Plasmids are autonomous circular oligonucleotides
Modern recombinant technology produces large numbers of identical DNA molecules. The technology relays on the formation of 3’ -> 5’ phosphodiester bonds to link DNA fragments to a vector molecule. When introduced into a host cell the DNA fragment together with the vector DNA is produced in large numbers. Two types of vectors are used most commonly: E. coli plasmid vectors and bacteriophage λ vectors.
Plasmids are circular double stranded DNA oligonucleotides that are separate from but can replicate independently of the hosts cell’s chromosome. These extrachromosomal circular oligonucleotides or DNAs occur naturally in bacteria, yeast, and some higher eukaryotic cells, and exist in a parasitic or symbiotic relationship with their host cell. The size of plasmids can vary from a few thousand base pairs to more than 100 kilobases (kb). Similar to chromosomal DNA, plasmid DNA is duplicated before every cell division. At least one copy of plasmid DNA is segregated to each daughter cell during cell division. These self-replicating circular oligonucleotides are maintained in the host cell in a stable and characteristic number of copies. The copy number remains constant from one generation to the next.
Plasmids contain genes that are beneficial to the host cell. Many of them contain ‘transfer genes” encoding proteins forming a pilus, or a macromolecular tube. Through this tube a copy of the plasmid can be transferred to other host cells of related bacterial species. However, most plasmid vectors contain just the essential nucleotide sequences required for their use in DNA cloning. Figure 1 shows diagrams of cloning vectors derived from plasmids.
Figure 1: Diagrams of cloning vectors derived from a plasmid. A diagram for a simple cloning vector is illustrated in A. A diagram for a pUC vector is depicted in B.
Plasmid vectors are approximately 1.2 to 3 kilobases (kb) in length and contain a replication origin (ori) sequence and a gene that permits selection. The gene used for the selection usually codes for a gene that is sensitive to antibiotics. The ampr gene that codes for the enzyme β–lactamase is an example. The enzyme β–lactamase inactivates ampicillin. Exogenic DNA can be inserted into regions that do not disturb the ability of the plasmid to replicate or express the ampr gene. pUC vectors such as the pUC18 and pUC19 vectors are small, high copy number, E.coli plasmids, 2686 bp in length and are identical except that they contain multiple cloning sites (MCS) arranged in opposite orientations. The physical properties including the topologies of these vectors have been investigated (http://pubs.acs.org/doi/abs/10.1021/ma0711689). A collection of plasmids can be found at the plasmid repository (https://dnasu.org/DNASU/Resources/Plasmid.jsp).
Proteins are involved in the partitioning of plasmid DNA
The replication origin (ori), a specific DNA sequence of 50 to 100 base pairs, must be present in a plasmid to allow for its replication. Proteins or enzymes in the host cell bind to this sequence motif and initiate replication of the circular plasmid.
Plasmids use two mechanisms for the replication of their DNA. These are the
- bidirectional replication of the plasmid and
- the rolling circle replication mechanisms.
Most plasmids replicate like small bacterial chromosomes. They possess an origin of replication where the DNA opens and replication begins. Two replication forks move around the circular oligonucleotide or plasmid DNA in opposite directions. Some tiny plasmids have only one replication fork that moves around back to the origin.
The rolling circle replication mechanism is shared by some plasmids and a few viruses. In this mechanism, one strand of the double stranded DNA is nicked at the origin of replication. The other circular stand starts to roll away from the broken strand. The result is two single stranded regions of DNA, one belonging to the broken strand and one circular strand. DNA is than synthesized starting at the end of the broken strand. This strand is now elongated and the circular strand is used as a template. The gap left where the original strands rolled apart is filled in. The process of rolling and filling continues and eventually the original broken strand is completely unrolled. Finally, the circular oligonuclotides are all paired with a new strand of DNA. The result is a single strand of DNA hanging loose. However, the next steps of this process depend on circumstances and the nature of plasmids involved. Some plasmids can transfer themselves from one bacterium to the next, as is the case for the F-plasmid.
