Under favorable conditions, as the cell-mass: cell volume ratio gains, the cell enters into cell division mode.
Replication origin called Ori-C is located at 84.3 mpu position of the genome. The oriC locus is about 250-350 bp long and located in between gidA and mio C gene
The topology of bi-directional replication of a circular prokaryotic chromosome. The continuous line is the DNA strand replicated as the leading strand; the dashed line is the DNA strand replicated as the lagging strand; Ori, the origin of replication; Ter, the terminus of replication. Ori and Ter divide the chromosome into two replichores, arbitrarily called left and right. Mackiewicz et al. Genome Biology 2001 2:interactions1004.1 doi:10.1186/gb-2001-2-12-interactions1004
This picture depicts their positions of 9-mer and 13-mers sequences called AT rich boxes; Fully methylated sites in 9-mer region are bound by Dna-A proteins cooperatively; it this binding leads to the DNA to wrap around the clusters that leads to torsion in opposite direction and brings about the unwinding of the DNA at 13-mer regions.
In previous round of replication, fully methylated DNA in the mentioned GATC regions becomes hemi-methylated. In this state initiation of replication is stalled for the lack of full methylation (methylation in both strands) in OriC region, but in other regions of DNA get methylated quickly. This is due to the binding of SeqA proteins to hemi methylated GATC sequences (called ‘A’ boxes) and masks the sequences from methylation. This time period is called eclipse period. Signals for activation of cell division leads to methylation of GATC at dnaA gene promoter as well as at other regions. This leads to the activation of dna-A gene and DnaA proteins are produced. Expression of dnaA gene is regulated by (it has two promoter regions) its own product DnaA-ATP. The DnaA is AAA+ protein and it is activated by the binding of ATP
In hemi-methylated state the newly formed circular DNA remains bound to mesosomal membranes at a site called attachment point. This attachment is facilitated by certain proteins that are bound to DNA, which in turn bind to membrane phospholipid head groups; the suspected proteins are SPoOJ and it prevents from releasing DNA and initiation of replication because of an inhibitor present in the membrane bound at hemi-methylated sites in the origin of DNA. Once it was believed that DNA binds to mesosomal membrane at specific DNA attachment sites. However it is not clear, but it is assumed that the DNA attachment site (there are twenty sites) to membrane is very close to replication origin. The inhibitor is called IciA (inhibitor of chromosome initiation). On full methylation, dna-A proteins release DNA from the inhibitors and from the membrane.
Activation of cell division process at cytoplasmic level involves many signal transduction pathways. In this process, the dnaA gene is activated; accumulation of DnaA protein to certain concentration is very important, till then the DnaA proteins are sequestered at dat-A region
GATC hemimethylation and A box masking:
Newly synthesized DNA lack methylation, however OriC and GATC remain unmethylated or hemimethylated for extended period. These sites are bound by SeqA proteins near DNA-A binding sites. The hemimethylated state remains for about 10 minutes.
DnaA titration by data: Multiple binding sites for DNA at data (Dna A titration) at 94.7mpu of E.coli gene map. As this site is the origin,it is replicated early causing the doubling of the sites thus DnaA protein level is kept low until the copy number builds by expression of weak constitutive promoter. Initiation of second round replication is kept in check till DnaA-ATP levels are high enough that weak R3 site is occupied (Katayama et al2001)
The gene dnaA expression is controlled by the binding of DnaA proteins to their respective promoter elements. The promoter consists of several sites of which one is strong binding site for DnaA, two are week binding sites and other two in flanking regions contain DnaA-ATP binding sites. Once the hemi methylated sites are fully methylated, proteins bind and dnaA gene is expressed.
The diagram depicts how the wrapping of the DNA around Dna-A proteins leads to opening of the DNA at 13-mer regions. ATP mediates the loading of Dna-A on to methylated Dna-Boxes. The opening also facilitates the loading of the Dna-B hexamer in ATP dependent manner, Dna-B is an Helicase-a ATP dependent motor protein.
