The genome of E. coli is about 4.6 million base pairs long and contains a single origin of replication (oriC), where initiation begins. This process relies on the initiation protein DnaA. First, DnaA must bind ATP. Then, the ATP-bound DnaA binds multiple specific sites within the ori called DnaA boxes. These boxes come in both high- and low-affinity versions, but binding of the low-affinity versions is required to start the replication process. When enough copies of ATP-DnaA are bound, including at the low-affinity sites, the proteins work cooperatively to separate nearby AT-rich regions of DNA.
In a test tube, the DNA helix can be separated by DnaA alone, as long as there are enough copies to bind every available DnaA box. In bacteria, however, the protein integration host factor (IHF) assists with the process: it bends the DNA to redistribute the DnaA from high-affinity boxes to low-affinity boxes, allowing lower levels of DnaA to get the job done.
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| Figure 1: DNA melting by DnaA and IHF is the first step of replication for the bacterial chromosome and for stringently controlled plasmids. Binding of IHF (green) redistributes DnaA (red) from binding primarily high-affinity DnaA boxes (dark red) to also binding low-affinity DnaA boxes (pale red). When most DnaA boxes are occupied by ATP-bound DnaA, the proteins cooperatively can separate the DNA strands. Created with BioRender.com. |
Once DnaA breaks the local hydrogen bonds holding the DNA together, it recruits helicases to continue unwinding the strands. This creates two replication forks that move in opposite directions away from the ori. In the elongation phase, a DNA polymerase synthesizes a new DNA strand at each replication fork. Termination sequences present in the chromosomal DNA ensure that the helicases are released from the DNA once the replication forks reach each other on the opposite end of the chromosome, ending replication.
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| Figure 2: Prokaryotic chromosomal replication. Adapted from BioRender template. |
Since DnaA binding is necessary to initiate replication, controlling the amount of active DnaA available is one of a cell’s best tools to regulate DNA replication. DnaA negatively regulates its own expression, so high levels of DnaA act as a feedback mechanism to reduce further transcription. Replication is also regulated by the balance of ATP and ADP available to bind DnaA.
Fun fact! As far as we can tell, DnaA doesn’t need to hydrolyze ATP to do its job — although ATP does get hydrolyzed over time, inactivating the protein binding it (Grimwade et al., 2018). Rather than serving as an energy source, ATP binding is one more way for the cell to control DNA replication.
A variety of other proteins also contribute to this regulation in different ways: methylating newly synthesized DNA, blocking binding sites, or trading out ADP for ATP to re-activate DnaA. These regulatory mechanisms allow E. coli to control chromosomal replication and balance population growth against sustainability.
Plasmid DNA replication and regulation
Plasmids must also strike a balance: replicating too slowly will eventually cause a plasmid to be lost from the population, while replicating too quickly can slow cell growth or even kill the host cell. For stringently controlled plasmids, replication is tightly coupled to the bacterial host's cell cycle, maintaining a stable concentration of plasmid. 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.
Let’s consider pSC101. This plasmid is stringently controlled and has a low copy number. Like the E. coli chromosome, it has a single ori and requires the host proteins DnaA and IHF to initiate replication. But pSC101 requires a third component: RepA, a protein encoded by pSC101 itself. RepA binds to directly repeated sequences called iterons located near the ori and directly interacts with DnaA, stabilizing its binding and contributing to DNA unwinding. This promotes initiation at the plasmid ori, after which replication of the plasmid proceeds (and terminates) similarly to the bacterial chromosome.
Since replication of pSC101 depends on host-encoded proteins, it can be regulated indirectly via the same mechanisms that affect DnaA and IHF. RepA also controls the copy number of pSC101 through a negative feedback cycle. As with DnaA, high concentrations of RepA negatively regulate its own expression and thereby the overall replication of pSC101 (del Solar et al., 1998). If that’s not enough to keep replication in check, high copy numbers can allow RepA to link iterons from two different plasmids, forming a pair of “handcuffs” that blocks replication of either of them (Park et al., 2001). The number of iterons encoded on a given plasmid influences how high the copy number needs to get before handcuffing occurs. These mechanisms to regulate plasmid replication through the dual functionality of RepA are efficient for the host, as no additional components are necessary.
