGGGGACTGACTGTG and its complementary strand (mutations are underlined). Mutant DnaA was purified as previously described for wild-type and other mutant DnaA proteins (22,34). Briefly, KA450 cells bearing pKW24-1 were incubated at 37°C in 7.2 liters of LB medium containing thymine (50 g/ml) and ampicillin (50 g/ml), expression of mutant dnaA genes was induced by adding 1% arabinose to log phase cells, and incubation was continued for 2 h. KA450 cells bearing pKW23-2 were similarly incubated, except at 30°C, the growth rate of this strain was normal at 30°C, but significantly reduced at 37°C.…”

A transcriptional response to replication status mediated by the conserved bacterial replication protein DnaA

“…The fluctuation of ATP-DnaA levels is one of the mechanisms that regulates the timing of initiation of DNA replication (57,63,64). We suggest that cycling of ATP-DnaA levels couples replication and other physiological processes, including cell division and development.…”