What is the significance of the origin of replication

For example, if the four query DNA sequences in the Example window are used as the queried data, the predictive results are the 1 st and 2 nd query sequences are replication origins, and the 3 rd and 4 th are non-replication origins. Publicly available datasets were analyzed in this study. BL provided the main idea of the manuscript and wrote the manuscript.

SC did the experiments and revised the manuscript. KY revised the manuscript and did the typesetting. FW did the experiments. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Chen, W. Prediction of replication origins by calculating DNA structural properties. Febs Letters 6 , — PseKNC: a flexible web server for generating pseudo K-tuple nucleotide composition. PseKNC-General: a cross-platform package for generating various modes of pseudo nucleotide compositions.

Bioinformatics 31 1 , — Efron, B. Feng, P. Frank, A. Asymmetric substitution patterns: a review of possible underlying mutational or selective mechanisms. Gene 1 , 65— Gao, F. Ori-Finder: a web-based system for finding oriC s in unannotated bacterial genomes. BMC Bioinform. Grigoriev, A. Analyzing genomes with cumulative skew diagrams. Nucleic Acids Res. Hastie, T. The elements of statistical learning 2nd ed.

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Li, W. Sequence analysis of origins of replication in the Saccharomyces cerevisiae genomes. Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Without transcription, replication in the purified system almost always initiates unidirectionally, although in vivo it is primarily bidirectional Schnos and Inman, ; Mensa-Wilmot et al.

How transcription significantly improves the frequency of bidirectional replication remains to be determined Learn et al. Transcription and negative supercoiling may also contribute in additional ways Szambowska et al. Thus, lowering the energy required for DNA strand separation may not be the only role of negative supercoiling.

The basic feature of the lambda origin, namely, an array of repeats of replicon-specific initiator binding sites iterons , can be found in the origins of a large family of bacterial plasmids Figure 5 ; Chattoraj and Schneider, Plasmid iterons are generally present in phase with the helical repeat of B-DNA, and disturbing the phasing can inactivate the origin Brendler et al.

Figure 5. Maps of iteron-bearing plasmids. Two P1 maps are shown, the top one being the wild type and the one below with the DnaA binding sites arrow heads deleted and one of them moved next to the iterons. Apart from iterons, the plasmid origins have binding sites for DnaA and a NAP, both of which are required for the origin function.

Plasmid replication is controlled instead by dimerization of plasmid specific initiators Paulsson and Chattoraj, Origin opening has been studied in several of the iteron-based plasmids, including P1 Mukhopadhyay et al.

In plasmid P1, DnaA alone can initiate opening, but it is greatly facilitated by the addition of RepA Mukhopadhyay et al. RepA alone is ineffective. Together, RepE and HU are efficient in opening. Addition of DnaA further increases the efficiency of opening and extends the open region.

The above studies indicate a direct correlation between the efficiency of origin opening and replication initiation. In P1 and F, situations that increase or decrease initiation due to changes in Rep or iteron concentration also correspondingly enhance or reduce opening Kawasaki et al. In pSC, a RepA mutant specifically defective in interactions with DnaA and replication initiation is also defective in origin opening Sharma et al.

The pi mutants that can open without the facilitators are also hyperactive copy-up for initiation. These results argue in favor of active anchoring mechanisms. While DnaA uses two different domains for binding to ds- and ss-DNA, it is not known whether that is also the case for plasmid initiators.

In many iteron-based plasmids, chaperones improve initiator—iteron binding that leads to opening. In plasmids, the chaperones increase the availability of initiator monomers in a form that binds to iterons Wickner et al.

The increase in monomer results from dimer dissociation in vitro and this results from refolding of misfolded subunits that apparently reduces dimerization affinity Giraldo et al. Although replication of these plasmids require the monomers, prevention of over-replication requires the dimers Paulsson and Chattoraj, The chaperones thus play an important role in maintaining the proper balance between the activator monomer and inhibitor dimer forms of the initiators.

Origins of iteron-based plasmids are generally not dependent on transcription. As in E. However, as in other systems, RNA polymerase has been shown to play a beneficial role in pSC replication in vivo. The plasmid has a locus, par , for gyrase binding that increases negative supercoiling specifically at the plasmid origin Miller et al. The localized changes are believed to improve initiator interactions with the origin and thereby its activity Ingmer and Cohen, Replication initiation of plasmid ColE1 differs from that of the replicons described above.

