The initiator is the family member who first recognizes a need or starts the purchase process.

3.1 Replication Initiation is a Stepwise Process that Begins with the Formation of a Complex of DnaA Assembled at oriC

DnaA protein initiates DNA replication by forming a specific DnaA-oriC complex. The types of high and low-affinity sites, the influence of the adenine nucleotide bound to DnaA on recognition of these sites, and evidence that describes the structure of this complex have been summarized above. Structure–function studies of DnaA indicate that it has four functional domains (Fig. 2). Domain IV is necessary and sufficient for binding to the DnaA box motif. Specific residues that confer DNA binding activity as well as specificity in DNA binding have been identified by molecular genetic methods [84,85]. Structures of this domain bound to the DnaA box complement the analyses described above [15,42], and also identify amino acid residues that contact nucleotides flanking the DnaA box sequence. As mentioned above, domains I and III contribute to DNA binding by promoting cooperative interactions between neighboring DnaA molecules assembled at oriC [24,25,86].

2.6.4 DnaA

The dnaA gene which encodes for DnaA protein is considered a cold-inducible protein and possesses both DNA binding/replication initiator properties and acts as a global regulator of transcription (Barria et al., 2013; Gualerzi et al., 2003). The DnaA protein is centrally involved in the initiation of chromosomal and mini-chromosomal DNA replication on oriC and appears to be important in the timing control of cell-cycle initiation (Atlung, Clausen, & Hansen, 1985; Messer & Weigel, 1997). It also autoregulates the dnaA gene and influences cell membrane structural properties (Atlung et al., 1985; Atlung & Hansen, 1999; Braun, O'Day, & Wright, 1985; Kaguni, 2006; Messer & Weigel, 1997; Wegrzyn & Wegrzyn, 2002; Węgrzyn, Wrobel, & Węgrzyn, 1999). Messer and Weigel (1997) have summarized the role of DnaA protein as a transcription factor which depending on the target gene promoter location can serve as a transcriptional activator, repressor, or terminator. Atlung and Hansen (1999) concluded that DnaA was involved in cold shock after demonstrating the levels of DnaA protein when E. coli was shifted from 37°C to 14°C were twofold higher even though there were indications some of the synthesized protein was irreversibly inactive or that all present at the lower temperature generally exhibited irreversible low activity. Less is known about DnaA in Salmonella, but when the dnaA sequences were compared between E. coli and S. Typhimurium they were relatively homologous and functionally were presumed to behave in a similar fashion by the authors (Skovgaard & Hansen, 1987). Further work on regulatory mechanisms associated with Salmonella dnaA has been done more recently. When Dadzie et al. (2013) conducted transcriptomic deep sequencing profiles of S. Typhi they identified a cis-encoded antisense RNA expressed primarily during stationary phase and contributed to the stability of dnaA mRNA. Expression of this antisense RNA was also observed in the presence of iron limitation and osmotic stress but cold shock was not examined in this study.

The DnaA–oriC Complex

As an adenine-nucleotide-binding protein, DnaA contains the amino acid sequence motifs shared by the AAA+ family of proteins. These motifs are the Walker A and B boxes, which respectively function in nucleotide binding and in coordinating magnesium chelated to the phosphates of the nucleotide, and sensor I, II, and box VII motifs that are considered to both promote and coordinate ATP hydrolysis with a conformational change. As predicted in the initiator titration model, initiation occurs at a particular cell mass when the abundance of DnaA reaches the level necessary for initiation. Thus, DnaA binds to the high-affinity sites early in the bacterial cell cycle. As ATP is more abundant than ADP in vivo, DnaA that is newly synthesized more likely binds ATP over ADP. As DnaA–ATP accumulates, it binds to the low-affinity sites, which include the R3 box and the I- and τ-sites, forming the DnaA–oriC complex. In support, in vivo footprinting experiments in synchronized cells reveal that DnaA is bound to R1, R4, and R2 throughout the cell cycle, and that R3 becomes protected at the time of initiation. When this study was performed, the other low-affinity sites had not been discovered.