Hemimethylation prevents initiation of replication, at the same time hemimethylation in promoter elements of dna-A gene also leads to repression. It is only on full methylation, the gene becomes active. Protein such as regulatory inhibitor of DnaA called RIDA and few others are involved in interacting with one another in binding and releasing the DNA from the membrane.
The release of the DNA is also assisted by full methylation of GATC sites in the origin region, it at this time SeqA are released. SeqA binding provides time for the synthesis and accumulation DnaA proteins; this happens in between two cycles of replication events. SeqA overlaps A-boxes thus mask the GATC sites from methylation.
---- Abox- SeqA-Abox-ori C-Abox-seqA-Abox--
There is another site called datA region (DnaA titration), which also contain four A-boxes to which DnaA-ADP proteins bind; the binding DnaA proteins to these sites acts as factor titration process. When the concentration of DnaA-ADP proteins is low they are all bound to data sites. When sufficient amount is produced, they used for initiation of replication by activating DnaA protein into DnaA-ATP.
The circular DNA detaches from the mesosome bound to attachment site. These two events are very important in initiating DNA replication cycle. Methylation of dna-A gene leads to transcription and translation of dna-A gene and Dna-A proteins build up in cytoplasm of the cell.
Wrapping of DNA around the proteins lead to negative twisting of ds DNA on its axis, which transmits as an unwinding force to the neighboring region, and the DNA sequence at 13 mer segments unwinds (all the 3 segments including in between spacers) and open into single stranded bubble of 60-68 base pair length.
Dna-B (hexamer ring) assisted by Dna-C in 1:1 ratio is loaded onto the fork in ATP dependent manner. During loading the circularly oriented helicase ring opens and clamps on to ss DNA at the joint of replication fork. One of the domains has ATPase activity. Dna-B, is an helicase, encircle the lagging stand at the head of the fork in 5’à3’ orientation. Helicase loading is assisted by Dna-C in ATP dependent manner. This motor protein is responsible for driving the fork opening further like unzipping. Dna-G, called primase or priming RNA polymerase, which is distinct from the pentameric regular RNA polymerase, associates with Dna-B on the ssDNA template.
The replication bubble shaped at the origin region shows direction of new strand synthesis.
Using 3’ GTC sequences it produces a short stretch of RNA strand complementary to the template strand, invariably starting with 5’ A G…ends with G 3’, an eleven ntds long RNA is assembled. Sequencing of the DNA in this region to find where the primer initiates and where it terminates, for DNA extension shows GTC at –11 and at -1, it has C nucleotide. The RNA and DNA joint sequence always starts with 5’CGG-’. The primer RNA -DNA junction shows the following sequence in several species, such as G4 phage, alpha 3 phages, and R 100 plasmid DNA, Ff6 DNA and R6k ori alpha. This region is 93% rich in purines.
The primase is a monomeric protein, resistant to Rifamycin, while the regular RNA-pol-III is sensitive to Rifamycin. The primase is responsible for laying short, 11 ntds long primers on leading strand once at the beginning and lay primers on lagging strand at frequent intervals of 1000 to 1500 nucleotides all along the length.
As the DNA opens into bubble ssDNA are immediately bound by SsBs
Stepwise process in opening of DNA by helicase into replication bubble and ssb binding and gamma complex assisted loading of DNA-pol.
DNA is threaded through a replication complex
A huge protein complex catalyzes the reactions of DNA replication.
This replication complex recognizes an origin of replication on a chromosome.
DNA replicates in both directions from the origin, forming two replication forks.
In DNA replication, both strands of DNA act as templates.
Until recently, it was believed that the replication complex moved along the strand of DNA.
Recent evidence suggests that the replication complex is stationary, and DNA threads through it.
Replication complexes consist of several proteins with different roles.
DNA helicase denatures the double helix.