ColE1 does not use a plasmid-encoded initiator. This serves to open the origin and provides the primer for DNA synthesis Figure 6. This diverges from the norm for bacterial replicons, where the primer is synthesized by the primase, DnaG, which is brought to the open origin by DnaB-DnaG protein-protein interactions Bell and Kaguni, Figure 6. Control of origin opening in plasmid ColE1. RNA II initiates and elongates normally up to nt, where it starts to form a persistent hybrid that increases the size of R-loop from a normal size of 10 nt to more than nt.

The non-template strand of the D-loop is then used for helicase loading. The persistence of hybridized RNA causes the R-loop to grow in size to even more than bp. In vivo , RNase H most likely prevents the R-loops to expand much in length, which needs to be at least 40 nt to allow helicase loading Masukata et al.

The three stranded D-loop synthesized by Pol I apparently provides sufficient opening for the helicase. The D-loop is a stable structure wherein the newly synthesized strand prevents the non-template strand from hybridizing back to the template strand.

As their concentrations increase with increasing plasmid copy number, hybridization becomes increasingly significant. Hybridization changes the secondary structure of RNA II that thwarts persistent R-loops formation, and hence, priming.

In sum, although the initiation of ColE1 replication is mechanistically distinct, it espouses the two features highlighted here: the need for stabilizing the open state and the use of the origin opening stage to control initiation. It should be noted that in ColE1, transcription plays a direct and essential role in initiation by providing the primer, whereas in other cases transcription helps indirectly by increasing mainly negative superhelicity and is not obligatory.

Finally, the study of ColE1 replication provided the first example of control by a non-coding antisense RNA, which is now recognized to be widespread in biology Tomizawa et al. The bacterial program of first opening the origin and then loading two hexameric helicases sequentially for bidirectional replication is not conserved in archaea and eukaryotes. In the latter, both the helicases are loaded together as a double hexamer to an unopened ds—origin Bell and Kaguni, The loading otherwise follows the basic bacterial paradigm: the helicase a hetero-hexamer, MCM in association with the helicase loader Cdt1, is recruited to the origin bound by the initiator ORC as a complex with another factor called Cdc6.

The double hexamer is loaded in the post-mitotic, early G1 phases of the cell cycle in an inactive state and as a ring that encircles the ds-origin in its central core. The helicase activation and strand separation occur later in S-phase where the double hexamer is converted to single hexameric rings, each encircling one single strand for bidirectional movement.

These major transitions require S-phase kinases and several additional factors, the details of which are under current investigation Yardimci and Walter, ; Bochman and Schwacha, ; Petojevic et al. Loading and activation of the helicase at different stages of the cell cycle help to restrict initiation to only once per cell cycle Nguyen et al. Since no new helicases can be loaded in S-phase, new origin firing cannot happen either. Thus, although the mechanisms of helicase loading have been largely conserved, the mechanisms of helicase activation and origin opening have diverged in different domains of life.

Here we have provided a few examples of how bacterial origins open, permitting loading of the replicative helicase. As some opening is possible without initiators, it is likely that the origin is inherently unstable Gille and Messer, ; Mukhopadhyay and Chattoraj, ; Polaczek et al. Initiator binding pushes the propensity of opening over the threshold. Of all the requirements for opening, the free energy of negative supercoiling of the origin region appears to be the most basic requirement Miller et al.

Many of the facilitators of opening e. Initiator multimerization appears to be a general contributor to origin opening. Stress from DNA bending can induce base-pair opening Kahn et al. The opening by bending is initially local but the unwinding may migrate. Reducing the number of initiator binding sites in DNA generally makes the origin inefficient or inactive, depending upon the degree of binding site reduction. Multimerization is also involved in ssDNA binding, which either stabilizes the open region or promotes actual unwinding or both; Figure 3A.

What triggers the conformational switch in initiators that allows them to bind ssDNA remains a challenging question Duderstadt et al. More structural studies of the complexes are in order to get further insights into the opening process.

This is now a realizable goal given the recent progress in cryo-EM Merk et al. Paradoxically, such extreme asymmetric distribution of purine and pyrimidines stiffens the DNA more than DNA with more random sequences Wells et al. In most cases, a specific strand is captured in the open region Mukhopadhyay et al. Strand capture preference is also observed in experiments where the single strands are supplied in trans Ozaki et al. A recent study has revealed a repeating trinucleotide motif that is conserved in bacterial DUEs and is required for origin opening Richardson et al.