Accurate and stable maintenance of DNA partition in bacteria requires proteins. This process can involve partition loci found on both chromosomes and plasmids. Bacterial low copy-number plasmids make simple DNA segregating machines that use an elongating protein filament between sister plasmids. The ParMR C system of Escherichia coli R1 plasmid, ParM, forms the spindle between ParR C complexes on sister plasmids. ParM filaments enable two ParR C–bound filaments to associate in an anti-parallel orientation to form a bipolar spindle. The spindle can elongate as a bundle of at least two anti-parallel filaments and pushes two plasmid clusters towards the poles.
During the partition of the Escherichia coli plasmid R1 a partition complex between the DNA-binding protein ParR and its cognate centromere site parC on the DNA is formed. This partition complex is recognized by a second partition protein, the ATPase ParM, that now forms filaments that allow the active bidirectional movement of DNA replicates.The plasmids P1and F are reported to employ a three-component system to partition replicated genomes. This system contains a partition site on the DNA target, typically called parS, a partition site binding protein, typically called ParB, and a Walker-type ATPase, typically called ParA. Par A also binds non-specific DNA. In vivo these protein family forms dynamic patterns over the nucleoid.
However, the process how the ATP-driven patterning works is not net very well understood and more research will be needed to decipherer all the details involved. The use of synthetic oligonucleotides, peptides and protein constructs may enable researchers to solve this puzzle in the future.
Figure 2: Crystal structures of the DNA-binding ParR protein which is a part of the plasmid partition complex (left) and the plasmid segregation protein Parm (right) are illustrated.
Moeller-Jensen et al. in 2007 reported the structures of a family of dimeric ribbon–helix–helix (RHH) 2 site-specific DNA-binding proteins. The reported crystallographic and electron microscopic data indicated that ParR dimers assemble into a helix structure with DNA-binding sites facing outward. In addition, genetic and biochemical experiments reported by this research group supported a structural arrangement in which the centromere-like parC DNA is wrapped around a ParR protein scaffold.
Gayathri et sl. In 2012 investigated the ParMRC system of the Escherichia coli R1 plasmid, ParM, an actin like protein. This protein assembly forms the spindle between ParRC complexes on sister plasmids. The researchers showed that ParRC is bound and could accelerate growth at only one end of polar ParM filaments. The architecture of ParM filaments enabled two ParRC-bound filaments to associate in an antiparallel orientation forming a bipolar spindle. The spindle elongated as a bundle of at least two antiparallel filaments that pushes two plasmid clusters toward the poles.
Reference
Gayathri P, Fujii T, Moller-Jensen J, Van Den Ent F, Namba K, Lowe J.; A bipolar spindle of antiparallel parm filaments drives bacterial plasmid segregation. Science (2012) 338 p.1334.
Ling Chin Hwang, Anthony G Vecchiarelli, Yong-Woon Han, Michiyo Mizuuchi, Yoshie Harada, Barbara E Funnell and Kiyoshi Mizuuchi; ParA-mediated plasmid partition driven by protein pattern self-organization. The EMBO Journal (2013) 32, 1249. www.embojournal.org.
Molecular Cell Biology. 4th edition. Lodish H, Berk A, Zipursky SL, et al. New York: W. H. Freeman; 2000.
Jakob Moeller-Jensen, Simon Ringgaard, Christopher P Mercogliano, Kenn Gerdes and Jan Loewe; Structural analysis of the ParR/parC plasmid partition complex. The EMBO Journal (2007) 26, 4413–4422.
Jeanne Salje, Pananghat Gayathri and Jan Löwe;The ParMRC system: molecular mechanisms of plasmid segregation by actin-like filaments. http://www2.mrc-lmb.cam.ac.uk/groups/JYL/PDF/nrmicro2425.pdf, http://www.ncbi.nlm.nih.gov/pubmed/20844556