Theta Plasmid Replication

August 24, 2021 Gaurab Karki Microbial Genetics 0




theta plasmid replication
theta plasmid replication

Theta Plasmid Replication

General Structure of Plasmid Origins of Replication

  • Replication starts from the origin of replication (ori).
  • It refers to the portion of the sequence that is targeted by replication initiation factors.
  • Origin of replication or orito refer to the cis-ori, and replicon to refer to basic or minimal replicons.
  • Rep protein helps in initiation during the replication process.
  • But some theta plasmids depend on the host initiation factors for replication.
  • Rep recognition sites typically consist of direct repeats or iterons.
  • Its specific sequence and spacing are important for initiator recognition.
  • Two Rep proteins are present:
  • π of R6K
  • RepA of ColE2

Replication Initiation: Duplex Melting and Replisome Assembly

  • Duplex melting is dependent on transcription.
  • It can be mediated by plasmid-encoded trans-acting proteins (Reps).
  • When the Rep protein binds the ori region then a nucleoprotein complex is formed.
  • At the A+T-rich segment, the DNA duplex is opened.
  • The opening of the two strands of the DNA is important.
  • In Theta-type plasmid, the assembly of the replisome can be:
    • DnaA-dependent
    • PriA-dependent
  • DnaA-dependent assembly closely resembles replication initiation at oriC. It is the site where the chromosomal replication initiates
  • PriA-dependent assembly parallels replication restart following replication fork arrest. It depends on D-loop formation with the extra DNA strand supplied by homologous recombination.
  • In the theta-type plasmids, the Rep protein unwinds the two strands.
  • The replication fork is formed where the DnaB is loaded in it. DnaA helps with this loading.
  • Some plasmids depend on transcription for duplex melting, i.e unwinding of the two strands.
  • The transcript itself can be processed in it and become the primer for an extension.
  • When the primer is extended continuously, it leads to the synthesis of a leading strand.
  • It facilitates  the formation of a Displacement loop or D-loop
  • The nascent ssDNA strand separates the two strands of the DNA duplex and hybridizes with one of them.
  • PriA (initiator of primosome assembly) can be recruited to the forked structure of the D-loop.
  • Alternatively, PriA can be recruited to a hairpin structure. It forms when the double-stranded DNA opens.
  • PriA helps in the unwinding of the lagging-strand arm.
  • It also helps in the assembly of two additional proteins (PriB, and DnaT) to load DnaB onto the lagging strand template.
  • The loading of DnaB is independent of DnaA in this case.
  • After loading DnaB, both DnaA-dependent and –independent modes of replication converge.
  • Other protein and enzymes involved in it are:
    • SSB (single-stranded binding protein)
    • DnaB (helicase)
    • DnaC (loading factor)
    • DnaG (primase)
    • DNA polymerase III (Pol III) holoenzyme.
  • SSB protein binds the single-stranded DNA and helps in its stabilization.
  • Then in the replication fork, DnaB is loaded in the form of a complex with DnaC.
  • Then, DnaG (the primase) synthesizes RNA primers for the synthesis of lagging-strand synthesis.
  • Then Pol III holoenzyme is loaded.
  • The holoenzyme contains:
    • a core (with α, a catalytic subunit, and ε, a 3’→5’ exonuclease subunit),
    • a β2 processivity factor
    • a DnaX complex ATPase.
  • DnaB helicase activity is stimulated through its interaction with Pol III and modulated through its interaction with DnaG.
  • It facilitates the coordination of leading-strand synthesis with that of lagging-strand synthesis during slow primer synthesis on the lagging strand.
  • In the Gram-negative bacteria, single replicative polymerase (Pol III) is present.
  • In the Gram-positive bacteria, two replicative polymerases are present:
    • PolC
      • PolC polymerase helps in the synthesis of the leading strand.
    • DnaE extends DnaG-synthesized primers before handoff to PolC at 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 can be generated by DnaG primase (in Gram – bacteria)
  • It is done by the concerted action of DnaE and DnaG primase (in Gram + bacteria)
  • It can also be done by alternative plasmid-encoded primases.
  • Discontinuous lagging-strand synthesis involves repeated priming and elongation of Okazaki fragments.
  • DNA polymerase I (Pol I) contributes to plasmid replication in several ways.
  • In ColE1 and ColE1-like plasmids, Pol I can extend a primer to initiate leading-strand synthesis.
  • Then it opens the DNA duplex.
  • This process can expose a hairpin structure in the lagging-strand, known as single-strand initiation (ssi) site or primosome assembly (pas) site, and/or generate a D-loop.
  • Both hairpins and forked structures recruit PriA. It is the first step in the replisome initiation complex.
  • Then, Pol, I help in the synthesis of the discontinuous lagging strand.
  • It removes RNA primers through its 5’→3’ exonuclease activity and fills in the remaining gap through its polymerase activity.
  • Pol I can functionally replace Pol III in  coli.
  • There are three modes of replication for circular plasmid replication. They are:
    • Theta
    • strand-displacement
    • rolling circle.