A high ATP concentration can cause a conformation change in DnaA that appears likely to be required for opening Saxena et al.

In closing, we prefer the view that the opening proceeds in steps rather than by a highly cooperative transition the two models in Figure 3A. The opening by initiators alone may not be sufficient for helicase loading. The involvement of multiple factors provides multiple opportunities for regulation. All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Abeles, A. A single DnaA box is sufficient for initiation from the P1 plasmid origin. Google Scholar. Abhyankar, M.

Alfano, C. Heat shock protein-mediated disassembly of nucleoprotein structures is required for the initiation of bacteriophage lambda DNA replication. PubMed Abstract Google Scholar. Ordered assembly of nucleoprotein structures at the bacteriophage lambda replication origin during the initiation of DNA replication.

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Bell, S. Helicase loading at chromosomal origins of replication. Cold Spring Harb. Bochman, M. DNA replication: strand separation unravelled. Nature , — Bowater, R. Biochemistry 30, — Bramhill, D. Duplex opening by DnaA protein at novel sequences in initiation of replication at the origin of the E. Cell 52, — A model for initiation at origins of DNA replication. Cell 54, — Brendler, T. Critical sequences in the core of the P1 plasmid replication origin.

The iteron bases and spacers of the P1 replication origin contain information that specifies the formation of a complex structure involved in initiation. Chattoraj, D. Replication control of plasmid P1 and its host chromosome: the common ground. Nucleic Acid Res. Cheng, H. Bacterial initiators form dynamic filaments on single-stranded DNA monomer by monomer. Nucleic Acids Res. Chodavarapu, S. Escherichia coli Dps interacts with DnaA protein to impede initiation: a model of adaptive mutation.

Conley, D. Effects of the pSC partition par locus on in vivo DNA supercoiling near the plasmid replication origin. Dodson, M. Specialized nucleoprotein structures at the origin of replication of bacteriophage lambda: localized unwinding of duplex DNA by a six-protein reaction. Specialized nucleoprotein structures at the origin of replication of bacteriophage lambda.

Protein association and disassociation reactions responsible for localized initiation of replication. Doran, K. Replication origin of the broad host range plasmid RK2. Positioning of various motifs is critical for initiation of replication.

Dorman, C. Nucleoid-associated proteins and bacterial physiology. Dove, W. Replicator activation in lambda. Japan J Genet 44, 11— Drlica, K. Histonelike proteins of bacteria. Duderstadt, K. DNA stretching by bacterial initiators promotes replication origin opening.

Origin remodeling and opening in bacteria rely on distinct assembly states of the DnaA initiator. Eguchi, Y. Antisense RNA. Erzberger, J. The structure of bacterial DnaA: implications for general mechanisms underlying DNA replication initiation. Friedman, D. Interactions of bacteriophage and host macromolecules in the growth of bacteriophage lambda.

Funnell, B. Complete enzymatic replication of plasmids containing the origin of the Escherichia coli chromosome. Furth, M. This might have been observed due to the different base composition of the particular strands.

From these data, it can be concluded that the initial opening of DUE is rather no shorter than c. The pattern of thermodynamic stability is different for various origins see Fig. The regions contain some guanine and cytosine residues and it is only natural that free energy will be accordingly higher for those parts of the AT-rich sequences.

In the origins of E. In other cases, as for example P1 or R6K plasmids Fig. The fluctuations and differences among the minima of free energy, which can be observed when analyzing the thermodynamic stability patterns of particular origins, exist due to the specific sequences of the regions and the existence of direct repeats within the sequence.

The internal stability pattern might also be related to the helical periodicity of some of the repeats within the AT-rich regions. This is the case in RK2 where the local minima and maxima of the free energy could be ascribed to the distance of one helical turn. Most likely the majority of the AT-rich regions of the prokaryotic origins could contain repeats of various lengths.

This phenomenon is of great importance for the origin functioning and the formation of protein complexes during replication initiation events described in more detail in the next paragraphs. The DNA sequence fragments taken for analysis were bp long. The red shading encloses the AT-rich regions with adjacent sequences. Black arrows depict repeats identified within the AT-rich regions of the given origins. Panels a-l present internal stabilities of selected chromosomal and plasmid origins of replication.

Within the AT-rich regions of many prokaryotic origins short repeated sequences have been identified; however, their exact role was not explained up to date. Most of the motifs in the AT-rich region are direct repeats, although there are exceptions like RK2 plasmid's oriV origin or T. This length and the number of repeats seem to be conserved among the chromosomal origins of replication, where the repeats were actually defined, as for example in P.

The E. Initially it was assumed that it is only the single-stranded structure and the helical instability of the mers that are important for the formation of the DUE and the origin activity. Replacing this motif with a sequence containing higher GC content completely destroyed the origin activity Asai et al. Recent studies demonstrated that the deletion of the L mer inactivates the heat-induced replication HIR and makes oriC inactive for cyclic replication in conditions of increased growth temperature Gonzalez-Soltero et al.

It was suggested that the strand opening for HIR initiation occurs due to heat-induced destabilization of the L mer Gonzalez-Soltero et al. Repeated sequences within the AT-rich regions of selected replication origins. The mers found in Pseudomonas origins are packed less tightly than those of E. The sequence composition of the P. The sequence of the Pseudomonas repeats differs from that in E. Only the P. When the whole sequence is compared, the repeats of pseudomonads differ from the E.

The AT-rich regions in V. This could be expected as earlier studies concerning replication in E. Besides the mer motifs, the AT-rich region of Vibrio origins also contains a short tract, AT cluster, preceding them and composed of adenine and thymine residues only Saha et al. The bp-long AT-rich region of C. When the sequences spacing the E. Therefore, the motif may have similar functional significance as its equivalents in E.

Concluding from the internal stability pattern of Cori fragment, containing the sequence of the AT-rich region where the mer is found in the region of the lowest thermodynamic stability Fig. At the replication origin of B. The importance of the mers was not determined, as KMnO 4 modifications upon binding of the DnaA protein to the origin demonstrated, that it is the mer that serves as the DUE for the Bacillus origin Moriya et al.

The comparative analysis of T. No mers similar to those of E. Based on the above finding, it can be observed that in the bacterial origins the repeats within the AT-rich regions are usually 13 nucleotides long and possess rather high sequence similarity with consensus similar to that determined for E.

A distinctive core consisting mostly of adenines and thymines can be distinguished in the sequence of the chromosomal repeats see Table , boxed in red. Most often there are three direct repeats in the region that play a direct role in the open complex formation at the origin during the replication initiation.

Those repeats seem to contain mostly adenine and thymine residues, which makes them c. It is rather difficult to determine a consensus sequence for the repeats found among different plasmids as they are of various lengths but a consensus can be established for the repeats within a particular origin.

Some of the repeats are shorter than the mer motifs of oriC, just like the 7-mers of P1 plasmid Chattoraj et al. In those repeats, as in E. They are tightly packed and their sequences partially overlap; three of them, L, M1, and R, are direct and situated on the top strand of DNA, the fourth, M2, is reversed and placed on the bottom strand Konieczny et al.

This arrangement of the repeats is crucial for origin functioning as introducing insertions between them disabled origin's activity in vivo Kowalczyk et al. The in vivo and in vitro experiments utilizing a group of mutants of the RK2 oriV AT-rich region also demonstrated that its role in replication initiation does not ensue only from its low inner thermodynamic stability.

It was shown that the position of each mer within the AT-rich region is critical for the activity of the plasmid's origin, as swapping or reversing the motifs had a negative effect on origin function Kowalczyk et al. As it was observed for mers in the chromosomal origins, the number and length of the repeats within the AT-rich region of an individual plasmid origin possibly also plays an important role in the origin's activity. For the RK2 plasmid, it was demonstrated that the deletion of one of the mer repeats present in the AT-rich region disabled oriV activity in vivo Kowalczyk et al.

The repeats, L and M mers, are spaced with relatively long sequence of 29 bp and the distance between M and R is 9 bp long. The lengths of the repeats and the spacers in between them approximately multiply the length of one helix turn c. In some cases, determining the repeats is more complicated and different publications give different data concerning the same replicon.

They account for the region of the lowest thermodynamic stability when compared with the surrounding sequences see Fig. However, Kawasaki et al. Further DNA unwinding at the F plasmid origin proceeds toward the left side of the AT-rich region, where the putative L and M1 motifs were suggested by the other group.

Differences also concern the origin of P1 plasmid, where various works identify five tandem 7-nucleotide repeats Chattoraj et al. Experiments utilizing mutants of the RK2 oriV AT-rich region demonstrated that the repeats are also origin specific. Those mutants where the sequence of a particular mer was substituted with a respective sequence of E.

