CH391L/S12/Origins of Replication
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Revision as of 14:48, 5 March 2012
In order for a piece of circular, dsDNA to be propagated in bacteria, it needs to be replicated by host machinery. There is a sequence in the plasmid that directs the cell to begin replication. Important considerations are host range, compatibly, and copy number. The host range refers to what species of bacteria will recognize the origin of replication and thus allow for replication. The compatibility refers to a plasmid's ability to coexist with another plasmid in the same cell. Copy number refers to the average or expected number of copies of the plasmid per cell.
There are three main mechanisms for plasmid replication: Rolling Circle, Strand Displacement, and Theta.
Strand displacement replication
RepC binds repeat sequences recruits RepA ( a helicase) to melt an AT rich region. This exposes two single stranded origins ssiA and ssiB. RepB polymerizes primers for these origins. DNA polymerization follows in each direction, meanwhile displacing the non-template stand.
Strand displacement is associated with broad host range vectors, possibly because it does not require any of the normal host machinery (DnaA, DnaB, DnaC, and DnaG)
Rolling circle replication
A nick is made by the Rep protein at the "double strand origin" of a dsDNA plasmdid. The free 3'OH is extended, displacing as it progresses. After one unit length of displacement, replication is terminated, yielding one dsDNA plasmid and ssDNA of one unit length. The displaced strand then serves as a template for replication from a "single strand origins." Since each strand is replicated independently, it is possible for the ssDNA form to accumulate.
This mechanism is found in gram-positive bacteria like Staphylococcus aureus and Streptomyces lividans as well as many bacteriophages.
DnaA (often with the help of other proteins) binds the origin at DnaA boxes. This promotes melting of the orgin. This allows DnaC to load to DnaB helicase, opening the origin further. DnaG is then recuited to form a short RNA primer.
DNA polymerase III extends this primter. If there is only one leading primer, a single fork circumnavigates the entire plasmid until the origin is reached, and daughter plasmids separate. In bidirectional replication, two forks propagate and meet on the far side of the plasmid before resolution.
Theta is the most common form of DNA replication, including most plasmids as well as chromosomes. It is particularly associated with gram-negative bacteria. ColE1, P15A, RK2, F, and P1 all use theta replication.
Plasmids are classified as having a narrow or broad host range.
- ColE1 and pMB1 are limited to E. coli and a few close relatives,
- RK2 plasmids can be used in most gram-negative bacteria.
- RSF1010 can use used in most gram-negative bacteria, and some gram-positive
- Plasmids from gram-positive bacteria tend to function well in other gram-positive bacteria.
If two plasmids have the same (or very similar) origins of replication, they will compete with each other for replication machinery. This results in an unstable situation. If the two plasmids posses different selectable markers, this can be maintained for several generations, but eventually one of the plasmids will be lost. For scenarios in which multiple plasmids are necesary, one must be careful to choose plasmids will compatible origins. The most common dual-plasmid pair is ColE1(or pMB1) and p15A. The most common plasmid triplet is ColE1 (or pMB1),p15A, and pSC101. Tolia and Joshua-Tor suggest the following groups:
- ColE1/pMB1 (eg pET, pUC, pBR322, pGEX, pMAL)
- P15A (eg pBad, pACYC)
An important consideration in choosing what plasmid backbone to use is the copy number. For example, cloning is best done with a high copy plasmid (e.g. pUC) as plasmid preps will have a higher yield. Expressing a toxic gene is better from a low to medium copy plasmid(e.g. pET which uses the pBR322 origin), as there are fewer copies.
- ColE1: 15-20 copies
- pMB1: 20-700 copies
- pUC: 500-700 copies
- pBR322: ~20 copies
- pSC101: ~5 copies
- P15A: 10-12 copies
- RK2: 4-7 copies
- F1: ~1 copy
- CloDF13: 20-40 copies
- ColA: 20-40 copies
- RSF1030: >100 copies
- P1: ~1 copy
Control of initiation/copy number
There are several mechanisms by which copy number is controlled. In all cases, some negative-regulating element (RNA or protein) is expressed from the plasmid. As the plasmid concentration increases, so too does the negative regulator. This provides a negative feedback, which stabilizes the copy number. Two plasmids that are regulated by each other's regulator will not be compatible.
ColE1/pMB1: The origin contains regions promoting the synthesis of RNA I and RNA II. RNA II hybrizes to the DNA, yielded a DNA/RNA hybrid which can serve as a substrate for RNaseH. Digestion of RNA II by RNaseH yields the primer for replication. RNA I binds and sequesters RNA II, so it is unavailable for RNAse H digestion. As the copy number increases, so does the concentration of RNA I. This provides a negative feedback for replication, and sets the average number of plasmids per cell.
Additionally, The Rop protein helps lower the copy number, by stabilizing the RNA I/ RNA II duplex. Deletion of Rop, as well as a point mutation that weakens the RNA I and RNA II duplex, accounts for the higher copy of pUC (a pMB1 derivatives)
P15A, ColA, RSF1030, and CloDF13 are similar, but with versions of RNA I and RNA II that sufficiently different to allow for compatibility.
RNA and protein regulation
On the R1 plasmid, OriR is bound by RepA, thus promoting replication by recruiting DnaA. RepA can be expressed from two different promoter. A proximal promoter (pRepA) drives only RepA while a distal promoter (pCopB) drives both CopB and RepA. CopB represses pRepA, thus once there are enough plasmids around, CopB levels become high enough to limit RepA expression to pCopB promoter. plasmid encoded CopA is completmentary, and thus binds to the 5' end of the transcript originating from the pCopB promoter. The dsRNA is a substrate for the processive RNase III.
Like the above examples, pSC101's replication is positively regulated by RepA binding the origin. RepA is also used to control copy number, by two mechanisms.
Firstly, RepA negatively regulates its own transcription, thus the RepA protein levels (and its ability to promote replication) is confined to narrow limits.
Secondly, The plasmid contains several (3-7) repeats of a 17-22bp sequence called iteron sequences. RepA binds the iterons, and at higher plasmid conncentration, this can lead to "handcuffing" of two plasmids. Interestingly, adding extra iteron sequences on other plasmids can reduce the copy number by this handcuffing mechanism.
F, RK6, P1, RK2, and RP4 also use iterons, but the regulating protein and origins differ.
pETcoco is an interesting plasmid, made by Novagen. It can be maintained as a single copy plasmid using the origin and positive regulator from the F plasmid (oriS and RepE). It can be swiched to a medium copy plasmid using the machinery from the RK2 plasmid (oviV and trfA). The switch is achieved by the induction of the trfA protein, which binds and iteron on oriV, thus promoting initiation from this origin by aiding in melting and recruitment of DnaB.
Othere extra-chromosomal bodies
BACs (bacterial artificial chromosomes) are based on the single-copy F origin. It is capable of maintaining inserts greater than 300kb. Key to its stability are the par elements. ParA and parB are plasmid encoded proteins that help ensure that the each daughter cell gets one copy of the BAC during binary fission.
Chromids are a "second chromosome" is about 10% of sequenced bacterial species. They contain essential genes and are composed much like the chromosome, but have replication and partioning machinery reminiscent of plasmids. By definition, chromids are the second largest
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Regarding the copy number of pUC vs pBR322
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regarding the differences between RNA I and RNA II sequences in P15A, ColE1, RSF1030, CloDF13, and pBR322.
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