Plasmids 101: Stringent Regulation of Replication

Posted by Jason Niehaus on Dec 3, 2015 10:30:00 AM


Plasmids utilize their host cell's replication machinery in order to replicate. As described in our previous Origin of Replication post, DNA replication is initiated at the ORI and may be synchronized with the replication of the host cell's chromosomal DNA or may be independent of the host's cell cycle. 

Plasmids are said to be under stringent control of replication when they are dependent on the presence of initiation proteins synthesized by the host cell in order to start their own replication. In general, these types of plasmids tend to be low copy number. Conversely, plasmids that can initiate DNA replication independently of the host's initiation proteins are said to be under relaxed control, as they only require the host's replication machinery for elongation and termination. These types of plasmids tend to be high copy number.

Here we will consider the replication mechanism of plasmids under stringent control in bacteria, such as pSC101. These types of plasmids share the same host proteins necessary for initiation of replication as the bacterial chromosome. Chromosomal replication in bacteria is carefully regulated to ensure that it occurs at the appropriate time in a given cell's life (i.e. the cell must have enough nutrients available to complete the entire round of replication). Since the primary control point of DNA replication in bacteria is initiation, the series of steps required to begin replication is a carefully controlled process balancing positive and negative regulators. Initiation begins at the ORI--a region of DNA where initiation proteins can bind and begin to unwind and separate the DNA strands, expose the unpaired nucleotides and permit other recruited proteins involved in DNA synthesis to form a stable DNA-protein complex that will continue the process.

DNA Replication and Regulation in Bacteria

Before looking at an example plasmid, let's see how E. coli bacteria normally replicate their chromosomal DNA. The genome of E. coli is about 4.6 million basepairs long and contains a single origin of replication (oriC). Three A-T rich repeats present in the ORI favor the separation of the DNA strands upon the binding of initiation protein, DnaA, at specific sites (DnaA boxes). Other regions of the ORI permit binding of regulatory proteins that form a DNA-protein complex that recruits a helicase which can unwind and break the hydrogen bonds between the DNA strands. Two replication forks are created by the action of the helicase and move in opposite directions away from the ORI. A DNA polymerase completes the formation of each initiation complex and synthesizes the new DNA strand. Termination sequences present in the chromosomal DNA ensure that, once the replication forks reach each other on the opposite end of the chromosome, the helicases are released from the DNA to end replication.

DNA replication in E. coli is primarily regulated via DnaA. Since DnaA binding to the ORI is necessary for initiating replication, a cell is able to control replication by the amount of available DnaA. Immediately after a replication cycle, the ratio of available DnaA to the number of available binding sites at the ORI is decreased so that the average DnaA level will need to increase before another cycle can begin. DnaA also negatively regulates its own expression, so that high levels of DnaA act as a feedback mechanism to reduce further transcription. Replication can also be regulated by the balance between active and inactive DnaA levels. The active form of DnaA necessary for initiating replication upon binding to the ORI is bound by ATP and is more likely to be present in actively growing cells with excess ATP. 

Replication is also regulated by DNA methylation state: both DNA strands must be methylated before initiation can proceed. Newly synthesized DNA is primarily methylated by Dam (DNA adenine methyltransferase), which likely begins methylating the newly synthesized daughter strand shortly after a short section of it is compelted by DNA polymerase. The E. coli origin contains 11 methylation sites; after replication, there is a refractory period during which the daughter strands are unmethylated at these sites and replication can only begin again once the daughter strands are methylated.

These regulatory mechanisms in E. coli permit controlled copying of chromosomes, balancing the necessity of replication for population growth with sustainability and overall health and fitness of the population.

Plasmid DNA Replication and Regulation

For stringently controlled plasmids, replication is tightly coupled to the bacterial host's cell cycle in order to maintain a stable concentration of plasmid. If a plasmid's replication rate is too slow it will eventually be lost; however, a high rate of replication is also undesirable as it is a burden to the host and can slow cell growth or, in the extreme case, lead to cell death. Similar to replication in E. coli, regulation primarily occurs by controlling the initiation of replication. Once the average copy number for a given plasmid has been reached, negative feedback circuits reduce replication of the plasmid to balance the total plasmid number. 


pSC101_replication_diagram.jpgThe replication of pSC101, a low-copy number plasmid, proceeds from a single origin of replication similar to oriC in E.coli and requires the same DnaA protein for the initiation of replication. The aptly named E. coli protein integration host factor (IHF) and RepA, a protein encoded by pSC101 itself, complete the three genetic components of the pSC101 replicon. As shown in the figure to the above, IHF binds to target sequences in the A-T rich region of the pSC101 ORI to bend the DNA and promote the binding of DnaA. RepA binds to directly repeated sequences called iterons that are located near the ORI and also interacts with DnaA directly. Both of these mechanisms stabilize DnaA binding and lead to breaking of the hydrogen bonds between the DNA strands (i.e. melting) to promote initiation of plasmid replication at the origin. The initiation complex formed by the action of IHF and the RepA-DNA-DnaA complex loads the helicase onto the replication forks at either end of the origin. The helicase proceedes to unwind the DNA from these replication forks and is followed by DNA polymerase which synthesizes the new daughter strands. Similar to E. coli, replication ends at termination sequences present in the plasmid where the replication complexes from the two replications forks meet one another.

Since replication of pSC101 depends on host-encoded proteins, such as IHF and DnaA, regulation of plasmid replication can occur indirectly via the concentration of these proteins. A similar negative feedback cycle involving the plasmid-encoded RepA protein permits copy number control for pSC101, as RepA not only binds to iterons to initiate replication but, at higher concentrations, RepA binds to inverted repeat sequences present in the RepA promoter to reduce its own expression and thereby the overall replication of pSC101. This mechanism to regulate plasmid replication by dual functionality of RepA is an efficient system for the host, as the iterons are directly proportional to the plasmid copy number and no additional host components are necessary.

Plasmids under stringent control of replication may differ in the specialized proteins or sequences necessary for initiation or the mechanisms utilized to regulate their replication, but their copy number is always limited by the use of the host's initiation proteins. These plasmids have cleverly evolved to ensure their propagation by balancing their copy number and compensating for the additional metabolic cost imposed on the host with a beneficial function (usually antibiotic resistance).


References

1. del Solar, Gloria, et al. "Replication and control of circular bacterial plasmids."Microbiology and molecular biology reviews 62.2 (1998): 434-464. PubMed PMID: 9618448. PubMed Central PMCID: PMC98921.

2. Rakowski, Sheryl A., and Marcin Filutowicz. "Plasmid R6K replication control."Plasmid 69.3 (2013): 231-242. PubMed PMID: 23474464. PubMed Central PMCID: PMC3691012.

3. Vocke, Cathy, and Deepak Bastia. "Primary structure of the essential replicon of the plasmid pSC101." Proceedings of the National Academy of Sciences 80.21 (1983): 6557-6561. PubMed PMID: 6579542. PubMed Central PMCID: PMC390392.

4. Feinbaum, Rhonda. "Introduction to plasmid biology." Current Protocols in Molecular Biology (2001): 1-5. PubMed PMID: 6579542. PubMed Central PMCID: PMC390392.

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