Mutagenesis at the AT-rich repeats was conducted within different plasmid replicons. Single-base substitutions in the P1 plasmid's AT-rich repeats demonstrated that the sequence of the 7-mers is essential for the functionality of the P1 origin Brendler et al. Those repeats contain GATC motifs see description in the paragraph below within their sequence. The negative effect of mutations in other positions was also observed when GATC motifs were left intact.

For each of the repeats, at least one single-base change that had a deleterious effect on replication was found. The integrity of the sequence of the 7-nucleotide motifs seems to be crucial for the origin activity but neither the sequence nor the length of the 3- to 6-bp spacing between the motifs is very important, although the requirement of minimal spacer length was suggested Brendler et al.

Point mutations within the AT-rich mers of the RK2 plasmid also negatively influenced the origin activity, decreasing it to 75 or even to 0. This suggests that the position of the adenine and thymine residues on each DNA strand may be critical for the interactions of proteins involved in the plasmid's replication initiation within this region. Changes simultaneously affecting the sequences of putative DnaA-box motifs within this region showed that their sequence is also important to the origin's activity Kowalczyk et al.

The activity of the oriV AT-rich region's mutants was similar in the in vivo experiments in E. It is possible that this particular motif is important for the plasmid's replication in E. This raises the possibility that host factors can specifically interact within the AT-rich region of the plasmid origin.

Moreover, it could be that the subtle changes of the internal stability resulting from the sequence change could affect replication activity in one host but not the other. Their length, number, and spacing are individual for each origin and the sequence of each is substantial for origin functioning.

Apart from the direct repeated sequences, there are other motifs that can be identified within the AT-rich regions of prokaryotic replication origins that play a crucial role in the process of replication initiation or are involved in its control see Table In the AT-rich region of E. It is difficult to comprehend if the ATP-DnaA-boxes are universal motifs that could be found in more origins or if they are unique only for some.

However, most likely the interaction of replication initiators with specific motifs within an AT-rich region of an origin could be widespread. In the table, proteins interacting or suspected to interact with the sequences of the AT-rich regions of replication origins are listed.

If a specific sequence was identified to be bound by a protein, it is given in the table see details in the text. SeqA binds to an origin tandemly as a dimer Lee et al.

The SeqA recognition sequences are found in similar positions of the repeats identified within AT-rich region of P.

In F plasmid, they overlap the 7-nucleotide repeated motifs and both the GATC sequence and its methylation are important for the functionality of the F origin in vivo Brendler et al. As GATC motifs are noticeably more frequent in the AT-rich regions of chromosomal origins of replication, their role in the regulation of replication initiation and chromosome segregation is of great importance.

This also suggests that there might exist specific sequences recognized by the protein within the mers. No data were found for the presence of IciA-recognized sequences within the AT-rich regions in other origins; however, such possibility cannot be excluded without proper biochemical analysis.

Sites recognized also by other proteins were shown to exist within the oriC AT-rich region. For example, a site for binding the ArcA protein that, among other sequences, interacts with the mers and blocks the open complex formation and therefore the replication initiation.

The protein binding does not affect the process once the ssDNA structure is formed Lee et al. Sequences within the mer region were also reported to bind a putative E. The sequences at the unwound AT-rich region of oriC were found to take part in the interaction with another factor — the CspD protein, belonging to the cold shock proteins family. Its binding to the ssDNA was shown to effectively inhibit both the initiation and elongation stages of the minichromosomal DNA replication in vitro Yamanaka et al.

The binding sites for nucleoid-associated proteins NAPs were also identified within AT-rich region of some replication origins. For example, Fis factor for inversion stimulation binding sites, which are present outside the AT-rich region of E. The IHF stimulates the unwinding of plasmid origins and E. Apart from the proteins described above also other factors were identified to be crucial for the regulation of replication activity of prokaryotic origins.

Some of them influence origin opening at the AT-rich region; however, none of them was demonstrated to interact directly with the motifs found in this region. This process, together with the dnaA gene autoregulation, acts to ensure homeostatic maintenance of the E. Rob protein binds the right side of oriC , the R2, R3, and R4 DnaA-boxes, which may influence the formation of nucleoprotein structure in this region Skarstad et al.

