Circular chromosome
A circular chromosome is a chromosome in bacteria, archaea, mitochondria, and chloroplasts, in the form of a molecule of circular DNA, unlike the linear chromosome of most eukaryotes.
Most prokaryote chromosomes contain a circular DNA molecule – there are no free ends to the DNA. Free ends would otherwise create significant challenges to cells with respect to DNA replication and stability. Cells that do contain chromosomes with DNA ends, or telomeres, have acquired elaborate mechanisms to overcome these challenges. However, a circular chromosome can provide other challenges for cells. After replication, the two progeny circular chromosomes can sometimes remain interlinked or tangled, and they must be resolved so that each cell inherits one complete copy of the chromosome during cell division.
Replication
The circular bacteria chromosome replication is best understood in the well-studied bacteria Escherichia coli and Bacillus subtilis. Chromosome replication proceeds in three major stages: initiation, elongation and termination. The initiation stage starts with the ordered assembly of "initiator" proteins at the origin region of the chromosome, called oriC. These assembly stages are regulated to ensure that chromosome replication occurs only once in each cell cycle. During the elongation phase of replication, the enzymes that were assembled at oriC during initiation proceed along each arm of the chromosome, in opposite directions away from the oriC, replicating the DNA to create two identical copies. This process is known as bidirectional replication. The entire assembly of molecules involved in DNA replication on each arm is called a "replisome." At the forefront of the replisome is a DNA helicase that unwinds the two strands of DNA, creating a moving "replication fork". The two unwound single strands of DNA serve as templates for DNA polymerase, which moves with the helicase to synthesise a complementary copy of each strand. In this way, two identical copies of the original DNA are created. Eventually, the two replication forks moving around the circular chromosome meet in a specific zone of the chromosome, approximately opposite oriC, called the terminus region. The elongation enzymes then disassemble, and the two "daughter" chromosomes are resolved before cell division is completed.Initiation
The E. coli bacterial replication origin, called oriC consists of DNA sequences that are recognised by the DnaA protein, which is highly conserved amongst different bacterial species. DnaA binding to the origin initiates the regulated recruitment of other enzymes and proteins that will eventually lead to the establishment of two complete replisomes for bidirectional replication.DNA sequence elements within oriC that are important for its function include DnaA boxes, a 9-mer repeat with a highly conserved consensus sequence 5' – TTATCCACA – 3', that are recognized by the DnaA protein. DnaA protein plays a crucial role in the initiation of chromosomal DNA replication. Bound to ATP, and with the assistance of bacterial histone-like proteins DnaA then unwinds an AT-rich region near the left boundary of oriC, which carries three 13-mer motifs, and opens up the double-stranded DNA for entrance of other replication proteins.
This region also contains four “GATC” sequences that are recognized by DNA adenine methylase, an enzyme that modifies the adenine base when this sequence is unmethylated or hemimethylated. The methylation of adenines is important as it alters the conformation of DNA to promote strand separation, and it appears that this region of oriC has a natural tendency to unwind.
DnaA then recruits the replicative helicase, DnaB, from the DnaB-DnaC complex to the unwound region to form the pre-priming complex. After DnaB translocates to the apex of each replication fork, the helicase both unwinds the parental DNA and interacts momentarily with primase.
In order for DNA replication to continue, single stranded binding proteins are needed to prevent the single strands of DNA from forming secondary structures and to prevent them from re-annealing. In addition, DNA gyrase is needed to relieve the topological stress created by the action of DnaB helicase.
Elongation
When the replication fork moves around the circle, a structure shaped like the Greek letter theta Ө is formed. John Cairns demonstrated the theta structure of E. coli chromosomal replication in 1963, using an innovative method to visualize DNA replication. In his experiment, he radioactively labeled the chromosome by growing his cultures in a medium containing 3H-thymidine. The nucleoside base was incorporated uniformly into the bacterial chromosome. He then isolated the chromosomes by lysing the cells gently and placed them on an electron micrograph grid which he exposed to X-ray film for two months. This Experiment clearly demonstrates the theta replication model of circular bacterial chromosomes.- See Autoradiograph of intact replicating chromosome of E.coli
- See Figure 4 of D. M. Prescott, and P. L. Kuempel : A grain track produced by an E. coli chromosome from cells labeled for 19 min with thymine, followed by labelingfor 2.5 min with thymine and thymidine. .