In vivo and in vitro evidence indicates that formation of the DnaA–oriC complex requires an interaction between and/or among DnaA molecules. Two regions within DnaA are required. Based on studies of a mutant DnaA carrying an alanine substitution for tryptophan 6 (W6A), one region appears to be a hydrophobic surface near the N-terminus. This mutant protein is defective in an assay (see later) that measures the assembly of DnaA at oriC in a supercoiled plasmid followed by the recruitment of DnaB complexed to DnaC. Other studies that rely on footprinting methods showed that this mutant DnaA fails to protect the I- and τ-sites within oriC. Together, these results suggest that the hydrophobic surface functions in DnaA oligomerization to stabilize the interaction between and/or among DnaA molecules bound to the I- and τ-sites. The second region was identified via the analysis of a mutant bearing an alanine substitution for a conserved arginine at residue 281 (R281A) in the box VII motif within domain III. Like the W6A-substituted protein, the R281A mutant was poorly retained on a supercoiled plasmid containing oriC, suggesting that this arginine is necessary for self-oligomerization of DnaA. The latter observations correlate with structural studies of Aquifex aeolicus DnaA (lacking residues 1–76), in which domain III of one molecule interacts with this domain in the adjacent molecule in the DnaA oligomer. In this structure, DnaA assembles as a right-handed helical filament in which a separate arginine at position 285 in box VII interacts with ATP bound between adjacent protomers.

The Bacterial Initiator DnaA and bORC

All bacterial origin firing depends on higher order assemblies of the DnaA protein initiator, but each oriC recognition site is occupied by a DnaA monomer (Speck et al., 1997; Weigel et al., 1997). DnaA's C-terminal domain (IV) with helix-turn-helix structure (Erzberger et al., 2002) is responsible for double-stranded DNA binding (Schaper and Messer, 1995), and signature amino acids of DnaA make base-specific contacts (Fujikawa et al., 2003; Fig. 2). DnaA is active in the ATP-bound state (Sekimizu et al., 1987) and the amino acids required for ATP-binding and hydrolysis are localized within an internal domain (III) (Fig. 2). Proteins that interact with DnaA usually contact the N-terminus (domain I) (Simmons et al., 2003), located on the end of a flexible stalk (domain II) whose length varies among bacterial types (Messer et al., 1999; Zawilak-Pawlik et al., 2005; Fig. 2).

Both ATP and ADP forms of DnaA are capable of self-oligomerization mediated by domain I-domain I interactions (Simmons et al., 2003; Zawilak-Pawlik et al., 2017). DnaA-ATP also self-oligomerizes when an arginine finger in one molecule's domain III contacts the ATP bound to an adjacent protomer, combined with additional domain III contacts (Erzberger et al., 2006; Fig. 2). The DNA-binding domain (domain IV) of DnaA-ATP also folds up toward domain III, helping to stabilize the intermolecular interactions, leading to the formation of a compact helical filament (Duderstadt et al., 2011). Compact DnaA-ATP filaments bind single-stranded DNA through a central channel formed by domains III and IV, and are reported to melt short DNA duplexes, presumably by stretching the helix (Duderstadt et al., 2011). However, the compact helical filament has very low affinity for double-stranded DNA (Duderstadt et al., 2011) and if a DnaA oligomer is formed on double-stranded DNA during pre-RC assembly, it must have an alternative structure that is not yet characterized.

DnaA does not readily form into complexes in the absence of DNA (Duderstadt et al., 2010), and for this reason, a true structural equivalent of eukaryotic ORC (see below) does not exist. However, it is possible to define the bacterial ORC (bORC) as the DnaA-oriC complex that, like the eukaryotic ORC, persists during extended periods of the cell cycle and serves as a scaffold for the recruitment of additional proteins that lead to origin DNA unwinding and helicase loading (Miller et al., 2009). Surprisingly, on native supercoiled DNA, the DUE of oriC spontaneously unwinds in the absence of associated protein (Kowalski and Eddy, 1989), and the bORC constrains oriC DNA to prevent unwinding until a complete pre-RC is assembled (Kaur et al., 2014). In E. coli, bORC comprises DnaA occupying the available three high affinity DnaA recognition sites (R1, R2, and R4) (Kd in the range of 4–20 nM) (Miller et al., 2009; Fig. 3). Since these recognition sites in E. coli are widely separated, DnaA-DnaA interactions within ORC are likely to form DNA loops. Clustered high affinity DnaA recognition sites are found within most bacterial oriCs (Gao et al., 2013) and these sites are also expected to be occupied in a similar fashion to E. coli, unless the availability of DnaA were extremely limiting. Each DnaA molecule in ORC is able to recruit additional DnaA molecules and nucleate the formation of DnaA oligomers during pre-RC assembly, see below Miller et al. (2009) and Rozgaja et al. (2011).

