Mechanism of Replication:

 

Initiation: 

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 .

 

 

Figure 1

 

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. Mickiewicz et al. Genome Biology 2001 2:interactions1004.1   doi:10.1186/gb-2001-2-12-interactions1004

 

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 SPoOJs and they prevent DNA release from membrane and initiation of replication is inhibited by the inhibitor present in the membrane at hemi-methylated sites at 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 site is recognized as OCB1 to which oriC binds.  This site is  perhaps the adhesion  region of outer and inner membranes. This region is located to the left of OriC where 10 out of 11 Dam methylation sites are concentrated. The probable protein binding site of DNA sequence can be   a/gCCa/tGGg. The inhibitor is called IciA (inhibitor of chromosome initiation).  On full methylation, Dna-A proteins release DNA from the inhibitors and from the membrane.

 

 

 

 

This picture depicts 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.

 

 

Figure 1.

The E. coli origin of replication bears five 9-mer DnaA-binding sites (R1, R2, R3, R4 and R5) as well as three 13-mer binding sites included in an A+T-rich DNA unwinding element (DUE). Although the 9-mers show no differential specificity between DnaA-ATP and DnaA-ADP, the 13-mers specifically recruit DnaA-ATP. In addition to DnaA-binding sites, oriC hosts-specific binding sites for IHF-Integration Host factor, SeqA and FIS-Factor for inversion, three proteins that regulate the activity of DnaA; Sylvain Zorman,1 H. Seitz,2 B. Sclavi,3 and T. R. Strick1,*

 

 

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 fully methylated strands (methylation in both strands) in OriC region.  This OriC region contains many GATC sequences and the hemi-methylated sequences are bound by  SeqA (called ‘A’ boxes) and masks the sequences from methylation. The SeqA is a tetramer protein has greater preference to hemi-methylated GATC sequences in newly replicated E.coli DNAs. This time period is called eclipse period. 

 

media/image3_w.jpg

 

The vast majority of chromosomal GATC sites are fully methylated until DNA replication generates two hemimethylated species, one methylated on the top strand and one methylated on the bottom strand. Within a short time after replication (less than 5 minutes), Dam methylates the nonmethylated GATC site, regenerating a fully methylated GATC site.

 

media/image2_w.jpg

 

Helically phased GATC sites can be bound by SeqA when they are in the hemi-methylated state. Binding of SeqA inhibits Dam methylation, maintaining the hemi-methylated state for a portion of the cell cycle. Dissociation of SeqA allows Dam to methylate the hemi-methylated DNA, thus generating fully methylated DNA. But SeqA spontaneously dissociate and reassociate.  During this period as more DnaA are produced due to activation of dnA gene.  The activation of dnaA gene is due to fully methylated state of the gene promoters. The dnaA gene is located at 82 min site.  Its promoter is ~945 bp.  And the gene codes for 54kDa protein.  It is located left of OriC.  It contains multiple promoters with abundance of GATC sequences (at least 9 of them, while OriC contains 11 of them.  The DnaA protein is involved in activation of its own gene by the binding of DnaA proteins to their promoter sequences or what is called DnaA box.  The gene is autoregulated by its own product DnaA at transcription level.  It is regulated by methylation of GATC sequences of both strands in the promoter region. The promoter consists of several sites, of which one is strong binding site to 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.

 

 

 

 

Two or more helically phased GATC sites can be bound by SeqA when they are in the hemimethylated state. Binding of SeqA inhibits Dam methylation, maintaining the hemimethylated state for a portion of the cell cycle. Dissociation of SeqA allows Dam to methylate the hemimethylated DNAs, generating fully methylated DNA.

 

Signals for activation of cell division leads to methylation of GATC at gene promoter dnaA as well as at other regions.  This leads to the activation of dna-A gene to produce DnaA proteins.  Expression of dnaA gene is regulated by  its own product DnaA-ATP (it has two promoter regions).  The DnaA is AAA+ protein and it is activated by the binding of ATP.  The DnaA protein contain four domain; domain III contains ATP binding, others have protein-proteins interaction and DNA binding domains. Proteins organize into right handed oligomeric forms around which the DNA wraps around in left handed form so as to render the DNA into negative supercoil (Bramhill and Kornberg). This has an effect on 13-mer region called DUE (DNA Unwinding Element) region opens up into single stranded templates.

 

 

Activation of cell division process at cytoplasmic level involves many signal transduction pathways similar to that of Arc two component systems.  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. Factor titration is done sequestering of DnaA proteins till adequate number of DnaA proteins is generated.

 

GATC hemi methylation 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 to 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.

 

 

---- Abox- SeqA-Abox-ori C-Abox-seqA-Abox--

 

 

 

 

 

 

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.

 

Hemi methylation prevents initiation of replication, at the same time hemimethylation in promoter elements of dna-A gene also leads to repression of it.  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--

 

 

 

The circular DNA 4x10^bp long is approximately 1mm long exceeds the size of the cell.  Thus the long DNA is compacted into supercoiled structured regions bound by proteins called nucleoids. They are like pearls on necklace. The bacterial SMCs (structural maintenance of chromosomes) act as molecular clamps.  Each of the nucleoids contains highly compacted supercoiled DNA (60%), nucleoid proteins NAPs (different from histones), topoisomerases and transcriptional factors and mRNAs.  These nucleoids are transcriptionally active. These regulatory NAPs are: Fis (factor for inversion stimulation), HU (histone-like protein), H-NS (histone-like nucleoid structuring protein), and IHF (integration host factor). The concentrations of these proteins vary in different growth phases, from 10,000 to 60,000 monomers per cell.  The said number of nucleoids can be more than 2063. The size of DNA can be approximately 1 to 1.5kbp.

 

     

50-100 kbp long loops studded with specific proteins; these organized structures are called Nucleoids.  Such loops are attached at the base by high salt insoluble nuclear matrix/scaffold like proteins.

 

Bacterial nucleoid-associated proteins, nucleoid structure and gene expression

 

The folded chromosome is organized into looped domains that are negatively supercoiled during the exponential phase of growth. In this phase, the abundant nucleoid-associated proteins histone-like nucleoid-structuring protein (H-NS) and factor for inversion stimulation (Fis) bind throughout the nucleoid and are associated with the seven ribosomal RNA operons. As shown here in two cases, these are organized into superstructures called transcription factories. b | In stationary phase the rRNA operons are quiescent and Fis is almost undetectable. The chromosome has fewer looped domains, and those that are visible consist of relaxed DNA, Shane C. Dillon & Charles J. Dorman

 

 

 

 

Bacterial nucleoid-associated proteins, nucleoid structure and gene expression

 

Once GATC sites are fully methylated at the region of ori and E.coli DNA membrane binding site, the circular compacted DNA detaches from the membrane. These two events are very important in initiating DNA replication cycle. Methylation of promoter region 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 13mer segments (DUE) 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.

 

 

sandwalk.blogspot.com/.../dna-replication-in-e-coli-proble...

 

 

  The replication bubble at the origin region shows direction of new strand synthesis.

 

 

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.

 

 

 

 

 

 

 

  

 

 

Stepwise process in opening of DNA into replication bubble and SSB loading and clamps and DNA-pol assembly

 

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 parental strands of DNA act as templates.

·         Until recently, it was believed that the replication complex perse moves along the strands of DNA.

 

 

Recent evidence suggests that the replication complex is stationary, and DNA strand is pulled and 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 uses the 3’OH of the RNA primer and adds complementary deoxy ribo nucleotides to the     growing strand, proofreads the DNA, and repairs it.

·         DNA ligase seals up if any 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 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 the base of DNA pol III provides firm binding to the DNA strand and helps processivity.  The gamma complex also facilitates the assembly of DNA-Pol III as dimers on to their respective beta clamps on to both strands.  DNA pol III, as a dimer complex, joins template at the joint of the fork.

 

Assembled components at one end of the replication fork.

 

So one finds one sets of two DNA-Pol dimers, i.e. at one end of the fork and the other set at the other end of replication fork.  Perhaps in the dimeric Holozyme configuration one that is bound to leading strand is oriented toward fork end and the other in opposite direction.   Beta clamps bind to complex assembles on to the template by displacing SSBs.   The new strands always grow in 5’ > 3’ direction.   One strand grows on leading  strand till the end of the DNA.  The other called lagging strand the DNA polymerase assembles nucleotides upto 1000 to 1500 ntds and dissociates. Then RNA primase adds primers on to it beta clamp with DNA pol is loaded with the help of Gamma complex.

 

 

 

DNA 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:DnaB 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

 

 

A 3-D model of Beta clamp encircled one of the DNA strands

 

 

 

Components of DNAPol and gamma complex with beta clamps

 

 

An illustration of the dimeric holozyme with all its components and a single holozyme

 

Assembled Holozyme components

 

 

Elongation:

 

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.

 

A ssDNA template with a primer

 

 

 

The diagram shows the addition of Nucleotides on the template strand

 

 

This figure shows a new strand formation on a template >>>.

 

 

 

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.

 

 

 

 

 

 

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’

 

While the replication is progressing with a zing, the RNA primers are removed and gaps are filled, and the ends are ligated by DNA pol -I and DNA ligase respectively.  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

 

 

Termination:

 

 

                                Location 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 decatenation of interlinked chromosomes.

An external file that holds a picture, illustration, etc., usually as some form of binary object. The name of referred object is ch13f22.jpg.

 

The role of terminator sequences during DNA replication in Escherichia coli:

(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 Tus 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 Tus, because the DnaB helicase that is moving the fork forwards can disrupt the Tus when it approaches it from this direction. The fork is then blocked by the second Tus, because this one has its impenetrable wall of β-strands facing towards the fork.

 

 

 

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.

Removal of catenation

 

 

Topoisomerase activities illustrated with on covalently closed circular DNA. Topoisomerase enzymes are able to form supercoils in DNA, and interconvert covalently 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.

0

A schematic view showing the replication of circular DNA

Step 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. 

 

 

A list of Genes Involved in Cytoplasmic Division:

 

Event

 

Gene

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

Septation

Fts-A = Transpeptidase

Fts-L

Fts-F

Fts-E

Fts-I

Fts-K

Fts-Q

Fts-N

Fts-W= transmembrane protein

Fts-Z= like tubulin proteins

Zip A

 

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

Env-A

 

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

Sfi-A

Lon

 

 

 

 

 

Muk-A,

Muk-B,

Muk-E,

Muk-F

Involved in segregation of chromosomes; they are like ‘smc’ proteins

 

Par-A,

Par-B

Par-C

They are involved in portioning of chromosomes

 

 

 

Cytoplasmic Division:

 

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.

 

 

 

Fig. 1.

 

 

 

 

 

 

 

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 Dividing cells 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.”

Figure 1

 

A simple illustration of Bacterial cell division using various components.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Fidelity of replication: