CH391L/S12/Origins of Replication
Origins of Replication, overview
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...
- In 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 Staphylococcus aureus and Streptomyces lividans as well as many bacteriophages.
- Theta replication is the most common form of DNA replication, including most plasmids as well as chromosomes. RNA serves a primer, DNA is polymerized in one or both directions. In the first case, 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.
Plasmids are classified as having a narrow or broad host range.
- ColE1 is 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/p15A. The most common plasmid triplet is ColE1/p15A/pSC101.
- ColE1: pUC, pMB1, pBR322, pGEM, pET, pUC, pQE, pMAL, pGEX
- p15A: pBad, pACYC
- ColE1: 20-700 copies
- pUC: 500-700 copies
- pBR322: ~20 copies
- pSC101: ~5 copies
- p15A: 10-12 copies
- F1: ~1 copy
- CloDF13: 20-40
- ColA: 20-40
- RSF1030: >100
Control of Initiation/copy number
- ColE1: Does not require plasmid encoded initiator protein, does require DnaA binding. 1kb. Includes (1) region promoting the synthesis of RNA II, which is the primer for the leading strand (2) sequences that promote stable hybridization of RNA II to DNA (3) sequences that promote RNAse digestion of RNA II, yielding primer for leading strand. RNA I is also transcribed, and sequesters RNA II, so it is unavailable for RNAse H.
- p15A: similar to ColE1 but different RNAs
- pSC101: iterons
- R1: oriR is bound by RepA promoting initiation. No iterons. Copy number controlled by copA and copB. copA is in RNA that binds the 5’ end of RepA RNA and prevents translation. CopB blocks repA transcription.