This appears to be the consequence of a handful of "initiator" or DnaA boxes within oriC which have a four-fold higher affinity for DnaA-ATP over DnaA-ADP |CITS: [14978287][16298387]|. In line with the model of these particular DnaA boxes being gatekeepers for initiation of replication, E. coli win which normal DnaA boxes have been replaced with additional "initiator" sequences are subject to asynchronous and overly frequent replication initiation |CITS: [17850252]|. More generally, all the DnaA boxes in oriC play a role in proper regulation of initiation, as does their spacing and orientation |CITS: [8858585][7667087][8022267][1603077][1291230][2830261]|. The transition between the "closed" and "open" origin is made sharper by the coordinated action of Fis, IHF, and DnaA. Fis initially blocks access by both IHF and DnaA, but as DnaA abundance increases, Fis is kicked off and IHF and DnaA bind the remaining DnaA boxes en masse, starting the opening process |CITS: [14982629]|. Once oriC has begun to unwind, DnaA-ATP binds with significantly higher affinity to the newly revealed single-stranded DNA than to the remaining double-stranded DNA, whereas DnaA-ADP shows no difference in affinity |CITS: [11250912]|. After unwinding the double helix at oriC, DnaA faciliates the loading of the replicative helicase DnaB onto two nascent replication forks. DnaA binds to DnaB, bringing it into the area of the unwound oriC |CITS: [8106460][627535]|. The complete "prepriming complex" involves 10 DnaA monomers and 2 DnaB hexamers per oriC, meaning that there is one replicative helicase (DnaB hexamer) per new replication fork |CITS: [11551962]|. Although the binding of DnaB and its loader to the oriC region depends on having available single-stranded DNA from the newly unwound helix, DnaA binding does not, meaning that in the prepriming complex DnaA monomers are bound to both the wound and unwound regions of the origin |CITS: [12161435]|. Although there will eventually be a helicase loaded onto each replication fork, loading that is directly attributable to the DnaA-DnaB interaction only occurs on one of the two strands |CITS: [12421318]|. Presumably the replication bubble is effectively "held open" by the first helicase loaded, allowing access for a second one. Following the generation of this oriC-DnaB prepriming complex, DnaA's role in initiation is complete and it is no longer required |CITS: [2153124]|. DnaA oligomerizes extensively as part of carrying out its role in initiating replication |CITS: [15878847]|. The final prepriming complex has been reported to comprise 10-30 DnaA monomers, with complete occupancy of all oriC DnaA boxes |CITS: [11551962][8663334][12864864]|. As described above, this oligomerization process appears to start with binding at high-affinity DnaA boxes followed by "spidering" out to lower-affinity sites across the whole of oriC. Proper oligomerization and subsequent opening of oriC depends on DiaA, which forms a tetramer that binds multiple DnaA monomers, suggesting that DiaA may act as a coordinator of DnaA oligomers |CITS: [15326179][17699754]|. DiaA appears unlikely to be part of some sort of DnaA-DiaA complex, as DiaA blocks DnaB loading, implying that at some point after oligomerization of DnaA, DiaA exits the picture |CITS: [19632993]|. Initiation of DNA replication has been proposed to be regulated by several mechanisms that operate through DnaA and oriC. These include control over the ATP/ADP status of DnaA, sequestering of the origin of replication, and titration of available DnaA. The ATP- versus ADP-bound status of DnaA has a major impact on its ability to operate as an initiator of DNA replication. Although DnaA binds both ATP and ADP with high affinity, only the ATP-bound form is able to initiate replication under normal circumstances, as described above |CITS: [3036372][9125116][2835363]|. This difference is not due to binding, as both forms can bind at oriC, but instead is a function of DnaA-ATP being the only form with the ability to prompt unwinding of the double helix |CITS: [8377183]|. Averaged across the entire cell cycle, some 15-30% of all DnaA is typically in ATP-bound form. This abundance rises across the course of the cell cycle, topping out at about 80% just ahead of DNA division and cell division |CITS: [10581238]|. The primary limiter of the abundance of DnaA-ATP is the DnaA regulator Hda, acting in concert with the beta clamp of DNA Pol III |CITS: [7721846][9473485][9674428][10581238][16882985]|. Hda acts as a bridge between the beta clamp of the polymerase and DnaA. Two Hda homodimers bind per clamp via the Hda amino-terminus, and then Hda interacts with DnaA via the DnaA amino-terminus. When this complexing occurs, ATP hydrolysis is stimulated, converting DnaA-ATP to DnaA-ADP |CITS: [15150238][15611053][11254969]|. ATP hydrolysis is inhibited when DnaA is bound to a DnaA box, but this inhibition is overcome by Hda-clamp binding and active DNA synthesis |CITS: [15189445][11309120]|. This suggests that either this interaction is tethering DnaA to non-DnaA-box DNA, or that it may let the DNA polymerase complex drag DnaA off of a DnaA box. This conversion of DnaA-ATP to its ADP state is required to prevent premature or asynchronous initiation of DNA replication |CITS: [15939703][16041320][16321957][12730188][11483528]|. DnaA-ADP is regenerated to its ATP-bound form via an interaction with phospholipids. Cardiolipin and various unsaturated fatty acids in relatively fluid membranes dissociate ADP from DnaA, leaving DnaA available for binding by new ATP |CITS: [2835364][8227025]|. Phospholipids lacking unsaturated fatty acids only poorly promote the release of ADP |CITS: [2845401]|. DnaA binding to oriC is important for stabilizing the protein during this process |CITS: [1512219]|. Conversely, the presence of various phospholipids in advance of binding blocks oriC binding by DnaA |CITS: [12366847]|. The carboxy-terminal portion of DnaA appears to actually enter the membrane during this regeneration process |CITS: [9478970]|. This aligns well with the observation that DnaA localizes to the cell membrane |CITS: [10762265]|. The regeneration of DnaA-ADP to DnaA-ATP is also promoted by two intergenic regions on the chromosome labeled DARS1 and DARS2. Each of these regions contains many DnaA sites, promoting the oligomerization of DnaA in such a manner as to enhance the release of ADP |CITS: [19401329]|. Newly synthesized DNA within oriC and the nearby dnaA locus remains hemimethylated significantly longer than other newly synthesized DNA. This was originally proposed as a mechanism of delaying initiation of new DNA replication, as hemimethylated DNA is not transcription competent |CITS: [1697508]|. Based on analyses carried out using oriC DNA on a plasmid SeqA was observed to bind this hemimethylated DNA and inhibit DnaA binding |CITS: [11122375]|. However, mutants lacking any seqA activity see only a modest increase in initiation of replication, and methylation state of sites within the dnaA promoter region has no impact on replication timing |CITS: [16041320][16740964]|. It does appear that SeqA facilitates oriC reset by blocking access of DnaA to the low-affinity DnaA boxes that are typically only occupied during the initiation of replication |CITS: [17114060]|. There are a number of other DnaA boxes throughout the E. coli genome beyond those within contained oriC and the DARS regions. One theory of regulation of DnaA activity as an initiator of DNA replication is that DnaA monomers are titrated out by binding to many of these alternate DnaA boxes. The major proposed DnaA titration site is datA. The datA site contains multiple DnaA boxes, and titrates out DnaA in a box-dependent manner. Mutations within this site lead to asynchronous and overly frequent initiation of DNA replicatio|CITS: [9765205][11690638][12068813]|. When additional datA sites are introduced into E. coli, DNA replication and cell division are delayed, followed by an induced SOS response and incomplete replication at especially high doses of datA |CITS: [14507385]|. However, given that cells are viable and DNA replication can be initiated in the absence of a datA site, it has been suggested that the role of datA titration of DnaA is in timing of initiation relative to cell growth, rather than in blocking incorrect initiation completely |CITS: [15939703][16041320]|. The DnaA boxes involved in titrating DnaA are structurally distinct from those involved in binding of oligomeric DnaA to oriC |CITS: [17316685]|. However, much like oriC, the DnaA boxes within datA contain an interstitial IHF binding region which must be intact to allow proper function of datA in regulating replication initiation |CITS: [19170757]|. DnaA is presented at approximately 800-2,100 molecules per cell. Although this number varies across different E. coli strains, it appears to be fairly consistent across differing growth conditions within a given strain |CITS: [1860829]|. The amount of DnaA per cell was decreased/increased in strains that were deleted for or overproduced RstA, respectively. RstA may regulate the expression of dnaA and indirectly affect DNA replication |CITS:[30011323]|. DnaA is involved in a number of processes beyond cellular DNA replication. DnaA is required for replication of many plasmids, including pSC101, pBR322, ColE1, F plasmid, R1, RK2, and mini-F plasmids such as pSC183, pKP1013, and pKV513 |CITS: [6091903][3020005][3931918][3037524][2820940][2844794]|. Mutations in dnaA can also lead to a decrease in linking number in resident plasmids |CITS: [8573119]|. It is also involved in the life cycle of phages such as f1, lambda, and Coliphage 186, as well as being required for robust Tn5 transposition |CITS: [4601460][7033562][2820938][7867947][9819062]|. DnaA belongs to the DnaA family of transcriptional regulators and it is subject to autoregulation, as increased abundance of DnaA represses transcription of dnaA |CITS: [2995766][2981626][3025553][2828882][8407830]|. This occurs via binding of DnaA to DnaA boxes at the dnaA locus, where the DnaA-DnaA box complex directly blocks transcription |CITS: [2854096][2548849][2088506][8995231]|. An internal DnaA box within the actual dnaA gene appears to play no role in modulating transcription, however, and it has been suggested that at least one other DnaA box at the locus is also dispensible for autoregulation |CITS: [7896719][9106220]|. DnaA and LexA may coordinate DNA replication with DNA repair, and the control mode may be conserved within Gram-negative bacteria |CITS:[29967594]|. A model for control of the SOS regulon by DnaA and LexA is proposed where both DnaA and LexA repress expression of the SOS genes when SOS is off |CITS:[29967594]|. Binding of DnaA to other DnaA boxes can similarly impact regulation of other genes, including rpoH and guaA |CITS: [2540187][1736096]|. The structure of DnaA has been examined in great detail, with an emphasis on how it relates to DnaA's function within the cell. The amino-terminal portion of DnaA is required for replication, but not for ATP or oriC binding |CITS: [9852089]|. This requirement for replication is likely due to the fact that the amino-terminal domain is both necessary and sufficient for oligomerization of DnaA, and is required for the interaction between DnaA and DnaB |CITS: [10540285][10972842]|. The amino-terminal region contains a number of residues whose hydrophobicity is required for proper DnaA function |CITS: [12132668]|. The flexible linker Domain II within DnaA has been evaluated via systematic deletion, revealing a minimum length requirement of 21-27 residues |CITS: [18957591]|. This domain is required for recruiting DnaB to oriC |CITS: [19546317]|. The Box VII domain within DnaA is required for oligomerization |CITS: [15371441]|. Phospholipid binding occurs via the region comprising residues 115-381 |CITS: [8670850][11102450]|. This region is an amphipathic helix that inserts into the membrane to activate the release of ADP |CITS: [9786858]|. ATP binding takes place near K415, R328, and K372 |CITS: [11700030][11853554]|. The carboxy-terminal 94 amino acids are both necessary and sufficient for binding to DnaA boxes in general and oriC in particular. This binding domain contains three α helices |CITS: [7744016][9417934][10844646]|. Circular dichroism and NMR analysis of the DNA-binding domain of DnaA has been carried out |CITS: [12435387]|. NMR analysis has also identified the specific binding surface within this DNA binding domain |CITS: [14523560]|. The DnaA amino-terminal region has also been evaluated via NMR |CITS: [17420252]|. The crystal structure of DnaA's DNA-binding domain complexed with an example DnaA box has been resolved to 2.1 Å resolution |CITS: [12682358]|. An experimental molecular weight of 48 kD has also been reported for DnaA |CITS: [6258023]|. The ectopic expression of a mutant of DnaA (L366K) restores the cellular growth that is caused by the accumulation of the unprocessed outer membrane |FRAME: CPLX0-8279| Lpp (pro-Lpp) |CITS: [34163454]|. This growth restoration appears to be a consequence of the repression of pro-Lpp expression by the |FRAME:RNA0-386 | |CITS: [34163454]|.