The initiator protein DnaA , chromosomal origin oriC and their interaction to generate origin recognition complex and pre-replication complex like nucleoprotein structures in Escherichia coli

Introduction The assembly of precisely timed nucleoprotein structures comprising an initiator protein bound to a chromosomal origin of replication ensures that initiation of DNA synthesis at each origin occurs only once per cell cycle. The origin recognition complex (ORC) present at a chromosomal origin in yeast Sachromyces cerevisiae is one of the well studied example of such nucleoprotein structure. Recruitment of other proteins to an ORC generates a pre-replication complex (pre-RC), which subsequently recruits a helicase and unwinds DNA duplex. Studies suggest that spatial and temporal similarities exist in the formation of nucleoprotein complexes for both eukaryote and prokaryote. This review briefly introduces the Escherichia coli initiator protein, DnaA, recent findings on the E. coli chromosomal origin, oriC and concludes with an overview of their interactions to produce the nucleoprotein complexes involved in forming bacterial ORC and pre-RC like structures. Conclusion The structural and functional insights of E. coli initiator protein, DnaA and chromosomal origin, oriC, have advanced our understanding of the complex process of replication The initiator protein DnaA, chromosomal origin oriC and their interaction to generate origin recognition complex and pre-replication complex like nucleoprotein structures in


Introduction
In both eukaryotes and prokaryotes, initiation of DNA synthesis at chromosomal origins of replication is a highly dynamic process and requires the functional assembly of several proteins.For example, in Saccharomyces cerevisiae, binding of the multiunit Orc proteins (Orc1-Orc6 proteins) within a chromosomal origin comprises an origin recognition complex (ORC) 1,2 , which is maintained throughout the cell cycle.At the time of initiation, recruitment of other proteins to an ORC results in the production of a replication efficient pre-replicative complex (pre-RC) 3,4 .The subsequent conscription of helicase protein by helicase loader leads to melting of the duplex DNA and sets a platform for chromosome replication 5,6 .Formation of nucleoprotein complexes at the Escherchia coli chromosomal origin (oriC) is mediated by DnaA, an initiator protein [7][8][9] , which is suggested to be a homologue of S. cerevisiae, Orc1/cdc6 protein 10 .DnaA and several Orc proteins are members of the AAA+ protein family (ATPase associated with diverse cellular activities), containing conserved features such as nucleotide binding and DNA binding motifs 10 (Figure 1).DnaA protein binds to multiple asymmetric DnaA binding 9mer sequences, within E. coli oriC, known as DnaA binding elements 8,11,12 (Figure 2).Similar to these, chromosomal origins present in S. cerevisiae contain 11bp conserved consensus sequences termed ' A' elements and nonconserved 'B' elements 13 .These findings cause wonder on whether there is a similar nature to the nucleoprotein assemblies generated at origins present in both S. cerevisiae and E. coli.This review summarises recent details about the structure and organisation of E. coli oriC and its interaction with DnaA to form ORC and pre-RC like specialised nucleoprotein assemblies during the bacterial cell cycle.

DnaA, the initiator protein
DnaA protein initiates replication at the E. coli chromosomal origin and is conserved throughout bacteria 14,15 .E. coli DnaA protein is composed of 467 amino acids comprising a multidomain (domains I-IV) structure 16 (Figure 1).Domain I consists of amino acid residues 1-86, which has been found to be involved in proteinprotein interaction between DnaA subunits as well as with the other DnaA-interacting proteins 16,17 (Figure 1).Domain II is a flexible linker region (amino acid residues 87-134) 16 that has recently been shown to be essential for cell viability 18 .Further, amino acid residues 135-374 constitute domain III, which contains several signature sequences, such as Walker A, B and sensor I, II motifs involved in nucleotide binding functions 14,16 (Figure 1).Lastly, the carboxyl Licensee OA Publishing London 2013.Creative Commons Attribution License (CC-BY)