The high content of adenine and thymine residues and consequent low internal stability of the AT-rich region facilitates DNA helix destabilization. It was observed in vitro that to some extent, the DUEs have a tendency toward spontaneous opening breathing without replication initiation factors Polaczek et al. This suggests that oriC unwinding could be a spontaneous event determined solely by the DNA sequence containing repeated motifs contributing to the specific internal stability pattern with periodical local minima of free energy see paragraphs above.

Therefore the replication proteins may function only to stabilize the ssDNA structure. Experiments conducted on other systems revealed that the DNA helix unwinding could be detected only in the presence of the replication initiation proteins Schnos et al. Moreover, a strict requirement of histone-like proteins indicates that the appropriate superhelicity of DNA within DUE is critical for its opening. Although the spontaneous destabilization of the AT-rich part of the origin is possible, no functional origin opening leading to the formation of replication complex and the initiation of DNA replication was observed without the initiator proteins.

Therefore the analysis of the nucleoprotein complexes formed at the replication origin is fundamental to our understanding of the mechanism of the initial origin opening. It is thought that the initial complex at E. The formation of the E. This indicates that the initial complex, consisting of Rep proteins and the wrapped DNA, is formed in the proximity but outside of the DUE element.

Experiments, demonstrating that the distance and helical spacing between the Rep binding sites and an AT-rich region is critical for origin activity see paragraphs above and Hsu et al. The last 10 years brought new data on the mechanism of the initial DNA unwinding and highlighted the contribution of the DNA sequence of the AT-rich region to the open complex formation.

Mutational analysis revealed that the E. This would position the protein to the region of unwinding. Crystallographic data on DnaA proteins, isolated from Aquifex aeolicus and T. It became clear that both the chromosomal DNA replication initiators, DnaA homologous proteins and the plasmid replication initiators, Rep proteins, form oligomers when interacting with their binding sites localized at the replication origin.

Structural analysis of the F plasmid's RepE Komori et al. The repeated sequences, iterons, and DnaA-boxes contribute to that specific nucleoprotein structure. It must be pointed out that due to the structural variations among the DnaA homologues and the Rep proteins, the nucleoprotein complexes they form with DNA differ.

The structure analysis of the A. The protein's C-terminal domain consists of a classic helix-turn-helix motif and an extra loop that could be in contact with DNA Erzberger et al. Based on the obtained structural data, it was proposed that the DnaA oligomer could conceivably accommodate either a closed ring or a helical filament arrangement of monomers. The DUE opening may occur spontaneously through a local strain induced by an assembly of the nucleoprotein complex in the presence of ATP Erzberger et al.

In a recent study, Berger's group reported that DnaA utilizes at least two different oligomeric conformations and that it plays distinct roles in controlling the progression of the replication initiation Duderstadt et al. Moreover, experiments on the T. ATP is not required for the E. The interaction of DnaA proteins with specific motifs located within the ssDNA of the AT-rich region is suggested to be a common mechanism for the origin opening of bacterial chromosomes.

Understanding the open complex formation at plasmid origins is more complex as two initiator proteins are involved during plasmid replication initiation. The plasmid and chromosomal origins exhibit similar positioning of the AT-rich region in respect to the binding sites of a replication initiator. The open complex formation at the plasmid origins is induced by Rep protein binding to the iterons; however, some plasmids still require DnaA to stabilize or enhance the origin unwinding Kawasaki et al.

The molecular basis for DnaA contribution to plasmid origin opening must be different comparing to mechanisms proposed for the chromosomal replication initiation by DnaA see above.

Moreover, it could not be excluded that during the opening reaction the Rep proteins could interact with the mer sequences within the AT-rich region. Both in vivo and in vitro tests revealed that specific alterations within the mer sequences result in inhibition of the RK2 oriV opening and consequently in the lack of origin activity Kowalczyk et al. As the introduced alterations did not change the internal stability pattern, these results indicate the possibility of specific protein—DNA interactions within the AT-rich region of plasmid origins.

The AT-rich region provides structural scaffold for the assembling of a replication complex. Regardless of a replicon analyzed, the formation of the complex involves helicase loading and subsequent assembly of primase and replicative polymerase. It is not clear if and how the nucleotide sequence of the opened region affects the formation of a replication complex.

Cryptic single-stranded-DNA binding activities of the E. Two E. After the helicase moves approximately 65 nucleotides, the primase synthesizes primers and two molecules of the DNA polymerase III holoenzyme are assembled on the ssDNA templates Fang et al. It is very likely that similar complexes, including also the plasmid Rep proteins, could be essential for the helicase loading at plasmid origins.