Iterons are not the only tool to stringently regulate plasmid replication. For example, the plasmid ColIb-P9 encodes the protein RepZ to drive its own replication. Like RepA, RepZ helps recruit host initiation proteins to the plasmid ori. In this system, however, RepZ concentration is controlled at the level of translation. RepZ mRNA must fold into a “pseudoknot” held together by intramolecular base pairs for the protein to be translated. An antisense RNA called Inc, also encoded by the ColIb-P9 plasmid, is complementary to part of the sequence forming this pseudoknot, so its binding prevents the mRNA from folding and blocks RepZ translation (Asano & Mizobuchi, 1998). This system keeps RepZ levels low even if plasmid copy numbers are high, restraining further replication.
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| Figure 3: Comparison of replication regulation mechanisms of stringently controlled plasmids pSC101 and ColIb-P9. pSC101 copy number is regulated by iterons. RepA binds to iterons and recruits host proteins to the ori to initiate replication. At high copy numbers, RepA proteins form complexes between iterons on different plasmids in a process called "handcuffing," which blocks replication. ColIb-P9 copy number is regulated by an lncRNA. RepZ recruits host proteins to the ori to initiate replication. RepZ is translated from an mRNA that forms a pseudoknot. At high copy numbers, high levels of the lncRNA bind to RepZ mRNA and prevent this pseudoknot from folding, blocking replication. Created with BioRender.com. |
Plasmids under stringent control of replication may differ in the specialized proteins or sequences necessary for initiation or the mechanisms that regulate their replication, but their copy number is always limited by the use of the host cell's initiation proteins. In nature, such plasmids ensure their propagation by limiting their copy number and compensating for the additional metabolic cost imposed on the host with a beneficial function (usually antibiotic resistance).
In the lab, low copy plasmids may result in low yields during DNA purification. On the other hand, they also impose a lower metabolic cost on cells and produce lower amounts of protein, which can be useful if the product is toxic in high amounts. These plasmids are also useful in certain in vivo studies where protein overexpression might skew the results. Thanks, stringent regulation!
This post was originally written by Jason Niehaus in December 2015 and was updated by Emily P. Bentley in June 2024.
References and resources
Further Reading
Quiñones-Valles, C., Espíndola-Serna, L., Martínez-Antonio, A., Quiñones-Valles, C., Espíndola-Serna, L., & Martínez-Antonio, A. (2011). Mechanisms and Controls of DNA Replication in Bacteria. In Fundamental Aspects of DNA Replication. IntechOpen. https://doi.org/10.5772/19313
References
Asano, K., & Mizobuchi, K. (1998). Copy number control of IncIalpha plasmid ColIb-P9 by competition between pseudoknot formation and antisense RNA binding at a specific RNA site. The EMBO Journal, 17(17), 5201–5213. https://doi.org/10.1093/emboj/17.17.5201
del Solar, G., Giraldo, R., Ruiz-Echevarría, M. J., Espinosa, M., & Díaz-Orejas, R. (1998). Replication and control of circular bacterial plasmids. Microbiology and Molecular Biology Reviews: MMBR, 62(2), 434–464. https://doi.org/10.1128/MMBR.62.2.434-464.1998
Grimwade, J. E., Rozgaja, T. A., Gupta, R., Dyson, K., Rao, P., & Leonard, A. C. (2018). Origin recognition is the predominant role for DnaA-ATP in initiation of chromosome replication. Nucleic Acids Research, 46(12), 6140–6151. https://doi.org/10.1093/nar/gky457
Park, K., Han, E., Paulsson, J., & Chattoraj, D. K. (2001). Origin pairing (‘handcuffing’) as a mode of negative control of P1 plasmid copy number. The EMBO Journal, 20(24), 7323–7332. https://doi.org/10.1093/emboj/20.24.7323
Resources at Addgene
- Read our blog post on the Origin of Replication
- Browse other Plasmids 101 Posts
- Get practical molecular biology help with our Molecular Biology Reference Pages
Topics: Plasmids 101, Plasmids
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