Macroinitiation

Several features regarding this region are notable. There are multiple binding sites for the DnaA protein (the DnaA boxes) (Figure 1). This is a nine-nucleotide sequence that has been shown to bind the DnaA protein. There are also multiple promoter elements, suggesting the involvement of RNA polymerase in macroinitiation. Possible DNA-bending sites for proteins that bend DNA, notably FIS and IHF, are also present. Another notable feature is the presence of multiple Dam methylase sites (GATC sequence). As Dam methlyases are not universally found throughout the prokaryotic world, the use of such sites for the regulation of initiation of DNA replication is a unique adaptation among E. coli strains and closely related bacteria.

The definition of the oriC region rests on cloning of this region into plasmids constructed so that replication of the plasmid depends on function of the oriC sequence. This has allowed development of an in vitro assay system for macroinitiation of DNA replication. The cloning of oriC confirmed that the specificity of macroinitiation in E. coli resides in the origin.

The primary protein actor in macroinitiation is the DnaA protein. DnaA belongs to a class of proteins termed AAA+ (ATPases associated with a variety of cellular activities), which generally oligomerizes and undergoes conformational changes as ATP is hydrolyzed to ADP. As noted below, the ‘clamp-loading’ γ complex (also referred to as the DnaX complex) of the Pol III holoenzyme also includes AAA+ proteins. DnaA binds at multiple sites within the oriC structure as noted. Its role in initiation is a change of conformation in the A-box region, leading to torsional stress on the adjacent AT-rich region, driven by the orchestrated oligomerization of DnaA–ATP bound to the DnaA boxes. The DnaA protein also binds in the promoter region of the DnaA gene itself, suggesting autoregulation, which is supported by genetic studies. Autoregulation of DnaA maintains steady-state levels of DnaA protein, rather than to directly regulate macroinitiation. In any event, it appears that the DnaA protein must act positively to initiate DNA replication in E. coli.

Both protein and RNA syntheses are required for macroinitiation to occur in E. coli. The macroinitiation phase may be further subdivided into stages. The earliest step involves the binding of the DnaA protein to the oriC structure. Again, it is the ATP-bound form of DnaA that participates in macroinitiation. ADP-bound DnaA is incapable of initiation, which provides one mechanism for preventing unscheduled reinitiation during the replication cycle. The conformational change of the origin, coordinated by the binding of DNA-bending proteins FIS, IHF, and possibly HU, opens up the adjacent AT-rich region, forming a small ssDNA bubble that allows loading of the hexameric DnaB helicase onto the origin by another AAA+ family member, DnaC, utilizing ATP. Once the DnaB hexamer is loaded, ATP bound to DnaC is hydrolyzed, and DnaC dissociates from DnaB. The dissociation of DnaC from DnaB frees the helicase to commence unwinding of DNA at the origin in an ATP-dependent fashion, further enlarging the ssDNA bubble. Concomitant with opening of the ssDNA bubble at the origin is the binding of single-strand binding (SSB) protein, which allows priming by DnaG primase, much as it occurs in the elongation phase of DNA replication. There is a trade-off of primase and the χ subunit of the γ complex, both of which bind SSB. This allows the concerted assembly of the Pol III catalytic core, the γ complex, and the β sliding clamp at the newly synthesized RNA primer terminus. The γ complex, as noted above, comprises subunits γ, τ, δ, and δ′, all of which are members of the AAA+ family of proteins, along with the additional subunits χ and ψ̃. The role of the γ complex is to open the dimeric β clamp and load it onto the DNA template. Specific roles of the individual subunits have been identified. The ATPases driving the ring-opening reaction are located in the γ and τ subunits, whereas the δ subunit ultimately pops the β clamp open. Various combinations of γ and τ subunits have been described in the γ complex, but the processive Pol III holoenzyme (Pol III*) is thought to contain two τ proteins and one γ, hence, τ2γδδ′χψ. The two τ subunits link DnaB and the catalytic α subunit of Pol III, thus coupling replication fork movement to both leading and lagging strand syntheses. After assembly of the complete replisome, this stage is followed by the propagation of microinitiation and elongation phases of replication.

Thus, the proteins required for the macroinitiation of E. coli DNA replication appear to include DnaA and DnaC in specific roles, as well as DnaB, SSB protein, gyrase, the DnaG primase protein, and the replicative apparatus of DNA polymerase III holoenzyme complex (see ‘Holoenzyme DNA polymerase III’).

The DnaA protein offers important support for the replicon hypothesis. Mutations in the DnaA protein demonstrate that all of the E. coli chromosome is under a unit control mechanism, defining it as a single replicon. Integration of certain low copy number plasmids into the chromosome suppresses the phenotype in DnaA mutants that were defective in macroinitiation. This ‘integrative suppression’ shows a general control of macroinitiation and supports the replicon hypothesis.

Prevention of reinitiation of replication at oriC is through tight regulation of available ATP-bound DnaA protein and restricted access to oriC once the origin has fired. Proposed mechanisms include a conformational change of DnaA to an inactive ADP-bound form once initiation has proceeded (termed ‘RIDA’, for regulatory inactivation of DnaA). Supporting this model, purification and characterization of yet another AAA+, Hda, has established that hydrolysis of ATP bound to DnaA is stimulated by Hda, and this is an integral event in RIDA. Moreover, while steady-state levels of DnaA protein remain fairly constant, the ratio of ATP–DnaA to ADP–DnaA changes over the E. coli cell cycle, peaking at about the time of macroinitiation and then falling. Once macroinitiation is under way, ATP is hydrolyzed, and oriC with the ADP-bound DnaA reverts to its closed conformation. Competition between the DNA-bending proteins IHF and FIS may also play a role in the assembly of oligomerized ATP–DnaA at oriC, with IHF proposed to enhance binding of DnaA to low-affinity sites, and FIS proposed to hinder binding of DnaA to low-affinity sites within oriC; at a certain threshold of ATP–DnaA, FIS would be displaced from oriC, allowing IHF to promote further binding and oligomerization of ATP–DnaA bound to DnaA boxes. In this model, the opposing influences of IHF and FIS keep oriC poised for a rapid single round of initiation.

Sequestration of the oriC region is a regulatory mechanism that relies on Dam methylase, which is unique to E. coli and related enteric Gram-negative bacteria. As noted above, oriC contains up to a dozen GATC sites, some of which are located within the AT-rich region. These Dam methylation sites are kept methylated on both strands during much of the cell cycle by Dam methylase. Just after initiation, however, the GATC sites are hemimethylated and are quickly bound by SeqA protein, which keeps oriC in a closed conformation until the nascent GATC sites are methylated.

Titration of DnaA by binding of the protein to low-affinity sites elsewhere on the chromosome also appears to regulate macroinitiation. As the chromosome is replicated, a region called datA becomes duplicated and doubles its capacity to bind up free DnaA protein, which reduces the available DnaA that could otherwise reinitiate unscheduled DNA replication at oriC.

All four mechanisms – RIDA, the IHF–FIS balance, sequestration of oriC, and titration of DnaA – are not mutually exclusive and may function sequentially or cooperatively. For example, sequestered, hemimethylated GATC sites are eventually methylated by Dam methylase, but by that time, the datA region has been replicated and is available to titrate DnaA. Lastly, it is evident that oriC is asymmetric, with priming and assembly of the bidirectional replisome in the single-stranded bubble that opens adjacent to the DnaA boxes, which are presumably obstructed by oligomerized DnaA protein. For replication to proceed bidirectionally, one replication fork must force its way through the DnaA-box region. For that reason, there has been some speculation that the fork movement into the DnaA box region displaces IHF and that concerted inactivation of DnaA by RIDA with fork progression leads to rapid disassembly of the DnaA-bound DNA-box complex. Further study of Hda protein may provide additional insights.

2.4 Bacteria

The loading of the bacterial replicative helicase (DnaB) is catalyzed by both the replication initiator protein (DnaA) and the helicase loader (DnaC) (Table 1 and Fig. 1A) [20–22] at the origin of replication (oriC) [23]. DnaA is a highly conserved protein among bacteria, which binds discrete regions of oriC through a helix-turn-helix motif within the CTD to form oligomers [24]. Active DnaA oligomers regulate bacterial DNA replication initiation primarily through binding ATP, changing conformation of the oligomer to open a ssDNA DUE bubble. DnaC is a monomeric protein that binds each subunit of the DnaB protein assembly in a 1:1 ratio to conform the ring into an open lock-washer shape before loading it onto the open DUE [25–27]. Two DnaB-DnaC complexes are recruited and loaded sequentially onto opposite strands, such that each helicase encircles one strand and excludes the other [28]. Post-loading, DnaC is displaced by the primase (DnaG), which stabilizes the N-terminal DnaB collar and stimulates helicase activity [29,30]. Once the active DnaB is released by DnaC, it begins translocating CTD first in the 5′-3′ direction on what will become the lagging strand at a rate of approximately 35 base pairs per second [31]. Once a suitable region of ssDNA has been exposed, DnaG synthesizes an RNA primer de novo, recruiting the replicative polymerase complex including the Pol III holoenzyme (HE), and DNA replication begins in earnest.

Initiation

E. coli chromosomal DNA replication initiates within a 245-bp region, termed oriC. This region contains four 9-bp binding sites for the E. coli initiator protein, DnaA. Nearby are three repeats of a 13-bp A/T-rich sequence. oriC also contains specific binding sites for two small histone-like proteins called HU and IHF. Replication is initiated with the cooperative binding of 10 to 20 DnaA monomers to their specific binding sites (Fig. 42.13). To be active, these monomers must each have bound ATP. Binding of DnaA permits unwinding of the DNA at the 13-bp repeats, in a reaction that requires the histone-like proteins. Next, DnaC binds to DnaB and escorts it to the unwound DNA. DnaB is the key helicase that will drive DNA replication by unwinding the double helix, but it binds DNA poorly on its own in the absence of its DnaC escort. Once DnaB has docked onto the DNA, DnaC is released, and the helicase can then start to unwind the DNA, provided that ATP, SSB, and DNA gyrase are present. SSB is a single-stranded DNA binding protein that stabilizes the unwound DNA, and DNA gyrase is a topoisomerase (see Chapter 8) that removes the twist that is generated when the two strands of the double helix are separated.

Initiation and Termination of Chromosome Replication

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In bacteria, there is one origin of replication that has three 13-base pair repeats and four 9-base pair repeats. A cluster of 30 DnaA proteins opens the helix at the 13-base pair repeats by first binding to the 9-base pair repeats and bending the DNA. DnaC proteins then help DNA helicase (DnaB) load onto the origin correctly.

As previously discussed in a Key Concept above, the origin of replication (oriC) is the site of replication initiation. The region is AT rich and comprises three 13 bp repeats and four 9 bp repeats. Several enzymes localize to the region, most of them involved in the initiation of each new strand. DnaA, DnaB, DnaC, DNA gyrase, and SSB protein are part of the initiation complex. Several DnaA proteins bind to the 9 bp repeats and then bend the DNA to gain access to the 13 bp repeats. Upon binding the larger repeats, the DnaA proteins melt the two strands and open the helix and allow for DnaB (helicase) to begin unwinding the DNA.

E. coli mark the parental strand of DNA by adding methyl groups onto GATC by Dam methylase. The newly synthesized strand of DNA is not methylated immediately, so that the mismatch repair enzymes can double-check for mistakes. This complex of enzymes removes the new incorrect nucleotide and replaces it with the correct one.

