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.

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• Those DNA molecules which can replicate autonomously are known as replicons. Example: Plasmids, phage DNA, chromosomes.
• Plasmid can replicate independently.
• Replicons have at least one origin of replication, or ori site, where replication begins.
• The cell also contains the proteins that help to initiate the replication.
• For their replication, plasmids encode only a few of the proteins.
• Many encode only one of the proteins needed for initiation at the ori
• Other required proteins are DNA polymerases, ligases, primases, helicases, etc.
• The plasmid replication origin is often named oriV for ori vegetative, to distinguish it from oriT. oriT is the site at which DNA transfer initiates in a plasmid.

Mechanism of Plasmid replication:

• There are two methods for the replication of plasmids. Among the two mechanisms, replication can occur by any one of the mechanisms:

1. theta mode for both unidirectional and bi-directional pathways
2. rolling circle mode.

1. Mechanism of Theta Plasmid Replication:

• DNA unwinds at the ori site from where the replication begins.
• It then creates the structure where the whole replicational machinery assembles.
• Since the structure resembles the Greek letter theta (θ), its name has been derived from it.
• The process gets initiated by the RNA primer.
• Then deoxyribonucleotides are added which extends the process.
• The replication process may proceed in one (unidirectional) or both directions (bi-directional).
• In the first case (unidirectional), a single replication fork moves around the circle until it returns to its point of origin. and then the two daughter DNAs separate.
• In the other case (bidirectional replicational) two replication forks begin at ori then it travels to the opposite until they meet at some point on the other side of the molecule.
• This is the most common mode of DNA replication.
• The theta mechanism is the most common form especially in Gram-negative bacteria like the proteobacteria.
• Commonly used plasmids, including ColE1, RK2, and F, as well as the bacteriophage P1, use this type of replication
• 

2. Mechanism of Rolling Circle Plasmid Replication

• It is called rolling-circle (RC) replication because it was first discovered in a type of phage where the template circle seems to “roll”.
• It is a unidirectional process (one direction only).
• Plasmids that replicate by this mechanism are sometimes called RC plasmids.
• This type of plasmid is found in the largest groups of bacteria, as well as in archaea.
• To perform this rolling-circle mode of replication, genetic material needs to be circular.
• In this method, one strand comes out while the other strand is being synthesized.
• Replication starts at the ori site that is the origin of replication where the Rep protein attaches one of the strands.
• Rep protein is the dimer that is formed of the two monomers.
• It has the tyrosine as the active group.
• First, the Rep protein recognizes and binds to the strand that contains the double-strand origin (DSO) on the DNA.
• Then the Rep protein can make a nick in the sequence.
• When the Rep protein has made a break in the DSO sequence, two ends will be formed in the DNA.
• At the 3’ end, there is the presence of OH group while at the 5’ end there is the presence of phosphate group.
• Rep protein will remain attached to the phosphate at the 5’ end of the DNA.
• Then the DNA polymerase III which is the replicative polymerase uses the free 3′ hydroxyl end at the break as a primer to replicate around the circle.
• For the separation of the strand, it may use a host helicase.
• The Rep protein itself may have the helicase activity, depending on the plasmid.
• Once the circle is complete, the 5′ phosphate is transferred from the tyrosine on the Rep protein to the 3′ hydroxyl on the other end of the strand. Then a single-stranded circular DNA is produced.
• This process is called a phosphotransferase reaction and requires little energy. The same reaction is used to re-form a circular plasmid after conjugational transfer.
• The displaced circular single-stranded DNA now replicates by a completely different mechanism using only host-encoded proteins.
• The RNA polymerase of the host cell recognizes the SSO ( single-strand origin) on the DNA.
• Then the RNA polymerase makes a primer.
• Then, replication occurs around the circle by DNA polymerase III.
• The RNA polymerase does not make this primer until the single-stranded DNA is completely displaced during the first stage of replication.
• When the entire complementary strand has been synthesized, the DNA polymerase I remove the RNA primer with its 5′ exonuclease activity while simultaneously replacing it with DNA.
•  DNA ligase joins the ends to make another double-stranded plasmid.
• Finally, The two new double-stranded plasmids are synthesized from the original double-stranded plasmid.
• 

3. Replication of linear plasmids:

• Some plasmids and bacterial chromosomes are linear rather than circular.
• The linear DNAs face two problems in all organisms.
• One problem is, at the end of linear fragments, the cell must distinguish the “normal” DNA ends from the ends formed when DNA double-strand breaks occur.
• This condition could be lethal to the cell and must quickly be repaired.
• A second problem with linear plasmids and chromosomes has to do with replicating the lagging-strand template.
• It is the strand that ends with a 5′ phosphate, all the way to the end of the DNA.
• Since DNA polymerases cannot initiate the synthesis of a new strand of the DNA, this has been the “primer problem”.
• They can only add nucleotides to a preexisting primer.
• In a linear DNA, there is no upstream primer on this strand from which to grow.
• The primer problem is solved by the different linear DNA in different ways.
• Some linear plasmids have hairpin ends. They have the 5′ and 3′ ends joined to each other.
• These plasmids replicate from an internal origin of replication to form dimeric circles.
• These dimeric circles are composed of two plasmids joined head to tail to form a circle.
• These dimeric circles are then resolved into individual linear plasmid DNAs with closed hairpins at the ends.
• The hairpin ends are previously not recognized as DNA double-strand breaks.
• It is because they are not targets for exonucleases in the cell.
• A completely different mechanism is also used to maintain linear plasmids in some systems.
• With this mechanism, a special enzyme called a terminal protein attaches to the 5′ ends of the plasmid DNA.

It is interesting that bacteria with linear plasmids also often have linear chromosomes, and the two DNAs replicate by similar mechanisms

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