Single-strand binding proteins keep the two strands separate.
RNA primase makes a primer strand that serves as a starting point for replication.
DNA polymerase adds complementary nucleotides to the growing strand, proofreads the DNA, and repairs it.
DNA ligase seals up breaks in the sugar–phosphate backbone.
This diagram shows how DNA-B helicase progresses the replication fork
Primase is always tagged on to the helicase. At the time of initiation primers are also laid on leading strand only once per cell cycle.
At this juncture Gamma complex loads beta-clamps not only on leading strand but also on lagging strand. The gamma complex binds to ATP and uses the energy to open the dimeric beta clamps and close on the DNA strand. The beta clamps are positioned on the template at 3’ position of the primer. The gamma complex also facilitates the assembly of DNA-Pol on to their respective beta clamps. DNA pol III, as a dimer complex, joins template at the joint of the fork.
Assembled component at one end of the replication fork.
synthesis at a replication fork(in case of E.coli):
Two new dsDNAs are created by using the two parent ssDNAs as templates near the replication fork. STEP1:naB helicase(blue) separates a dsDNA into two ssDNAs, as cutting the hydrogen bands between base pairs. It seems to as if unzip. Topoisomerase(lime green) has a role in rewinding the twist of double helices which was generated by the dsDNA separation. STEP2:The separated ssDNA has a tendency of annealing. For preventing annealing, Single-Stranded DNA Binding Protein : SSB(brown) bind to separated ssDNA.STEP3:DnaG primase(purple) is activated by binding to DnaB helicase, and synthesizes a short RNA primer approximately 10 nucleotides long using a ssDNA as a template. STEP4:
DNA polymerase III elongates a new ssDNA strand by adding a deoxyribonucleotide at a time in the 5'-3' direction to the RNA primer, using a ssDNA as a template.
The E.coli polymerase III is a complex consisting of multiple different protein subunits. The core part(green) consists of three subunits(alpha, epsilon, delta). The clamp part is beta subunit(navy blue), and clamp-loader part is gamma complex(yellow).
A simple representation of the components at replication fork is figure represents the assembled components in action
The structure represents two subunit (homodimer); acts as clamp around the ssDNA at the base of DNA pol III Holozyme
Dimeric Holoenzyme with clamp and clamp loader gamma complex
An illustration of the dimeric holozyme with all its components and a single holozyme
Assembled Holozyme components
Let us clarify certain points. At the replication fork, which can accommodate just two pairs of enzyme complexes, one pair at each fork joint, they are bound in such a way; they are positioned back to back.
The core enzyme bound on leading strand moves in 3’à 5’direction and the other core enzyme bound to lagging strand moves in 5’à 3’ direction. Perhaps this movement is propelled and facilitated by the Helicase complex that moves on the lagging strand in 5’à3’ direction. The Helicase is known as the motor protein, it uses ATP energy as driving force for movement. The Helicase protein is endowed with DNA dependent ATPase activity.
The DNA polymerase performing synthesis and editing
The diagram shows the formation of Phosphodiester bond formation between the incoming triphospho nucleotide and primer nucleotide between alpha phosphate with 2’OH group
The diagram illustrates positioning of core enzymes in opposite direction.
The diagram shows a different model for the synthesis of a new DNA strand on leading and lagging strand as well.
Orientation; The extension of the primer as DNA in 5’à3’ direction is absolutely perfect for the enzyme for the enzyme is traversing the leading strand in 3’à 5’ direction. On the contrary the core enzyme, which is moving on, the lagging strand is in 5’à3 direction. The primers are laid in the opposite direction. In order to synthesize the new strand on lagging strand one of the two core enzyme has to move in the opposite direction to obey the direction of new strand synthesis. This is what is called a paradox.
In this process whatever may the orientation of the template strands, the new strand is synthesized in 5’-3’ direction.
A perfect catalytic site formation with proper geometrical shape is an important conformational event and such conformational changes are induced by the nucleotide geometry of the template.
At catalytic site where the arriving nucleotide sits, contains a pocket, where the gamma and beta phosphate chain of triphosphate is drawn into and the alpha phosphate group is positioned against the 3’OH group of the primer nucleotide or any other nucleotide that already exists. Thus provides electronegative rich surrounding for the catalysis, to break a bond and make a bond.
Then the core complex moves forward to continue to synthesize the complementary strand.
The movement of replication fork ahead and the movement of enzyme, while polymerization ensuing is well coordinated events.
The above diagram and the diagram below show how leading and lagging strand are copied simultaneously.
This diagram is self explanatory
The diagram shows the 3-D folding of Klenow large fragment bound to DNA
The diagram is DNA pol I large fragment performing 5’>3’ polymerization and 3’>5 exonuclease activity’
As DNA pol-I has both 3’à5’ and 5’>3’ exonuclease and 5’à3’ polymerase activity, the enzyme can recognizes the 3’OH end of primer RNA and removes RNA nucleotides by strand displacement till the entire RNA is removed. While the enzyme is engaged in progressive removal of RNA primer, it can also use the 3’OH of the DNA and extends till it reaches 5’end of next Okazaki fragment, at which it stops and dissociates from the dsDNA. DNA pol-Is’ processivity is not great and also its fidelity, yet its activity is very important for it is also involved damage repair.
The diagram illustrates the chemical mechanism of joining DNA fragments
Site of TER region
TUS protein by binding to TER sequences prevent reinitiation of DNA replication in the newly replicated DNA
Replication of the DNA separating the opposing replication forks, leaves the completed chromosomes joined as ‘catenanes’ or topologically interlinked circles. The circles are not covalently linked, but cannot be separated because they are interwound and each is covalently closed. The catenated circles require the action of topoisomerases to separate the circles [decatanation]. In E.coli, DNA topoisomerase IV plays the major role in the separation of the catenated chromosomes, transiently breaking both DNA strands of one chromosome and allowing the other chromosome to pass through the break. Confusion arises when some scientific literature state that DNA gyrase is the sole enzyme responsible for decatanation. In an experiment conducted by Zechiedrich,Khodursky and Cozzarelli in 1997, it was found that topoisomerase IV is the only important decatenase of DNA replication intermediates in bacteria. In this particular experiment, when DNA gyrase alone were inhibited, most of the catenanes were unlinked. However, when Topoisomerase IV alone was inhibited, decatenation was almost completely blocked. The results obtained suggest that Topoisomerase IV is the primary decatenase in vivo, and although DNA gyrase does play a role in decatenation, its function is not as essential as topoisomerase IV in the decatentation of interlinked chromosomes.
(A) The positions of the six terminator sequences on the E. coli genome are shown, with the arrowheads indicating the direction that each terminator sequence can be passed by a replication fork. (B) Bound proteins allow a replication fork to pass when the fork approaches from one direction but not when it approaches from the other direction. The diagram shows a replication fork passing by the left-hand , because the DnaB helicase that is moving the fork forwards can disrupt the when it approaches it from this direction. The fork is then blocked by the second , because this one has its impenetrable wall of β-strands facing towards the fork.
The TER sequences, however, not all together required for termination of replication. There are many plasmid DNAs that complete replication without TER sequence; such DNA are too many and too far to understand. If a plasmid containing TER sequences are deleted replication is not affected.
Removal of catenation
Topoisomerase activities illustrated with on covalently closed circular DNA. Topisomerase enzymes are able to form supercoils in DNA, and interconvert covalentely closed circular DNA and their catenated forms.
Second round of DNA replication has to wait till the cell mass increases with cell volume (size).
As the two daughter DNA molecules are segregated they are sequestered to mesosomal membrane at attachment point. At this point the cytoplasm divides to generate two daughter cells which grow in time.