During the RK2 plasmid replication initiation, the E. Specific alterations within the mer sequences, which do no affect origin opening, significantly disturb the E. In case of five mutants, this process showed that the helicase loading at the unwound single-stranded AT-rich region depends strictly on the activity of the plasmid's Rep protein and the sequence of the mer repeats Rajewska et al. This adds to the notion of the AT-rich region being important to replication initiation not only due to its low internal stability.

However, whether it is the sequence itself or the sequence-dependent formation of secondary structures at the unwound origin still remains ambiguous. Experiments on the E. The potential formation of secondary structures within the open region of origin Pearson et al.

The AT-rich part of a replication origin containing mer repeats and sequences overlapping them, including GATC sites, ATP-DnaA boxes, and other more or less specified motifs for binding various regulatory proteins see paragraphs above could be considered as a major region for controlling the DNA replication initiation. This control is achieved mainly by managing the unwinding of an AT-rich region. The binding of the regulatory proteins changes the DNA architecture or prevents the formation of specific nucleoprotein complexes at the DUE.

That results in the stimulation or inhibition of origin opening. This prevents an immediate ATP-dependent activity of DnaA and as a result an uncontrolled origin opening. The replication initiation could possibly be also regulated by controlling the formation of a helicase complex at the unwound AT-rich structure.

It was recently proposed that the DiaA dynamics is coupled with changes in the initial complexes at the origin, leading to helicase loading Keyamura et al. The AT-rich region of a replication origin accommodates multiple protein interactions and conformations.

The formation of oligomeric structures composed of replication initiators and architectural proteins as well as specific interactions of regulatory factors seem to be the key factors for origin activities. A specific nucleotide sequence of DUE element is essential and provides specificity of nucleoprotein interactions during the process of replication initiation and its control. A common feature of many bacterial origins of replication is the presence of a region rich in adenine and thymine residues.

They are usually characterized by low thermodynamic stability in comparison with the overall origin stability and contain repeated sequences of various lengths that are crucial for the proper functioning of the replicons. Patterns of the internal stability may also be related to the helical periodicity of some of the repeats within the AT-rich regions.

In the bacterial chromosomal origins of replication, the AT-rich regions are usually located at one side of a cluster of DnaA-box sequences. In plasmids, they are usually followed by one or two DnaA-boxes and precede the binding sites for plasmid initiator protein.

Such arrangement of the motifs seems to be of great importance for the efficient functioning of replication origins. It is possible that proper organization of the motifs might be connected with the function of initiator proteins and the mechanism of replication initiation at a particular origin.

The AT-rich regions are of different sizes, depending on the origin; however, what seems to be common for them is the size of the initial DNA opening at DUE, which is very rarely longer than 50 bp and no shorter than 20 bp. This extent of origin opening seems to be sufficient for the assembly of the prereplication complex at the unwound site. The great majority of the AT-rich regions of prokaryotic origins contain repeats of various lengths. They appear to be absolutely critical for the origin functioning and the formation of protein complexes during replication initiation events.

In the bacterial origins, the repeats within the AT-rich regions are usually 13 nucleotides long and possess high sequence similarity with consensus determined for E. They also contain a distinctive core consisting mostly of adenines and thymines at the right-hand side of each repeat. Most often, there are three direct repeats in the region that play a direct role in the open complex formation at the origin during the replication initiation. In plasmids, the repeats vary both in length and sequence and establishing a general consensus for them is rather difficult.

However, the length, number, and spacing between the repeats are individual for each plasmid origin and the specific sequence of each is substantial for origin functioning.

Biochemical analyses of replication initiation at different origins revealed that the sequence or, possibly, the secondary DNA structure resulting from it is absolutely critical for the replicating activity of a replicon. However, whether it is the linear or secondary structure of DNA in the AT-rich region, which is crucial for origin's activity and multiprotein interactions in this region, still requires investigation.

Within the repeats of the chromosomal and plasmid AT-rich regions overalapping motifs for binding proteins engaged in replication initiation and its regulation were also identified. However, the exact role of the particular motifs is also being investigated. It is highly interesting whether the multiprotein interactions within the AT-rich region are competitive or maybe the accumulation of motifs for protein interactions plays a role at different stages of replication and allows separation of particular events of the process.

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