Dam methylase recognizes GATC and attaches a methyl group to the adenine base of both strands prior to replication. After replication, though, only the parental strand is methylated, thus, the DNA is hemi-methylated. Any erroneous base inserted during replication is recognized by the mismatch repair enzymes. These enzymes recognize the methylated parental strand and use it as a template to remove the incorrect base and insert the correct base on the complementary, new strand.

Hemi-methylation of the origin of replication helps control how often bacterial chromosomes are replicated.

DNA methylation not only serves a role for repair enzymes to recognize the parental strand, but methylation also is critical in the regulation of DNA replication. Hemi-methylated DNA is present for a short period of time after replication. The cell takes several minutes to fully methylate the new DNA, and even longer to methylate the origin of replication and the promoter region of the DnaA protein, which initiates replication. Hemi-methylated DNA cannot be replicated, but it can be sequestered to the cell membrane with the help of the SeqA protein.

Although methylation of DNA and SeqA play important roles in regulation replication, they are not essential. E. coli dam and seqA mutants are viable.

Replication finishes at the terminus, which has several termination sites and the Tus protein. These block the movement of DNA helicase.

When the two replication forks encounter the terminus, replication ends. The terminus contains several Ter sites that stall the forward movement of DNA helicase. There are different Ter sites for counterclockwise and clockwise movement of the replisome on the circular, bacterial chromosome. The outermost sites likely serve as a backup in case the first Ter sites fail to stop the replication fork from moving forward. Also in the Ter sites are sequences that are recognized by Tus protein. Tus binding at the terminus physically blocks helicase from moving forward, thus stalling the replication forks.

Circular chromosomes of bacteria can tangle during replication or even become covalently joined due to odd numbers of crossovers. Topoisomerase IV decatenates tangled circles and resolvase separates circles with odd numbers of crossovers.

Circular chromosomes may become catenated after replication, that is, interlocked, which would prevent the chromosomes from separating into the daughter cells. Several enzymes work together to overcome this possible outcome. Topoisomerase IV normally slides along the DNA behind the replication fork and prevents tangling. Once replication is finished, Topo IV can decatenate the two circular chromosomes.

Similar to eukaryotes, prokaryotic chromosomes can undergo recombination between the two daughter chromosomes while replication is proceeding. An even number of crossing over events is ideal as this leaves two distinct chromosomes. However, if an odd number of crossovers occurs, the two chromosomes can become covalently linked (see textbook, Fig. 10.20). Separation of the covalently linked chromosomes occurs with the help of crossover resolvase, XerCD, which forces a final crossover.

DNA Replication within the Cell

DNA replication is initiated at particular points within the DNA known as ‘origins’. The oriC origin in E. coli contains an A–T rich sequence recognized by the replication initiator protein, DnaA. DnaA subsequently recruits other proteins beginning a series of reactions that result in the separation of the DNA strands at the origin, forming a bubble with two replication forks at either end. In bacteria, which have a single origin of replication on their circular chromosome, the bidirectional DNA synthesis eventually creates a theta structure because the DNA resembles the Greek letter theta, θ. Termination of replication occurs when the two replication forks meet each other on the opposite side of the parental chromosome. E. coli regulate this process through the use of the Tus protein and termination sequences that allow replication forks to pass through in only one direction, but not the other. Thus, the two replication forks are constrained to always meet within the termination region of the chromosome. Regulation of DNA replication in E. coli is achieved through several mechanisms, including the ratio of ATP to adenosine diphosphate (ADP) and the levels of DnaA within the cell, as well as the hemimethylation and sequestering of oriC. DNA synthesis results in hemimethylated DNA recognized by the SeqA protein. SeqA binds the hemimethylated DNA and sequesters the origin sequence preventing newly replicated origins from immediately initiating another round of DNA replication before cell division.

Who is the family member who first recognizes a need or starts the purchase process?

What four things help determine the roles of the members of a family in making purchase decisions?

Which of the following statements is true regarding the nature of family purchases in the roles played by various family members?

Which type of family consists of a married couple and their own or adopted?