Immediate replication is prevented for one of the newly produced strand at ori site and the promoter region of dnaA gene is yet hemi methylated at GATC sites.
Hemi methylated GATC sites in ori c region makes the DNA bind to membrane. The region of origin has several GATC sequences; in hemi methylated state is covered by proteins called SeqA, which prevents methylation.
At the same time the promoter region of dnaA gene, as it is hemi ethylated fails to initiate transcription. The promoter has sequences which contains strong and weak sites for the binding of DnaA binding and DnaA-ATP binding respectively.
Complete methylation of hemi ethylated sites at promoter region makes the gene active.
Complete methylation at attachment site release the DNA from the membrane.
With time methylation at other GATC sites make SeqA to dissociate as more and more DnaA protein are formed and active Dna-ATP subunits are produced. This leads to the binding of DnaA-ATP to 9-mer sites initiates another round of replication. Between the events, completion and initiation there is a time lapse called eclipse period. It is at this point of time cell acquires the competency for the second round replication.
Segregation, Partioning of Chromosomes and Cytokinesis:
Daughter DNA molecules produced in general move away from the central region, but at the end of replication the daughter DNA molecules also have chances of recombination before partitioning or segregation. If they undergo recombination by Xer-C and Xer-D, which are recombinases, they generate Holliday junctions. This leads to problems in segregation. Xer-C and Xer-D dependent recombination takes place in a region determined by ‘Dif’ site, which is about 30kbp long near Ter region. However resolution of Holliday junction by the enzyme Fts-K releases circular DNA from catenated condition into independent circles; this function is essential for segregation. Fts-K is a membrane protein; its Carboxyl end causes Xer to resolve this. The Fts-K has ATPase activity and it can traverse along the DNA.
Mutation that affects partioning of DNA is also found in Topoisomerases. These enzymes are required for separation of daughter DNA molecules; otherwise they will be tangled at TER regions. Such mutations can result in cell containing 2n DNA in some cells and some cells without DNA. Mutations that are cis-acting and trans-acting have been identified, but gene products that are transacting have been isolated. If protein synthesis is blocked before termination of replicated DNA molecules fail to segregate, but if protein synthesis is allowed to resume chromosomes segregate and move away from the middle region. It is now known that segregation requires Muk-genes. Mutations affect this function. Muk-A is identical to an envelope protein called Tol-C that is involved in chromosomal attachment. Gene Muk-B produces a protein, which is 180KD; it works like structure maintenance chromosome (SMC) proteins. Muk-B has some sequence relation to that of Dynamin a motor protein. Muk-BEF proteins act on chromosomal DNA in condensing into individual nucleoids which helps in proper segregation, higher super coiled density helps in proper segregation.
“The replication of E. coli circular DNA begins from the region called oriC. Replication forks run to both directions and they are stopped by the Tus protein at the region of ter which is opposite to oriC. The dimer of double-stranded DNA (dsDNA) which finished replication is recomposed in the region’ dif ‘and separates into two monomers. The enzyme recognizing the dif sequence and making recombination is a complex of XerC and XerD. Furthermore, it is noted that XerCD divides the dimer into two monomers with the help of the FtsK protein. FtsK is a membrane-bound DNA translocase found in many eubacteria, such as E. coli.
The DNA translocase is an adenosine triphosphate (ATP)-dependent molecular motor which move DNA rapidly in the case of chromosome division, DNA recombination, and DNA transport. T7gp4, DnaB, SV40 SpoIIIE, etc. belong to this type of protein. The structure of FtsK may be divided into three regions (N, linker, C). The N domain is locates on cell membrane which invaginates in cell division. The length and structure of the linker region varies depending on bacterial species. The C domain transposes DNA to the direction of a daughter cell at the rate of more than 6.7 kbp/s. At the same time, the C domain proceeds to the dif region and separates dsDNA by activating XerCD complex. FtsK expresses the function by forming a hexamer-ring. A large channel is formed in the center of each subunit and dsDNA passes through this channel.