Critical review
terminus domain IV (amino acid residues 375-467) carries DNA binding functions 14,19 (Figure 1).Present in between domains III and IV is a small amphipathic helix (amino acid residues 354-374), identified to be associated with acidic phospholipids present in the bacterial membranes 20,21 .
DnaA protein has tight affinity for adenosine nucleotides, ADP and ATP (KD of 0.03 and 0.1 µM, respectively) 9 .Whereas, ADP-DnaA is replicatively feeble form of the protein, ATP-DnaA efficiently promotes initiation of DNA replication at the E. coli chromosomal origin 9 .In vitro acidic phospholipids such as phosphotidylglycerol (PG) and cardiolipin (CL) present in a fluid bilayer promote the conversion of inactive ADP-DnaA to replicatively proficient ATP-DnaA 22,23 .In vivo evidence links proper cellular levels of acidic phospholipids such as PG and CL, with continued cell growth 24,25 and normal chromosomal replication in E. coli 26 , whereas reduced levels of acidic phospholipids, result in arrested-growth 24,25 and inhibited chromosomal replication in otherwise wild-type E. coli 26 .
High resolution X-ray crystal structures of truncated ADP-DnaA 14 and ATP-DnaA 27 protein (domains III-IV) from thermophilic bacteria Aquifex aeolicus, have provided insight into the conformational differences between the two nucleotide forms of DnaA protein and explain how ATP-DnaA protein can accommodate and stabilise DnaA protomer interaction 14,27 .These crucial rearrangements introduced into DnaA protein by ATP binding are not sterically compatible within ADP-DnaA, which therefore is unable to oligomerise 14 at oriC.More recently, glutradehyde cross-linking suggests ADP-DnaA remains as a monomer in solution, while on the other hand, higher oligomeric structures within DnaA protein can be induced in presence of ATP as well as its non-hydrolysable analog like AMPPCP, ATPγS and ADP-BeF3 28 .The structural details obtained for an ORC complex in Drosophila melanogastor also suggest the characteristic of ATPdependent conformational changes in the Drosophila DnaA homologue 29 .

Chromosomal origin of replication in Escherichia coli
E. coli oriC is composed of a unique 245 base pair region, which can be further subdivided into a DNA unwinding element (DUE), which contains three

Critical review
present in DnaA protein and DNA is revealed by an X-ray crystal structure of DNA binding domain (DBD) of the E. coli DnaA protein in complex with a high affinity DnaA binding sites 19 .The DBD contains a helix-loop-helix motif for DNA binding, similar to the winged-helix motifs present in Orc proteins 10 .Certain amino acid residues present in the DBD, including Arg407, Gln408, Lys415, Ser421, Arg432, Asp433 and Thr436 (Figure 1) are shown to be important in formation of non-covalent bonds with the phosphodiester backbone of the major groove of DNA 19 .Substitution of these amino acid residues causes deficiencies in the DNA binding ability of DnaA protein 19.In addition to these residues, Arg399 (Figure 1) also forms a hydrogen bond with the phosphodiester backbone of the minor groove of DNA and has been identified as an essential residue in performing DnaA-dependent chromosomal replication 19 .
It is also evident from mutagenesis studies that Arg227 and Leu290 present in the AAA+ domain of DnaA protein play an important role in the formation of a DnaA-oriC complex required for oriC unwinding origin 30 .Interestingly, the subsequent conversion of dsDNA to ssDNA in the DUE region requires two distinct assembly states of DnaA protein on the chromosomal origin 28 .These events mark the generation of an open complex, a pre-requisite for the recruitment and assembly of other proteins required for replication.