Theta Plasmid Replication:

  • Theta mode of replication is similar to chromosomal replication.
  • There is the synthesis of leading- and lagging-strand.
  • Lagging-strand is discontinuous.
  • No DNA breaks are required for this mode of replication.
  • There is the formation of bubbles in the early stages of replication.
  • It resembles the Greek letter θ.
  • Theta replication is of 4 types:
    • θ class A
    • θ class B
    • θ  class C
    • θ  class D

Class A Theta Replication

  • Class A theta plasmids include:
    • R1
    • RK2
    • R6K
    • pSC101
    • pPS10
    • F
    • P
  • For the replication initiation, all these plasmids depend on Rep protein:
    • RepA for R1, pSC101, pPS10, and P1
    • Trf1 for RK1
    • π for R6K
  • Rep proteins bind interons (direct repeats) in the plasmid origin of replication.
  • In plasmid P1, RepA monomers contact each iteron through two consecutive turns of the helix.
  • It leads to in-phase bending of the DNA, which wraps around RepA.
  • In R6K plasmids, the π binding of its cognate iterons bends the DNA and generates a wrapped nucleoprotein structure.
  • The two exceptions to the presence of multiple iterons in class A theta plasmid origins of replication are:

(a) Plasmid R1, which features two partial palindromic sequences instead of iterons. R1 palindromic sequences are recognized by RepA.

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

  • γ (with 7 iterons)
  • second origin (α) features a single iteron
  • third origin (β) only has half an iteron.
  • γ oriis an establishment origin. It allows replication initiation immediately the following mobilization when levels of π protein are low.
  • α and β oris would be maintenance 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 transfer of π.
  • α and β oris are still dependent on the multiple iterons present in ori γ.
  • Rep binds the ori region and duplex DNA melting occurs.
  • Rep-DnaA interaction is frequently involved.
  • In plasmid pSC101, RepA helps to stabilize DnaA binding to distant dnaA It leads to strand melting.
  • Plasmid P1’s ori has two sets of tandem dnaA boxes at each end.
  • DnaA binding loops up the DNA which leads to preferential loading of DnaB to one of the strands.
  • RK2’s TrfA mediates the open complex formation and DnaB helicase loading in the absence of dnaA
  • The presence of DnaA protein is still required.

