Where is replication in a cell

The first of these is the recognition and binding to its target sites. The interaction between RctB and the mer and mer is dependent of the DNA methylation state, while its binding to the mer and the mer is methylation independent Demarre and Chattoraj, ; Venkova-Canova et al.

It was first proposed that RctB binds to the methylated mer both as a monomer and a dimer Jha et al. However, the head to head dimeric form of RctB is incompatible with the head to tail arrangement of mer within ori2-min Orlova et al. Furthermore, mutations in the three domains do not exhibit the same behavior regarding binding activity to the 11—mers and to the 29—mers.

Indeed, all three domain I, II, and III, seem to be involved in the methylation dependent DNA binding mer and mer , while only domain II is involved in the methylation independent binding mer and mer Orlova et al. Main regulatory mechanisms controlling Chr2 replication initiation. A Representation of the two different models of RctB binding to the iterons.

Both dimers and monomers are able to binds to the iterons. B Representation of the mechanisms involved in ori2 replication initiation. RctB binding sites within the ori2 are indicated and color codes are identical to those of the A. A black arrow illustrates RctB binding to its binding sites. The handcuffing of the mer with iterons within ori2-inc has a positive control on ori2 replication initiation since it competes with the mer handcuffing with ori2-min iterons bar blue arrow.

In the iteron-plasmids mechanism of replication initiation, DnaK and DnaJ enhance initiator binding to the origin Wickner et al. DnaK and DnaJ were first discovered as factors required for the bacteriophage lambda replication and later as enhancers for the replication of plasmids containing iterons within their origin Friedman et al. Plasmid initiators can dimerize, but in general bind to the origin only as monomers. In solution the RctB dimeric form is the most stable, this implies that monomerization of the protein has to be triggered to permit DNA binding Jha et al.

For Chr2 replication initiation, DnaK and DnaJ are strictly required to promote ori2 replication initiation, and were shown to promote RctB binding to both activating and inhibiting sites mers and mers Jha et al. That being said, the elucidation of the precise characteristics of the RctB-DNA interaction needs further structural and biochemical studies, for example, to experimentally show the incapacity of RctB dimer to bind DNA.

RctB mutants reducing the dimerization e. Once bound to the ori2-min mer, RctB has to oligomerize to open the adjacent A-T rich region unwinding activity. The nature of this last process remains obscure. Thus, experimental data determining the role of DnaK and J, the identification of the RctB domain s involved in its oligomerization, as well as the precise role of A-T rich sequences needed to stabilize the opening of ori2 are still missing.

Vibrio cholerae Chr2 replicate once per cell cycle, pointing to a tight control through the balance between positive and negative effectors Egan and Waldor, ; Egan et al. To summarize, RctB acts on two major types of sites, the mer iteron to promote the replication initiation by unwinding the AT-rich region, and the mer to inhibit it Figure 3B.

Furthermore, the addition of the mer to a plasmid containing ori2-min drastically reduced the plasmid copy number in the cell Venkova-Canova and Chattoraj, ; Koch et al. The two main mechanisms of inhibition correspond to 1 the RctB titration and 2 the handcuffing between the mer and the ori2-min mer mediated by RctB Figure 3B Venkova-Canova and Chattoraj, The regulatory function of the iterons found in the ori2-inc region is dual.

Indeed, they have a titration activity, similar to the mer, but, additionally, they help to restrain the mer inhibitory activity by enhancing the handcuffing inside the ori2-inc region, thus releasing the ori2-min mers Venkova-Canova and Chattoraj, Figure 3B. Furthermore, the ParB2 protein, which binds Chr2 specific centromeres localized closer to the ori2-inc , serves as RctB competitor for the mers binding by two mechanisms: 1 spreading from the parS2 site closer to the leftmost mer and 2 direct interaction with the central mer Yamaichi et al.

In addition, as the leftmost mer is covered by the rctA transcript, this also interferes with the RctB binding at this site and thus impede its inhibitory activity Venkova-Canova et al. Furthermore, as found for DnaA, the concentration of available RctB in the cell controls the Chr2 replication initiation. Thus, RctB gene expression is also tightly controlled. RctB auto-regulates its own expression through binding to the mer located in the rctB promoter, where it plays a role of transcriptional repressor and exerts a negative feedback regulation Pal et al.

This mer is also implicated in the ori2 iterons handcuffing and is able to functionally replace the mer Venkova-Canova et al. In addition to this transcriptional regulation, the RctB concentration available to initiate the replication is also significantly controlled by its titration on various regulatory sites. As introduced above, the ori2-inc iterons together with the mers and mer can titrate RctB and reduce RctB binding to the ori2-min replicative iterons.

