Plasmid Replication – Mechanism With Diagram

What are plasmids?

In addition to the chromosome (nucleoid) of bacteria, the cytoplasm of bacterial cells typically contains genetic components. These genetic components live independently of the chromosome and proliferate as plasmids.

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Plasmids are double-stranded, self-replicating DNA segments with a few kilobases that are often found in gram-negative and gram-positive bacterial strains, as well as various fungi, including unicellular yeasts. Although plasmids are typically circular, there have also been reports of linear plasmids.

• Lederberg discovered the existence of plasmids in bacterial cytoplasm in 1952 while researching the bacterial conjugation process.
• Lederberg created the name plasmid to refer to the transmissible genetic material that were transferred from one bacterial cell to another and defined bacterial maleness.
• There are literally thousands of known plasmids; over 300 naturally occurring plasmids have been recovered from Escherichia coli strains alone.
• Many artificially modified plasmids have been generated and utilised as vectors in the process of gene cloning, in addition to naturally occurring plasmids (genetic engineering).
• Despite the fact that plasmids are not required for bacterial survival, they encode essential genetic determinants that enable bacteria to adapt and resist unfavourable conditions for better survival and to face external threats with other microorganisms occupying the same position in an ecological food chain.
• Plasmid replication processes are host-specific and influence plasmid copy number.
• In the 4 kilobase region of the DNA fragment, plasmid replicons contain one or more origins of replication (ori) and a small number of regulatory components, such as Rep proteins.
• Moreover, plasmids contain a few important genes that aid in DNA replication.
• The molecular mechanism of bacterial plasmid replication resembles the beginning of E. coli chromosome replication.

Copy Number and Physical Nature of Plasmids

• Plasmids have a straightforward physical makeup. They are tiny DNA molecules with two strands.
• The majority of plasmids are circular, although there are also numerous linear plasmids known.
• The size of naturally occurring plasmids ranges from roughly 1 kilobase to over 1 megabase, with the average plasmid DNA being less than 5% the size of the bacterial chromosome.
• The majority of plasmid DNA extracted from bacterial cells exists in the supercoil configuration, the most compact form for DNA to exist in the cell.
• The copy number refers to the fact that various plasmids are present in cells in varying quantities. Some plasmids may be present in the cell in as few as one to three copies, while others may be present in greater than 100 copies.
• Genes on the plasmid and interactions between the host and plasmid regulate copy number.

Properties of Plasmids

• They are particular to one or a few distinct microorganisms.
• They replicate independently of the chromosome of bacteria.
• They are responsible for coding their own transfer.
• They function as episomes and integrate reversibly into the bacterial genome.
• They may pick up and transfer specific genes from the bacterial chromosome, and they may alter some cellular properties.
• Plasmids are distinct from viruses in two ways.
• They do not cause cell harm and are often advantageous.
• They lack extracellular forms and exist only as free, usually circular DNA within cells.

Plasmid Replication Mechanism

• During cell division, bacterial plasmid replication is independent of its nuclear genome replication, with lengthy pauses occurring between replication events.
• The exact amount of plasmid copies depends on the plasmid type, host organism, and growth conditions.
• Unintended deviations from the regular number of copies are adjusted. There do exist dominant and recessive copy mutants of the wild type.
• Rolling circle, Col E1 type, and Iteron contain replication are the three kinds of plasmid replication.

1. Rolling circle Mode of Replication

• The rolling circle replication method is unique to the bacteriophage family m13 and the fertility F factor, which codes for the production of sex pili during conjugation-based recombination.
• Typically, fragments smaller than 10 kilo bases multiply by this process, as found in certain gram-positive bacteria.
• It enables the rapid transmission of single-stranded replication products to the recipient cell’s pilus in the case of reproductive factor or to the membrane in the case of phage.
• Rolling-circle (RC) replication was initially identified in a form of phage in which the template circle seems to “roll.”
• It is a one-way procedure (one direction only).
• Plasmids that reproduce using this technique are occasionally referred to as RC plasmids.
• This form of plasmid is prevalent in the major bacterial and archaeal groupings.
• For this rolling-circle mode of replication, circular genetic material is required.

Mechanism of Rolling circle replication in plasmid

1. A covalently closed circular segment of double-stranded DNA undergoes rolling circle.
2. Enzyme nickases create a nip in one of the strands by producing a 5′ phosphate and a 3′ hydroxyl.
3. DNA polymerase will employ free 3′ hydroxyl to build new DNA, displacing the old nicked strand from the template DNA.

2. Col E1 type replication

• Col E1 replication is a negative regulation process involving RNA type I, RNA type II, Rom protein, and the plasmid itself that enables the plasmid to control its own copy quantity.
• Col E1 replication is initiated by RNA-RNA interactions and does not rely on the plasmid-encoded replication initiation protein to regulate its copy quantity.

Mechanism

1. The transcription of RNA type II that originates 555 base pairs upstream of the replication origin of the Col E1 plasmid indicates the beginning of Col E1 replication.
2. A confirmed hybrid with the DNA strand is created by a G-rich loop at position 290 of RNAII and a C-rich area 20 nucleotides upstream of the origin on the template strand.
3. The freshly created secondary structure displays several stems and loops.
4. The enzyme RNase recognises a DNA/RNA hybrid and dissociates the RNA hybrid from the 3′ end of RNAII.
5. The generated RNA primer is attached to the plasmid via a 3′ hydroxyl group that is free.
6. This RNA promotes DNA replication by providing a particular location for DNA polymerase to initiate nucleotide synthesis.
7. Therefore, the leading strand initiates DNA synthesis.

3. Iteron-containing replicons

• This replicon contains a gene encoding Rep protein for plasmid replication initiation, a series of direct repeat sequences called iteron, an adjacent AT-rich region, and Dna boxes, a protein essential for the commencement of bacterial chromosome replication.
• However, a replicon’s surrounding AT-rich region length and number of iterons and DnaA boxes vary.

Mechanism

1. Iteron contain replication starts when Rep proteins attach to the iteron in the same way that the DNA helix is set up.
2. By binding to the DnaA boxes in the replicon, the Rep-DnaA-DNA assembly helps melt the strand in the nearby AT-rich region. This lets host replication factors get to the region and start making the leading and lagging strands in a way similar to how replication starts at the chromosomal origin, oriC.
3. The number of copies of a plasmid is mostly controlled at the start of replication.
4. Part of what controls how often replication of iteron-containing plasmids starts is that the origin region is locked away in nucleoprotein complexes and that complexes on different plasmids pair up with each other in a process called “handcuff.”

Linear Plasmids Replication

• Some plasmids and chromosomes in bacteria are straight instead of round.
• In all living things, linear DNAs have to deal with two problems.
• One problem is that at the end of linear fragments, the cell needs to be able to tell the difference between “normal” DNA ends and ends that are made when a double strand of DNA breaks.
• This could kill the cell, so it needs to be fixed as soon as possible.
• The second problem with linear plasmids and chromosomes is that the lagging-strand template is hard to copy.
• All the way to the end of the DNA, it is the strand that ends with a 5′ phosphate.
• This has been the “primer problem” because DNA polymerases can’t start making a new strand of DNA.
• People can only add nucleotides to a primer that already exists.
• In a linear strand of DNA, there is no upstream primer to grow from.
• Different types of linear DNA solve the primer problem in different ways.
• Some ends of linear plasmids look like a hairpin. The 5′ and 3′ ends are connected to each other.
• These plasmids copy themselves from an internal source to make circles with two parts.
• The two plasmids that make up these dimeric circles are joined head to tail to make a circle.
• Then, each of these two-membered circles is separated into a single, linear plasmid DNA with a closed hairpin at each end.
• Before, the hairpin ends were not thought to be DNA double-strand breaks.
• This is because exonucleases in the cell don’t go after them.
• In some systems, linear plasmids are also kept together by a completely different method.
• With this method, a special enzyme called a terminal protein attaches to the 5′ ends of the plasmid DNA.

So, controlling the number of copies and compatibility of plasmids in bacteria and other organisms is best done by rolling circle, Col E1 type, and Iteron contain replication.

Applications of Plasmid Replication

Plasmid replication plays a crucial role in various applications in the field of molecular biology and biotechnology. Here are some key applications of plasmid replication:

1. Gene Cloning: Plasmid replication is widely used in gene cloning techniques. By inserting a specific DNA fragment into a plasmid, scientists can replicate the plasmid and produce multiple copies of the DNA of interest. This allows for the amplification of the desired gene or DNA sequence for further analysis or use in downstream applications.
2. Recombinant Protein Production: Plasmid replication is commonly utilized for the production of recombinant proteins. Researchers can engineer plasmids to contain the gene encoding the desired protein, along with regulatory elements for expression. By introducing these plasmids into host cells, such as bacteria or yeast, the cells can replicate the plasmids and produce large quantities of the recombinant protein.
3. Genetic Engineering: Plasmid replication is a fundamental tool in genetic engineering. It enables the introduction of foreign DNA into host cells, allowing scientists to manipulate the genetic makeup of the organisms. Plasmids can carry various functional elements, such as selectable markers and promoters, which facilitate the integration and expression of foreign genes in the host organism.
4. Gene Therapy: Plasmid replication has significant implications in gene therapy research. Plasmids can be engineered to carry therapeutic genes, such as those encoding proteins that can correct genetic disorders. By introducing these plasmids into target cells, the therapeutic genes can be replicated, leading to the production of therapeutic proteins that can potentially treat or alleviate the symptoms of genetic diseases.
5. Molecular Diagnostics: Plasmid replication is employed in various molecular diagnostic techniques. Plasmids can serve as templates for PCR (polymerase chain reaction) amplification, allowing for the detection and quantification of specific DNA sequences. Plasmids can also be used as positive controls in diagnostic tests or to generate standard curves for accurate quantification of target DNA.
6. DNA Sequencing: Plasmid replication has been historically important in DNA sequencing methods. Plasmids, such as those derived from bacteriophage M13, have been used as vectors for DNA sequencing reactions. The replication of these plasmids allows for the production of single-stranded DNA templates, which can be used for sequencing reactions using Sanger sequencing or other sequencing technologies.

FAQ

References

• Dmowski, Michal & Jagura-Burdzy, Grazyna. (2013). Active Stable Maintenance Functions in Low Copy-Number Plasmids of Gram-Positive Bacteria I. Partition Systems. Polish journal of microbiology / Polskie Towarzystwo Mikrobiologów = The Polish Society of Microbiologists. 62. 3-16. 10.33073/pjm-2013-001. 
• del Solar G, Giraldo R, Ruiz-Echevarría MJ, Espinosa M, Díaz-Orejas R. Replication and control of circular bacterial plasmids. Microbiol Mol Biol Rev. 1998 Jun;62(2):434-64. doi: 10.1128/MMBR.62.2.434-464.1998. PMID: 9618448; PMCID: PMC98921.
• Kües U, Stahl U. Replication of plasmids in gram-negative bacteria. Microbiol Rev. 1989 Dec;53(4):491-516. doi: 10.1128/mr.53.4.491-516.1989. PMID: 2687680; PMCID: PMC372750.
• https://www.frontiersin.org/articles/10.3389/fmicb.2015.00242/full
• https://www.longdom.org/open-access/mechanisms-of-plasmid-replication-jpb-1000444.pdf
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