Rolling Circle Replication Of Circular DNA (RCR),Prokaryotic DNA/Genome Replication By RCR; RCR In conjugal Transfer,Vegetative DNA Replication And Transposition

Rolling circle mechanism:

RCR was identified during the analysis of replication in bacteriophage ØX104 of E.coli, with the passage of time many other organism are identified which replicate their DNA by RCR mechanism including plasmids of gram positive and gram negative bacteria, archeal plasmids, bacteriophages and many eukayotic viruses.Characteristic feature of rolling circle mechanism is its asymmetric character due to the separation of leading strand synthesis from the lagging strand synthesis.Replication on both leading and lagging strand is initiated at different origins of replication. RCR mechanism is employed in following biological processes

· RCR mechanism in vegetative DNA replication

· RCR mechanism in conjugal transfer

· RCR mechanism in transposition

Rolling circle mechanism in vegetative DNA replication

Most known RCR replicons including bacterial plasmids and bacteriophages replicate their genome by RCR process.RCR replication is completed in 3 stages, which are discussed below:

Initiation:

Process of replication start when the initiator protein also called nick enzyme- RepA interact with the origin of replication which has two different regions : binding site and nick site.RepA protein cut one of strand due to its catalytic tyrosine activity at specific origin of replication such as “dso” (double strand origin, while other strand remains intact(no nick is created in it).

Elongation:

After causing break in double stranded DNA two ends are produced 5’phosphate and 3’OH, RepA protein remained attached to the 5’ whereas free 3’OH end is released which then act as primer for DNA Pol III to synthesize new strand of DNA..Here no additional primer is needed as 3”end is already available Halicase enzyme of host is used to epen the strand.5’end remain attached to RepA protein until circle is completed, after completion of circle 5’ end form RepA protein is transferred to 3’OH group of second strand.This process require energy and termed as phosphotransfersase reaction. of leading strand synthesis occur and circular single stranded DNA molecule is released.

Later this ssDNA is converted into dsDNA.At lagging strand origin of replication sso(single strand origin )is present where ezymetic machinery of host is required rather then initiator protein to synthesize that strand. Primase complex is recruited at sso site and DNA polymerse began the synthesis of DNA using the unnicked strand as a template, releasing the nicked strand as single stranded DNA in the by host helicase enzyme such as pcrA .

Termination:

In this step linear DNA molecule is converted to the circular DNA through polymerization by DNA polymerase,for this initiator protein creates another nick in leading strand to terminate its synthesis as a result one dsDNA molecule is released,first circle.After that DNA polymerase III And RNA polymerse replicate second strand which is released as linear single strand and ligated by DNA ligase into circular ssDNA, replication start at “sso “ site and Primer is removed by DNA polymerase I and nick sealed by DNA ligase and 2nd double stranded circular DNA is released.

Diagrammatic representation of Rolling Circle Replication

Rolling circle mechanism in conjugal transfer:

Conjugal transfer is the process in which bacterial plasmids are transferred from one bacteria to another.Two gene modules are involved in this process

· MPF: Mating pair formation, it encodes type-4 secretion system (T4ss) which is involved in transport as well as biogenesis of bacterial conjugated pairs connected by pillus.

· DTR: DNA transfer and replication, it encodes mob proteins responsible to create nick in one DNA strand to initiate RCR replication

Relaxase (mob protein) is replication initiator protein, consists of two conserved tyrosine residues in its catalytic site i.e , Y18 and Y26 , it creates nick at specific DNA region known as origin of transfer “oriT” , cleavage on one strand to initiates RCR replication. OriT contains both nick and bind site, analogous to “dso” region present in RCR plasmids as discussed in above mechanism. Most important role which RCR plays in conjugal transfer is simultaneous replication of both strands in different bacterial cells, replication of leading strand occur in donor cell by first tyrosine residueY18 whereas lagging strand is replicated in the recipient cell.

After Initiation of RCR mob protein remain attached on the 5” end of origin of transfer , on the other hand3”OH end act as primer to synthesize complementry strand in the donar cell. Nucleoprotein complex known as relaxosome is formed by DNA relaxation, and targeted by T4ss which form pores in the membrane and transfer leading strand along with relaxosome from donor to the receipient cell and round of replication is completed. Mob protein is also transferred in recipient cell with relax some complex , it’s a multifunction protein having different domains:

· Y18 tyrosine residue, initiate replication at oriT in donar cells

· Y26 tyrosine residue ,which is responsible for termination of replication on lagging strand in recipient cell

· Additional domain having primase activity to synthesize primer in recipient cell and convert ssDNA into dS DNA, even in those strains where origin of replication on lagging strand is not recognized so, host range is increased.

Rolling circle mechanism in transposition:

Bacteria also contain transposable elements “TEs “which are mobile genetic elements may integrate in host genome by trasposase enzyme and change their location by recombination such as as transposition.Presence of inverted repeats at terminal region is their most Important feature of TEs.Members of IS91family constitute rolling circle transposition mechanism RCT, is 5’P identified in bacterial genome of different taxanomic groups many TEs other then IS91family are reported in plant and animal genomes which follow rolling circle transposition.

They encodes transposase TnpA which contain two conserved tyrosine residues same like mob protein Y249 and Y253.RCT mechanism one strand of ISN1 is exicied from donor and then integrated at specific target site within host genome.Terminal region of IS91 conatin ori91 and ter91 for initiation as well as termination of first transposition step. In first step of transposition, nick is created within ori91 region by TnpA and resulting 3”OH group act as a primer for initiation of leading strand replication whereas the displaced DNA strand is converted into RCR specific single stranded circular DNA as a result of cleavage at ter91 region by tnpA protein.In 2 nd step of transposition this circular ssDNA is incorporated at specific new target site by tranposase .