Chromatin immunoprecipitation Chip-chip experiments have revealed that RctB also binds to a number of sites clustered within a 74 Kbp sequence on the Chr2 located 40 Kbp away from the ori2 Baek and Chattoraj, This 74 Kbp sequence contains six RctB binding sites: five iterons and one mer like sequence, which also negatively regulate the ori2 replication initiation.

This locus titrate RctB and inhibit the ori2 replication initiation, its activity and localisation suggest that it is comparable to the E. The mechanisms of control also involve the methylation state of ori2 , which prevents the replication restart during the same cell cycle Demarre and Chattoraj, Contrary to the Chr1 origin, ori1 , the Dam methylation of ori2 is strictly required for its replication initiation Demarre and Chattoraj, ; Val et al.

Indeed, a dam mutant of V. The SeqA sequestration prevents the immediate re-initiation of the replication, as in the case of Chr1, by temporally inhibiting the full-methylation of the DNA and initiator binding. Thus, the RctB binding to the iterons, which is dependent on the DNA methylation, is integrated to the cell cycle contrary, to its binding to the mers and mer. This methylation binding balance is involved in the cell cycle control of the Chr2 replication initiation.

Marker frequency analysis MFA of a wide selection of Vibrios, with large variations in Chr1 and Chr2 sizes, suggests that there is a selective pressure for a termination synchrony, despite the fact that the control of Chr2 replication is at the initiation level Kemter et al.

Furthermore, in mutants where Chr2 finishes replicating earlier than Chr1, no impact on fitness was detected Val et al. However, in these mutants the Chr2 terminus region ter2 was shown to relocate earlier to mid-cell than in the wt , and remained localized at mid-cell until late in the cell cycle Val et al.

Despite early Chr2 replication termination, ter2 retention at mid-cell suggests a secondary safeguard. How and why ter2 segregation is delayed and results in re-synchronization with the Chr1 terminus region ter1 is unknown.

The mechanism coordinating the synchronous termination of the two replicons is driven by a locus found on the main chromosome. It is localized in the right replichore at around Kbp downstream from ori1 , and presents no homology with previously described RctB binding sites e.

Interestingly, moving the V. Replication of this Chr1 site triggers the replication of Chr2, which initiate after a short delay corresponding to the time needed for the replication of Kbp. Val et al. Besides, by employing chromosome conformation capture 3C experiments, it has further been demonstrated that ori2 and crtS are in a physical contact. These observations suggest that this ori2 replication initiation regulatory mechanism could involve a structural interplay between Chr1 and Chr2 Val et al.

However, the copy number of plasmids containing the ori2 of Photobacterium profundum , Vibrio vulnificus , or Vibrio harveyi , is not increased when crtS from other species e. These discrepancies could be due to the independence of the P.

Thus, the crtS control activity is conserved, and crtS sites of divergent Vibrio species seem, to a certain extent, to be interchangeable for triggering the ori2 replication initiation, showing a loose crtS species-specific activity Kemter et al. The Chr1 origin ori1 brown circle starts its replication initiation first. Once the crtS locus is replicated, Chr2 replication is triggered and occurs at o ri2 orange circle. Chr1 and Chr2 termination of replication is synchronous.

The controlled replications of one Chr1 and one Chr2 of a mother cell lead to the formation of two Chr1 and two Chr2, which are equitably distributed in the daughter cells not represented. The crtS chaperone activity, remodeling RctB to promote the ori2 replication initiation, is represented by a black curved arrow from the light blue to the light green form.

The in vivo effects of crtS on the RctB binding to the iterons mer and the mer are indicated: a black arrow oriented to the top represents the increasing interaction between RctB and mer and a black arrow oriented to the bottom represents the decreasing interaction between RctB and the mer. The RctB binding to crtS is hardly detected in vitro by DnaseI footprint experiments or by electrophoretic mobility shift assay Baek and Chattoraj, It was proposed that, in E. This was drawn from in vivo data, but the in vitro experiments electrophoretic mobility shift assay did not allow obtaining clear results.

Indeed, the authors observed only an in vitro decrease of RctB affinity to the mer in presence of crtS , which could also reflect the competition between two types of RctB binding sites Baek and Chattoraj, Thus, from these results it is difficult to differentiate a simple competition from an in vitro crtS remodeling activity. Moreover, in E. The crtS activity triggering ori2 replication initiation is independent on methylation state of its GATC sites de Lemos et al.

However, the crtS form responsible for the DNA chaperone activity is still unknown. The passage of the replication fork across crtS would induce the formation of transient hemimethylated GATC sites, and the hemimethylated crtS may impact the RctB binding. Passage of the replication complex also generates single stranded DNA on the template of the lagging strand synthesis and could allow the formation of DNA hairpin.

Nevertheless, the replication of crtS could simply lead to the duplication of the site, which could change the balance of free active RctB to catalyze the ori2 opening. When already two copies of crtS were inserted on Chr1, Chr2 copy number was doubled suggesting that it is the presence of two crtS sites after replication that is important Val et al. Indeed, a recent paper shows that the crtS duplication, without active replication, is sufficient to initiate ori2 replication initiation Ramachandran et al.

However, it seems difficult to explain the crtS DNA chaperone activity solely from doubling its gene dosage. Further experimental data are needed to understand if either the active replication or the duplication of crtS is the signal controlling Chr2 replication initiation. In conclusion, the molecular mechanisms by which the replication of crtS triggers the initiation of Chr2 through RctB are largely unknown.

All these mechanisms control the availability of the active form of DnaA in initiating replication from oriC. If the control of ori2 initiation by crtS was performed only by controlling the availability of the RctB active form, we would expect a similar synchrony in the firing of multiple ori2 and this would be observed by cells containing only 2 n ori2 foci e. However, using cells with two chromosomal copies of crtS , the duplication of one crtS triggers the firing of only one ori2 Val et al.

This suggests that Chr2 initiation firing may necessitate a contact between crtS and ori2. The contacts between ori2 and Chr1, introduced above, may be caused by the simultaneous binding of RctB to ori2 and crtS Val et al.

The most frequent contacts between ori2 and Chr1 occur immediately downstream of crtS. A possible explanation is that, following the duplication of the crtS locus, the replication machineries of Chr1 and Chr2 are maintained in the vicinity of each other until the end of replication of the two chromosomes. Non-replicating cells i. Overall, the 3C analysis of the V.

How trans topological contacts would drive a functional interaction between the two chromosomes remains unknown. The genetic information of alpha-proteobacteria is commonly carried by a multipartite genome Landeta et al. Whatever their nature, megaplasmids or chromids, the replication and segregation of those replicons involve, in most cases, three genes organized in operon: repA, repB, and repC Galibert et al.

The proteins encoded by the repABC operon are involved in two distinct mechanisms; RepC is essential for replication, and RepA and RepB are dispensable for replication but required for the partition.

The repA , repB , and repC genes are expressed from promoters found upstream of repA. Most of our knowledge about the transcriptional regulation of the repABC operon comes from the A. The promoter P4 ensures the basal expression of the operon, but this promoter can be activated by the regulator VirG once phosphorylated by VirA, in response to plant pheromones Cho and Winans, Furthermore, the pTiR10 four promoters are activated by the LuxR-family quorum sensing system Pappas and Winans, a.

The partition system is composed the genes repA light brown and repB dark blue , and their cognate parS sites are represented by small dark blue boxes. The replication system is only composed of the repC gene green , containing the RepC binding site light gray. For all the represented repABC operons the gene orientation corresponds to the black arrows.

Same color code as in A. For further information, see text. RepC proteins are considered as the initiator protein of the repABC replicons and are found only in the alpha-proteobacteria Palmer et al. The repC gene alone is able to replicate a plasmid, showing that the origin is localized inside repC Cevallos et al.

This sequence is localized in the middle of the repC coding sequence Figure 5A Cervantes-Rivera et al. RepC binds it cooperatively with a high specificity. Indeed, overexpression of RepC in A. Thus, RepC functions only in cis. The same phenomenon is observed for the RepC protein of the R. RepC exhibits no homology with other replication initiators. Its predicted secondary structure suggests that RepC is divided in two domains: an amino-terminal NTD domain from residues 1 to and a carboxy-terminal CTD domain from residues to Finally, in the case of the p42d RepC, the last 39 amino acids residues are shown to be involved in the incompatibility phenotype Cervantes-Rivera et al.

Inside the NTD domain, the region spanning residues 26— exhibits a structural similarity with the MarR family of transcription factors and is sufficient to bind the DNA. The control of replication initiation catalyzed by RepC is dependent of two major mechanisms, which both act on the repC expression level. These sites are essential for plasmid stability and are involved in the incompatibility mechanism between parental plasmids MacLellan et al. Indeed, point mutations in the parS sites upstream the repA2 of pSymA reduce the RepB binding and impede the incompatibility between pSymA parental plasmids.

This incompatibility is presumably due to the competition between the two parental plasmids for the same partitioning system. RepA and RepB, together with parS sites, also participate to the negative transcriptional regulation of the operon, and thus act on the replication control of repABC replicons. Some bacteria belonging to the alpha-proteobacteria may have up to six repABC replicons; and the question of the RepA and RepB specific activity at their cognate sites and not at heterologous sites is still open.

It seems that the RepD protein is not involved in the replication and partition of pTiRlike replicons Chai and Winans, b. The RepE action model, proposed for the A. In the absence of RepE, the intergenic region repB-repC is predicted to fold in a large stem-loop, leaving the repC Shine-Dalgarno sequence and its initiation codon single stranded, thus permitting the repC translation.

In presence of RepE, its interaction with the target mRNA induces the re-folding of the sequence downstream of the interaction site, and creates two new stem-loops.

One of the new stem-loops forms a Rho-independent termination site upstream of the repC ribosome-binding site leading to a premature termination Figure 5B Chai and Winans, a ; Cervantes-Rivera et al. Mutations reducing the RepE expression or remodeling its structure have been indeed shown to decrease the incompatibility Chai and Winans, a ; Venkova-Canova et al. All together, these mechanisms, i. The replication and segregation of the alpha-proteobacteria multipartite genomes containing a repABC chromid is poorly documented.

Nevertheless, the comparison of the data obtained for the bacteria A. The genome of B. The two replicons of B. This last observation is similar to the results obtained for the repABC replicons of A.

Furthermore, the origin duplication of the B. In the tripartite genome bacterium, S. The replication of the three replicons occurs once per cell cycle, and the segregation pattern is such that the chromosome segregates first, followed then by pSymA, and then by pSymB Frage et al.

Interestingly, the pSymA repABC region is sufficient to confer the spatiotemporal behavior of this replicon to a small plasmid. Besides, alterations of the DnaA activity, either positively or negatively, only impact the chromosome replication, and have no effect on the secondary replicons replication Frage et al. Thus, it is likely that the strict timing of replication and segregation of repABC replicons only involve genetic components located within the repABC operon.

Finally, compared to the V. In the alpha-proteobacteria C. CcrM is functionally related to the E. Indeed, compared to Dam, which is active throughout the cell cycle, CcrM is synthesized and active only in predivisional cells. However, CcrM overexpression results in abnormal chromosomes content per cell in C. Thus, CcrM is essential for normal chromosomal replication.

CcrM is conserved across the alpha-proteobacteria and its orthologs has been studied in S. Interestingly, with the notable exception of C. CtrA is involved in the regulation of ccrM expression in both C. In order to permit a faithful transmission of the genetic information, but also to avoid any problems due to polyploidy, chromids have to be replicated once and only once per cell cycle. In this review, we gave a short overview of chromid domestication history, and further focused our analysis on their replication and how they became integrated in the bacterial cell cycle.

Most of our knowledge on chromid replication initiation comes from the repABC and iteron models, where controls mainly occur at the initiation step. These controls are mostly centered on the initiator proteins both at the gene expression level and through the regulation of their specific activities.

This first step is already controlled by diverse and numerous mechanism. Movement of replicating DNA through a stationary replisome. Cell 6, — Leonard, A. Regulation of DnaA assembly and activity: taking directions from the genome. The orisome: structure and function. Lewis, J. Single-molecule visualization of fast polymerase turnover in the bacterial replisome.

Li, Y. Super-resolution imaging of DNA replisome dynamics in live Bacillus subtilis. The segregation of the Escherichia coli origin and terminus of replication. Liao, Y. Single-molecule DNA polymerase dynamics at a bacterial replisome in live cells. Lin, L.

Lin, Y. The chromosomal DNA of Streptomyces lividans 66 is linear. Lioy, V. Multiscale structuring of the E. Cell , Liu, X. Replication-directed sister chromosome alignment in Escherichia coli. Livny, J. Distribution of centromere-like parS sites in bacteria: insights from comparative genomics.

Initiation of chromosomal replication in predatory bacterium Bdellovibrio bacteriovorus. Mangiameli, S. The replisomes remain spatially proximal throughout the cell cycle in bacteria. Masai, H. Eukaryotic chromosome DNA replication: where, when, and how? Eukaryotic DNA replication origins: many choices for appropriate answers.

Mettrick, K. Stability of blocked replication forks in vivo. Midgley-Smith, S. Chromosomal over-replication in Escherichia coli recG cells is triggered by replication fork fusion and amplified if replichore symmetry is disturbed. Migocki, M. The midcell replication factory in Bacillus subtilis is highly mobile: implications for coordinating chromosome replication with other cell cycle events.

Moolman, M. Nielsen, H. The Escherichia coli chromosome is organized with the left and right chromosome arms in separate cell halves. Dynamics of Escherichia coli chromosome segregation during multifork replication.

Nielsen, O. Once in a lifetime: strategies for preventing re-replication in prokaryotic and eukaryotic cells. EMBO Rep. Niki, H. Possoz, C. Tracking of controlled Escherichia coli replication fork stalling and restart at repressor-bound DNA in vivo. Postow, L. Topological domain structure of the Escherichia coli chromosome. Genes Dev.

Raaphorst, R. Chromosome segregation drives division site selection in Streptococcus pneumoniae. Ramachandran, R. Chromosome 1 licenses chromosome 2 replication in Vibrio cholerae by doubling the crtS gene dosage. Chromosome segregation in Vibrio cholerae. Reyes-Lamothe, R. Chromosome replication and segregation in bacteria. Independent positioning and action of Escherichia coli replisomes in live cells.

Cell , 90— Stoichiometry and architecture of active DNA replication machinery in Escherichia coli. Richardson, T. Nature , — Replisome localization in vegetative and aerial hyphae of Streptomyces coelicolor. Samadpour, A.

Santi, I. Single-cell dynamics of the chromosome replication and cell division cycles in mycobacteria. Chromosome organization and replisome dynamics in Mycobacterium smegmatis. Shaner, N. A guide to choosing fluorescent proteins. Methods 2, — Skarstad, K. Strand separation required for initiation of replication at the chromosomal origin of E.

Timing of initiation of chromosome replication in individual Escherichia coli cells. Regulating DNA replication in bacteria. Cold Spring Harb. Srivastava, P. Selective chromosome amplification in Vibrio cholerae. Stokke, C. Replication patterns and organization of replication forks in Vibrio cholerae. Stracy, M. In vivo single-molecule imaging of bacterial DNA replication, transcription, and repair. FEBS Lett. The replicase sliding clamp dynamically accumulates behind progressing replication forks in Bacillus subtilis cells.

Cell 41, — Sukumar, N. Exploitation of Mycobacterium tuberculosis reporter strains to probe the impact of vaccination at sites of infection. PLoS Pathog. The nucleotides that make up the new strand are paired with partner nucleotides in the template strand; because of their molecular structures, A and T nucleotides always pair with one another, and C and G nucleotides always pair with one another.

This phenomenon is known as complementary base pairing Figure 4 , and it results in the production of two complementary strands of DNA. Base pairing ensures that the sequence of nucleotides in the existing template strand is exactly matched to a complementary sequence in the new strand, also known as the anti-sequence of the template strand. Later, when the new strand is itself copied, its complementary strand will contain the same sequence as the original template strand.

Thus, as a result of complementary base pairing, the replication process proceeds as a series of sequence and anti-sequence copying that preserves the coding of the original DNA. In the prokaryotic bacterium E. In comparison, eukaryotic human DNA replicates at a rate of 50 nucleotides per second.

In both cases, replication occurs so quickly because multiple polymerases can synthesize two new strands at the same time by using each unwound strand from the original DNA double helix as a template. One of these original strands is called the leading strand, whereas the other is called the lagging strand.

The leading strand is synthesized continuously, as shown in Figure 5. In contrast, the lagging strand is synthesized in small, separate fragments that are eventually joined together to form a complete, newly copied strand.

This page appears in the following eBook. Aa Aa Aa. How is DNA replicated? What triggers replication? Figure 1: Helicase yellow unwinds the double helix. The initiation of DNA replication occurs in two steps. First, a so-called initiator protein unwinds a short stretch of the DNA double helix. Then, a protein known as helicase attaches to and breaks apart the hydrogen bonds between the bases on the DNA strands, thereby pulling apart the two strands.

As the helicase moves along the DNA molecule, it continues breaking these hydrogen bonds and separating the two polynucleotide chains Figure 1. How are DNA strands replicated? Figure 3: Beginning at the primer sequence, DNA polymerase shown in blue attaches to the original DNA strand and begins assembling a new, complementary strand.

Mobile Newsletter chat subscribe. Prev NEXT. Life Science. The double helix of DNA unwinds and each side serves as a pattern to make a new molecule. Image courtesy U. Department of Energy Human Genome Program. An enzyme called DNA gyrase makes a nick in the double helix and each side separates An enzyme called helicase unwinds the double-stranded DNA Several small proteins called single strand binding proteins SSB temporarily bind to each side and keep them separated An enzyme complex called DNA polymerase "walks" down the DNA strands and adds new nucleotides to each strand.

The nucleotides pair with the complementary nucleotides on the existing stand A with T, G with C. Animal vs.