Class B Theta Replication:

  • Class B theta plasmids include ColE1 and ColE1-like plasmids.
  • Class B plasmids rely on host factors for both double-strand melting and primer synthesis.
  • The DNA duplex is opened by transcription of a long (~600 bp) pre-primer called RNA II.
  • It is transcribed from a constitutive promoter P2.
  • The 3’ end of the pre-primer RNA forms a stable hybrid with 5’ end of the lagging-strand DNA template of ori.
  • This stable RNA-DNA hybridization (R-loop formation).
  • The pairing of the G-C between the transcript and lagging strand DNA template facilitates it.
  • It forms a hairpin structure between the G- and C-rich stretches.
  • Then the RNA pre-primer is processed by RNAse H producing a free 3’ -OH end
  • It recognizes the AAAAA motif in RNAII.
  • Extension of this RNA primer by Pol I initiates leading-strand synthesis.
  • The point where the RNA primer is extended (known as RNA/DNA switch) is considered the replication start point.
  • The nascent leading-strand separates the two strands of the DNA duplex and can hybridize with the leading-strand template, forming a D-loop.
  • PriA is recruited to the forked structure of the D-loop.
  • Alternatively, PriA can be recruited to hairpin structures forming on the lagging-strand template when the duplex opens.
  • priA strains do not support ColE1 plasmid replication.
  • The hypomorphic mutations in priA priBresult in a reduced 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 toward RNA II sequence is arrested 17 bp upstream of the DNA/RNA switch, at a site known at terH.
  • It is unidirectional replication.
  • Lagging-strand replication by Pol III appears to end a few hundred nucleotides upstream of the terH site, leaving a gap that is filled by Pol I.

R-loop formation:

  • R-loop formation is essential in process of replication initiation.
  • Deficits in RNAse H and/or Pol I do not prevent initiation.
  • In the absence of RNAse H, unprocessed transcripts can still be extended with some frequency.
  • In the absence of Pol I, the Pol III replisome can still be loaded on an R-loop formed by the transcript and lagging-strand template.
  • R-loop formation occurs as a result of local supercoiling in the trail of the advancing RNA polymerase during transcription and is highly deleterious.
  • It is because R-loops block transcription and the elongation step during translation.
  • So, the unscheduled R-loop formation is suppressed by the cell.

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

  • The specialized priming mechanisms are present in these two classes which are combined with elements of class A and class B replication.
  • Rep protein is present in class C and D plasmids.
  • They initiate the leading-strand synthesis by Pol I extension of a free 3’-OH.
  • They have termination signals in the 3’ direction of lagging-strand synthesis.
  • Replication of these plasmids is unidirectional.
  • Class C and D have evolved the specialized priming mechanisms.
  • Class C includes ColE2 and ColE3 plasmids.
  • The oris for these two plasmids are the smallest and differ only at two positions.
  • One of them determines plasmid specificity.
  • ColE2 and ColE3 oris have two iterons and show two discrete functional subregions.
  • One is specializing in the stable binding of the Rep protein (region I)
  • another one specializing in the initiation of DNA replication (region III).
  • The Rep protein in class C plasmids has primase activity.
  • It synthesizes a unique primer RNA (ppApGpA) which is extended by Pol I at a fixed site in the origin region.
  • Class C replication is unidirectional.
  • The Rep protein may stay bound to the ori after initiation of replication, blocking the progression of the replisome synthesizing the lagging strand.

Class D:

  • It includes large, low-copy streptococcal plasmids.
  • Replication occurs in a broad range of Gram-positive bacteria.
  • Examples:
  • Enterococcus faecalispAMβ1
  • pIP501 from Streptococcus agalactiae
  • pSM19035 from Streptococcus pyogenes
  • It requires transcription across ori sequence, Pol I extension, and PriA-dependent replisome assembly.
  • The transcript is generated from a promoter controlling expression of rep.
  • Replication depends on transcription through the origin.
  • Rep binds specifically and rapidly to a unique site.
  • Denaturation of AT-rich sequence occurs and forms the open complex.
  • This binding denatures an AT-rich sequence immediately downstream of the binding site to form an open complex.
  • RepE also has an active role in primer processing.
  • As melting increases RepE binding and RepE can cleave transcripts from the repE operon close to the RNA/DNA switch.
  • Class D replisome assembly is PriA-dependent.
  • A replisome assembly signal can be found 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 caused by collision with a site-specific resolvase, Resb.
  • It is a plasmid-borne gene responsible for plasmid segregation stability.