Formation of ORC and pre-RC structures at Escherichia coli oriC
The nucleoprotein complexes produced by binding of ADP-DnaA and ATP-DnaA to high affinity DnaA recognition sites generate a structure similar to eukaryotic ORC 12,31 that remains present throughout the bacterial cell cycle 41 (Figure 3).Only at the time of initiation does ATP-DnaA occupy the low affinity recognition sequences, thereby to convert AT-rich 13mer repeats and the DnaA assembly region (DAR) 30,31 (Figure 2).Unlike DUE, which is functionally important for helicase loading and subsequent opening of DNA duplex, DAR contains high affinity DnaA recognition elements and low affinity DnaA binding sites 30,31 .The high affinity DnaA recognition elements harbor the 9mer consensus sequence, TGTG-GATAA and are termed 'R boxes', R1-R5 11,12,32 (Figure 2).In addition to R boxes, DAR also contains other DnaA recognition sequences known as 'I sites'-I1, I2 and I3, which are low affinity sites with nucleic acid sequence TG/TGGATCAG/A 12,32 (Figure 2).Additional low affinity sequences, tau sites (τ1 and τ2) and C1-C3 sites are also found within E. coli oriC 33,34 (Figure 2).
It has been well established that both ADP-DnaA and ATP-DnaA can bind to high affinity DnaA binding elements 8,11 .However, substitutions at the first two positions within high affinity R boxes weaken their interaction with either ADP-DnaA or ATP-DnaA and thus fail to promote origin unwinding 35 .In contrast to high affinity DnaA recognition sequences, low affinity sites prefer interaction with only ATP-DnaA 12,32 .Interestingly, this specificity can be eliminated by mutating the guanine at the third position of I2 and I3 12 .
Further, DAR also houses elements that can bind to the nucleoid associated protein factors, initiation host factor (IHF), histone like protein HU and FIS protein (factor for inversion stimulation) 30,31 (Figure 2).DAR has been further differentiated into four sub-regions 30,31 : (i) DUE-flanking (DF) that confers R1 and IHF binding site, (ii) left low affinity (LL) that includes τ1, R5, τ2, I1 and I2 sites, (iii) right low affinity (RL) that contains R2, C3, R3, C2 sequences and FIS binding elements and (iv) right edge (RE), which carries I3, C1 and R4 DnaA binding sequences (Figure 2).The DUE, DF and LL regions constitute left half, whereas RL and RE compose right half of the E. coli oriC 30,31 .A recent finding established that the right half of the DAR-DnaA sub-complex is important for stimulating DnaB helicase loading 30 , while the DUE flanking left half produces a DnaA-oriC sub-complex competent in DUE unwinding and DnaA-DnaC assisted loading of helicase protein DnaB onto single stranded DUE 30 .Interestingly, it has been observed that deletion of the right half of E. coli oriC does not affect function of the origin present in bacterial cells growing on minimal media 36 .This is due to the fact that the time needed for the completion of chromosomal replication is shorter than the cell generation time 36 .However in rich media, the generation time of fast growing cells is approximately one third to the time required to complete the replication process and therefore the requirement of a full length oriC to support initiation on a multi-forked chromosome 36 .Indeed the growth conditions for E. coli determine the cell mass, DNA content and more particularly the number of DnaA recognition boxes required to form adequate nucleoprotein structures respectively at single versus multi-forked chromosomes so as to compensate with the pace of initiation of replication 36 .

DnaA-DNA interaction
Filter retentions 9 , gel mobility shift 37 , Dnase I footprint 32,38 and di-methyl sulphate footprint 12,32 assays have revealed different functional aspects of the interaction between DnaA protein and chromosomal origin.It is to be noted that the stoichometery of DnaA bound to oriC varies depending on the method of detection 8,9,37,39 .Electron microscopy 8,40 and filter retention 9 studies suggest 15-30 DnaA protein molecules per oriC, while gel mobility 37 and gel filtration 39 experiments support 5-8 DnaA molecules at an origin.
Detailed insight into the nature of interactions between amino acids ORC into a higher order nucleoprotein structure that has been compared to a eukaryotic pre-RC 12,32 (Figure 3).The formation of pre-RC in E. coli occurs in the presence of nucleoidassociated DNA bending proteins, HU and IHF, which produce replication proficient assemblies 42,43 (Figure 2).HU protein exists as either a homodimer (αα or ββ) or a heterodimer (αβ) and remains bound to DNA non specifically.The alpha subunit of HU protein has recently been shown to interact with the N-terminal region of DnaA and HUαα-homodimer is found to be more active in promoting DNA strand opening 44 .Aided by the helicase loader DnaC, which interacts with oriC bound ATP-DnaA, DnaB helicase is loaded onto the nucleoprotein structure, enabling subsequent bi-directional unwinding and replication fork formation 30 .
It has been recently established that the propagation of DnaA protein from high affinity sites to weak affinity sites, occurs during the conversion of ORC to pre-RC 45 , Interestingly, occupation of specific sequences present in the right and left half of oriC is sequential and polarised 34 .The binding of DnaA protein emanates from R4 to R2 within the right half and assembly expands from R1 to R2 in the left half of oriC 34 .Notably, the array of sequences present in the left (DF and LL) and right half (RL and RE) of oriC are placed in a similar orientation 34 .It has also been reported that any changes in the orientation or spacing of these low affinity DnaA binding sites in either array disrupt DnaA binding and thus prevent ORC to pre-RC conversion 34 .
Beside the specific arrangement of DnaA binding elements present within oriC, a mutation, DnaA(L366K) 46 , present in the membrane binding domain of DnaA protein (Figure 1) also prevents ORC to pre-RC conversion 32 .In-fact in vitro, an ADP-DnaA ORC can not support the loading of ATP-DnaA(L366K) to low affinity sites 32 .On the other hand, an ADP-DnaA(L366K) ORC can successfully be converted to a pre-RC in the presence of ATP-DnaA protein 32 .These properties of DnaA(L366K) protein has been attributed to its altered interaction with low affinity recognition sequences.

Discussion
The author has referenced some of its own studies in this review.The protocols of these studies have been approved by the relevant ethics committees related to the institution in which they were performed.
Initiation of replication at chromosomal origins in both eukaryotes and prokaryotes is a complex and highly regulated process.This involves the interaction of an initiator protein with a chromosomal origin to produce several specialised nucleoprotein assemblies as precisely timed events during the cell cycle.
One might think that replication initiation in eukaryotes and prokaryotes would have major differences, such as the presence of a single chromosomal origin in E. coli compared to multi chromosomal origins in higher organisms necessitated by the larger genomes of the latter.However, with recent advancements in the understanding of initiation mechanisms, the Critical review process of initiation is now considered to be well conserved in all genera of life, particularly in terms of the characteristic of the nucleoprotein complexes generated at origins.In fact, initiator proteins present in archeae to bacteria to multi-cellular organisms belong to the AAA+ super family of proteins containing a conserved core with characteristic nucleotide binding motifs.Moreover, the DBD domain of E. coli DnaA protein contains helix-loop-helix motifs similar to winged helix domains of eukaryote initiator proteins, therefore suggesting a similar nature in the macromolecular assemblies generated at chromosomal origins.

Conclusion
Remodelling of the E. coli origin proceeds in three different steps: (i) occupancy of high affinity DnaA recognition sequences to form ORC, (ii) extension of ATP-DnaA molecules from ORC to low affinity DnaA recognition sites, thereby generating pre-RC and (iii) melting of the DNA duplex in the DUE region producing ssDUE for helicase loading.The structural and functional insights of E. coli initiator protein, DnaA and chromosomal origin, oriC, have advanced our understanding of the complex process of replication initiation in bacteria and helped in highlighting the similarities and differences that exist between replication processes in prokaryote and eukaryote.

Figure 1 :
Figure 1: Schematic representation of the functional domains of Escherichia coli DnaA protein (domains I-IV) and important regions (walker A, walker B, Sensor I and Sensor II motifs) involved in nucleotide binding.The membrane binding amphipathic helix is also indicated.

Figure 3 :
Figure 3: (A) Replication inefficient ADP-DnaA remains bound to high-affinity sites on oriC to form an ORC that persist throughout bacterial cell cycle.(B) At the time of initiation, replication proficient ATP-DnaA accumulates on lowaffinity sites following its donation from high affinity sequences, generating a pre-RC, a prerequisite for DUE unwinding.(C) The helicase loader, DnaC, interacts with ATP-DnaA, with the DnaC-DnaA complex assisting the loading of DnaB helicase on to the chromosome to produce bi-directional replication forks.