A schematic view showing the replication of circular DNA
A: The replication of circular DNA proceeds with two replication forks from the
region of oriC
to the region of ter
in opposite directions.
Step B: When replication has finished at ter, XerCD complex bind to dif site located in the ter.
Step C: One replicated circular DNA is dragged into the daughter cell through a channel in the center of the FtsK(C-domain). At the same time, this means dif move toward the FtsK(C-domain).
Step D: When dif approaches FtsK(C-domain), XerCD is activated by the FtsK.
Step E : The circular DNA dimer is completely resolved by the activated XerCD site-specific recombination.
(*) Although only two FtsK is shown in the Fig.1, many FtsK exist on invaginated region of cell membrane.”]
Bacterial DNA has been found to be in association with membrane fraction, the membrane fraction have been found to be enriched with genetic markers associated with Origin, replication fork and Ter regions. Protein in this region is involved in initiating replication at origin, but mutations affect this process. Association of origins to membranes is the key for segregation of daughter chromosomes in condensed state.
A similar situation is also found in the case of single copy plasmids, when the single copy plasmid replicates it has to be segregated equally otherwise the plasmid is missing in one of the daughter cells. But there are genes involved in segregation of single copy plasmids, they are called Par- genes; par-A and par-B whose products are transacting components.
The plasmids also contain one cis acting site called “par-S”. Par-A is an ATPase binds to Par-B protein and forms dimmers. Meanwhile a dimer protein called Integration Host Factor (IHF) binds to ‘S’ , a 24 bp long site, which is located in between par box-A and par box-B. These boxes are the sites to which Par-A and par-B proteins bind as dimmers. Binding of IHF to “S” bends DNA in such a way the par boxes are brought very close to one another. This facilitates the binding of Par-A and Par-B proteins as dimmers to these boxes. This complex proteins is called partitioning complexes which assemble on each plasmid DNA. These Complexes some how are associated with partioning membranes, which are segregated by septal membranes.
Bacterial cell division involves cleavage of the cytoplasm in the middle and cell separation. A set of genes is involved in septal formation that leads to cell division; they are many such genes, such as min genes, PBP2, PBP3 and Rod-A, Fts-genes, Xer and Muk and other genes. Information on the details, though scanty, conditional mutants have provided some significant information.
A list of Genes Involved in Cytoplasmic Division:
Gene product n function
Maintains rod shape
Rod-A and pBP2
Rod –A is a member of SEDS family proteins; involved in periseptal ring formation
Fts-A = Transpeptidase
Fts-W= transmembrane protein
Fts-Z= like tubulin proteins
Fts-Z is Involved in mid septal ring formation not polar septa, it is temperature sensitive, mutation produces long filamentous cells.
Cell separation or splitting the septal structure
Inactivation of septation site
min B, min-C, min- D, and min-E
Mutation in min-B leads to septation even at poles leading to produce mini cells; mutation in C and D leads to filamentous morphology.
Regulation of septation
Involved in segregation of chromosomes; they are like ‘smc’ proteins
They are involved in portioning of chromosomes
Cell division in bacteria is dependent on the precise placement of components of the division machinery at the cell center, a process initiated by assembly of a medial ring of the tubulin analog FtsZ.
In E. coli, movement of the growing fork is about 1000 bp per second. It takes about 42 minutes to duplicate the entire genomic DNA. In eukaryotic DNA, the fork movement is only about 100 bp per second. This is probably due to the association of DNA with histones, which may hinder the fork movement. In humans, replication of the entire genome requires about 8 hours. In fruit flies, it takes only 3 - 4 minutes.”
A simple illustration of Bacterial cell division using various components One interesting point is that the enzymes are in the center of the cell and DNA new strands are pulled along the enzyme instead of enzymes moving along the DNA.
Fidelity of replication: