Theta Plasmid Replication – On-line Biology Notes

Common Construction of Plasmid Origins of Replication

• Replication begins from the origin of replication (ori).
• It refers back to the portion of the sequence that’s focused by replication initiation components.
• Origin of replication or orito consult with the cis-ori, and replicon to consult with primary or minimal replicons.
• Rep protein helps in initiation through the replication course of.
• However some theta plasmids rely on the host initiation components for replication.
• Rep recognition websites sometimes encompass direct repeats or iterons.
• Its particular sequence and spacing are essential for initiator recognition.
• Two Rep proteins are current:
• π of R6K
• RepA of ColE2

Replication Initiation: Duplex Melting and Replisome Meeting

• Duplex melting depends on transcription.
• It may be mediated by plasmid-encoded trans-acting proteins (Reps).
• When the Rep protein binds the ori area then a nucleoprotein complicated is shaped.
• On the A+T-rich phase, the DNA duplex is opened.
• The opening of the 2 strands of the DNA is essential.
• In Theta-type plasmid, the meeting of the replisome will be:
  • DnaA-dependent
  • PriA-dependent
• DnaA-dependent meeting intently resembles replication initiation at oriC. It’s the website the place the chromosomal replication initiates
• PriA-dependent meeting parallels replication restart following replication fork arrest. It relies on D-loop formation with the additional DNA strand provided by homologous recombination.
• Within the theta-type plasmids, the Rep protein unwinds the 2 strands.
• The replication fork is shaped the place the DnaB is loaded in it. DnaA helps with this loading.
• Some plasmids rely on transcription for duplex melting, i.e unwinding of the 2 strands.
• The transcript itself will be processed in it and develop into the primer for an extension.
• When the primer is prolonged constantly, it results in the synthesis of a number one strand.
• It facilitates  the formation of a Displacement loop or D-loop
• The nascent ssDNA strand separates the 2 strands of the DNA duplex and hybridizes with one among them.
• PriA (initiator of primosome meeting) will be recruited to the forked construction of the D-loop.
• Alternatively, PriA will be recruited to a hairpin construction. It varieties when the double-stranded DNA opens.
• PriA helps within the unwinding of the lagging-strand arm.
• It additionally helps within the meeting of two further proteins (PriB, and DnaT) to load DnaB onto the lagging strand template.
• The loading of DnaB is impartial of DnaA on this case.
• After loading DnaB, each DnaA-dependent and –impartial modes of replication converge.
• Different protein and enzymes concerned in it are:
  • SSB (single-stranded binding protein)
  • DnaB (helicase)
  • DnaC (loading issue)
  • DnaG (primase)
  • DNA polymerase III (Pol III) holoenzyme.
• SSB protein binds the single-stranded DNA and helps in its stabilization.
• Then within the replication fork, DnaB is loaded within the type of a posh with DnaC.
• Then, DnaG (the primase) synthesizes RNA primers for the synthesis of lagging-strand synthesis.
• Then Pol III holoenzyme is loaded.
• The holoenzyme incorporates:
  • a core (with α, a catalytic subunit, and ε, a 3’→5’ exonuclease subunit),
  • a β₂ processivity issue
  • a DnaX complicated ATPase.
• DnaB helicase exercise is stimulated by its interplay with Pol III and modulated by its interplay with DnaG.
• It facilitates the coordination of leading-strand synthesis with that of lagging-strand synthesis throughout sluggish primer synthesis on the lagging strand.
• Within the Gram-negative micro organism, single replicative polymerase (Pol III) is current.
• Within the Gram-positive micro organism, two replicative polymerases are current:
  • PolC
    • PolC polymerase helps within the synthesis of the main strand.
  • DnaE extends DnaG-synthesized primers earlier than handoff to PolC on the lagging strand.
• In theta plasmids, lagging-strand synthesis is discontinuous and coordinated with leading-strand synthesis.
• The replicase extends a free 3’-OH of an RNA primer, which will be generated by DnaG primase (in Gram – micro organism)
• It’s accomplished by the concerted motion of DnaE and DnaG primase (in Gram + micro organism)
• It can be accomplished by various plasmid-encoded primases.
• Discontinuous lagging-strand synthesis includes repeated priming and elongation of Okazaki fragments.
• DNA polymerase I (Pol I) contributes to plasmid replication in a number of methods.
• In ColE1 and ColE1-like plasmids, Pol I can prolong a primer to provoke leading-strand synthesis.
• Then it opens the DNA duplex.
• This course of can expose a hairpin construction within the lagging-strand, referred to as single-strand initiation (ssi) website or primosome meeting (pas) website, and/or generate a D-loop.
• Each hairpins and forked constructions recruit PriA. It is step one within the replisome initiation complicated.
• Then, Pol, I assist in the synthesis of the discontinuous lagging strand.
• It removes RNA primers by its 5’→3’ exonuclease exercise and fills within the remaining hole by its polymerase exercise.
• Pol I can functionally substitute Pol III in  coli.
• There are three modes of replication for round plasmid replication. They’re:
  • Theta
  • strand-displacement
  • rolling circle.

Theta Plasmid Replication:

• Theta mode of replication is just like chromosomal replication.
• There may be the synthesis of leading- and lagging-strand.
• Lagging-strand is discontinuous.
• No DNA breaks are required for this mode of replication.
• There may be the formation of bubbles within the early levels of replication.
• It resembles the Greek letter θ.
• Theta replication is of 4 varieties:
  • θ class A
  • θ class B
  • θ  class C
  • θ  class D

Class A Theta Replication

• Class A theta plasmids embody:
  • R1
  • RK2
  • R6K
  • pSC101
  • pPS10
  • F
  • P
• For the replication initiation, all these plasmids rely on Rep protein:
  • RepA for R1, pSC101, pPS10, and P1
  • Trf1 for RK1
  • π for R6K
• Rep proteins bind interons (direct repeats) within the plasmid origin of replication.
• In plasmid P1, RepA monomers contact every iteron by two consecutive turns of the helix.
• It results in in-phase bending of the DNA, which wraps round RepA.
• In R6K plasmids, the π binding of its cognate iterons bends the DNA and generates a wrapped nucleoprotein construction.
• The 2 exceptions to the presence of a number of iterons at school A theta plasmid origins of replication are:

(a) Plasmid R1, which options two partial palindromic sequences as an alternative of iterons. R1 palindromic sequences are acknowledged by RepA.

(b) The R6K plasmid, which has three origins of replication:

• γ (with 7 iterons)
• second origin (α) includes a single iteron
• third origin (β) solely has half an iteron.
• γ oriis an institution origin. It permits replication initiation instantly the next mobilization when ranges of π protein are low.
• α and β oris could be upkeep origins in cells inheriting the plasmid by vertical transmission.
• γ ori acts as an enhancer which favors the long-range activation of α and β oris by switch of π.
• α and β oris are nonetheless depending on the a number of iterons current in ori γ.
• Rep binds the ori area and duplex DNA melting happens.
• Rep-DnaA interplay is steadily concerned.
• In plasmid pSC101, RepA helps to stabilize DnaA binding to distant dnaA It results in strand melting.
• Plasmid P1’s ori has two units of tandem dnaA bins at every finish.
• DnaA binding loops up the DNA which ends up in preferential loading of DnaB to one of many strands.
• RK2’s TrfA mediates the open complicated formation and DnaB helicase loading within the absence of dnaA
• The presence of DnaA protein remains to be required.

Class B Theta Replication:

• Class B theta plasmids embody ColE1 and ColE1-like plasmids.
• Class B plasmids depend on host components for each double-strand melting and primer synthesis.
• The DNA duplex is opened by transcription of an extended (~600 bp) pre-primer known as RNA II.
• It’s transcribed from a constitutive promoter P2.
• The three’ finish of the pre-primer RNA varieties a steady hybrid with 5’ finish of the lagging-strand DNA template of ori.
• This steady RNA-DNA hybridization (R-loop formation).
• The pairing of the G-C between the transcript and lagging strand DNA template facilitates it.
• It varieties a hairpin construction between the G- and C-rich stretches.
• Then the RNA pre-primer is processed by RNAse H producing a free 3’ -OH finish
• It acknowledges the AAAAA motif in RNAII.
• Extension of this RNA primer by Pol I initiates leading-strand synthesis.
• The purpose the place the RNA primer is prolonged (referred to as RNA/DNA change) is taken into account the replication begin level.
• The nascent leading-strand separates the 2 strands of the DNA duplex and may hybridize with the leading-strand template, forming a D-loop.
• PriA is recruited to the forked construction of the D-loop.
• Alternatively, PriA will be recruited to hairpin constructions forming on the lagging-strand template when the duplex opens.
• priA strains don’t assist ColE1 plasmid replication.
• The hypomorphic mutations in priA priBend in a decreased ColE1 plasmid-copy-number.
• When the Pol III holoenzyme is loaded, this polymerase continues leading-strand synthesis.
• Then it initiates lagging-strand synthesis.
• Pol III replication of the lagging strand towards RNA II sequence is arrested 17 bp upstream of the DNA/RNA change, at a website identified at terH.
• It’s unidirectional replication.
• Lagging-strand replication by Pol III seems to finish just a few hundred nucleotides upstream of the terH website, leaving a niche that’s crammed by Pol I.

R-loop formation:

• R-loop formation is important in strategy of replication initiation.
• Deficits in RNAse H and/or Pol I don’t stop initiation.
• Within the absence of RNAse H, unprocessed transcripts can nonetheless be prolonged with some frequency.
• Within the absence of Pol I, the Pol III replisome can nonetheless be loaded on an R-loop shaped by the transcript and lagging-strand template.
• R-loop formation happens because of native supercoiling within the path of the advancing RNA polymerase throughout transcription and is very deleterious.
• It’s as a result of R-loops block transcription and the elongation step throughout translation.
• So, the unscheduled R-loop formation is suppressed by the cell.

Hybrid Lessons of Theta Replication (Class C and D):

• The specialised priming mechanisms are current in these two courses that are mixed with components of sophistication A and sophistication B replication.
• Rep protein is current at school C and D plasmids.
• They provoke the leading-strand synthesis by Pol I extension of a free 3’-OH.
• They’ve termination alerts within the 3’ route of lagging-strand synthesis.
• Replication of those plasmids is unidirectional.
• Class C and D have advanced the specialised priming mechanisms.
• Class C contains ColE2 and ColE3 plasmids.
• The oris for these two plasmids are the smallest and differ solely at two positions.
• Certainly one of them determines plasmid specificity.
• ColE2 and ColE3 oris have two iterons and present two discrete practical subregions.
• One is specializing within the steady binding of the Rep protein (area I)
• one other one specializing within the initiation of DNA replication (area III).
• The Rep protein at school C plasmids has primase exercise.
• It synthesizes a novel primer RNA (ppApGpA) which is prolonged by Pol I at a set website within the origin area.
• Class C replication is unidirectional.
• The Rep protein might keep certain to the ori after initiation of replication, blocking the development of the replisome synthesizing the lagging strand.

Class D:

• It contains giant, low-copy streptococcal plasmids.
• Replication happens in a broad vary of Gram-positive micro organism.
• Examples:
• Enterococcus faecalispAMβ1
• pIP501 from Streptococcus agalactiae
• pSM19035 from Streptococcus pyogenes
• It requires transcription throughout ori sequence, Pol I extension, and PriA-dependent replisome meeting.
• The transcript is generated from a promoter controlling expression of rep.
• Replication relies on transcription by the origin.
• Rep binds particularly and quickly to a novel website.
• Denaturation of AT-rich sequence happens and varieties the open complicated.
• This binding denatures an AT-rich sequence instantly downstream of the binding website to kind an open complicated.
• RepE additionally has an energetic position in primer processing.
• As melting will increase RepE binding and RepE can cleave transcripts from the repE operon near the RNA/DNA change.
• Class D replisome meeting is PriA-dependent.
• A replisome meeting sign will be discovered 150 nt downstream from ori on the lagging-strand template.
• Replication arrest is induced by Topb, a plasmid-encoded topoisomerase.
• A second replication arrest is brought on by collision with a site-specific resolvase, Resb.
• It’s a plasmid-borne gene answerable for plasmid segregation stability.