Deoxynucleotides are then added to this primer by a single DNA polymerase III dimer, in an integrated complex with DnaB helicase. Leading strand synthesis then proceeds continuously, while the DNA is concurrently unwound at the replication fork. In contrast, lagging strand synthesis is accomplished in short Okazaki fragments. First, an RNA primer is synthesized by primase, and, like that in leading strand synthesis, DNA Pol III binds to the RNA primer and adds deoxyribonucleotides.
When the synthesis of an Okazaki fragment has been completed, replication halts and the core subunits of DNA Pol III dissociates from the β sliding clamp . The RNA primer is removed and replaced with DNA by DNA polymerase I and the remaining nick is sealed by DNA ligase, which then ligates these fragments to form the lagging strand.
A substantial proportion of the replication forks originating at oriC encounter a DNA damage or strand break when cells are grown under normal laboratory conditions. The encountered DNA damages are ordinarily processed by recombinational repair enzymes to allow continued replication fork progression.
Termination
Termination is the process of fusion of replication forks and disassembly of the replisomes to yield two separate and complete DNA molecules. It occurs in the terminus region, approximately opposite oriC on the chromosome. The terminus region contains several DNA replication terminator sites, or "Ter" sites. A special "replication terminator" protein must be bound at the Ter site for it to pause replication. Each Ter site has polarity of action, that is, it will arrest a replication fork approaching the Ter site from one direction, but will allow unimpeded fork movement through the Ter site from the other direction. The arrangement of the Ter sites forms two opposed groups that forces the two forks to meet each other within the region they span. This arrangement is called the "replication fork trap."- See locations and sequences of the replication termini of E. coli. Map showing the ori and the 10 Ter sites. The consensus sequence of Ter.
- The crystal structure of the Ter DNA-Tus protein complex showing the nonblocking and the fork-blocking faces of Tus. A cross-sectional view of the helicase-arresting surface.
There has been some confusion about the role DNA gyrase plays in decatenation. To define the nomenclature, there are two types of topoisomerases: type I produces transient single-strand breaks in DNA and types II produces transient double-strand breaks. As a result, the type I enzyme removes supercoils from DNA one at a time, whereas the type II enzyme removes supercoils two at a time. The topo I of both prokaryotes and eukaryotes are the type I topoisomerase. The eukaryotic topo II, bacterial gyrase, and bacterial topo IV belong to the type II.
We often forget that DNA gyrase does in fact have topoisomerase type II activity; thus, with it being a homologue of topoisomerase IV we expect similarity in the two proteins' functions. DNA gyrase preliminary role is to introduce negative super coils into DNA, thereby relaxing positive supercoils that come into play during DNA replication. Topoisomerase IV also relaxes positive supercoils, therefore, DNA Gyrase and topoisomerase IV play an almost identical role in removing the positive supercoils ahead of a translocating DNA polymerase, allowing DNA replication to continue unhindered by topological strain.
Confusion arises when some scientific literature state that DNA gyrase is the sole enzyme responsible for decatanation. In an experiment conducted by Zechiedrich, Khodursky and Cozzarelli in 1997, it was found that topoisomerase IV is the only important decatenase of DNA replication intermediates in bacteria. In this particular experiment, when DNA gyrase alone were inhibited, most of the catenanes were unlinked. However, when Topoisomerase IV alone was inhibited, decatenation was almost completely blocked. The results obtained suggest that Topoisomerase IV is the primary decatenase in vivo, and although DNA gyrase does play a role in decatenation, its function is not as essential as topoisomerase IV in the decatentation of interlinked chromosomes.