This lecture deals all about DNA replication of bacteria
DNA replication is necessary for cells to divide, whether to reproduce new organisms, as in unicellular microorganisms, or to produce new cells as part of a multicellular organism. DNA replication must be sufficiently accurate that the daughter cells are genetically identical to the mother cell (or almost so). This involves a host of special enzymes and processes.
DNA exists in cells as a double helix with complementary base pairing. If the double helix is opened up, a new strand can be synthesized as the complement of each parental strand. DNA replication is semi-conservative, referring that the two resulting double helices consist of one new strand and one parental strand. The DNA strand that is used to make a complementary daughter strand is called the template, and in DNA replication each parental strand is a template for one newly synthesized strand.
The precursor of each new nucleotide in the DNA strand is a deoxynucleoside 5`-triphosphate. The two terminal phosphates are removed and the innermost phosphate is then attached covalently to a deoxyribose of the growing chain. This addition of the incoming nucleotide requires the presence of a free hydroxyl group, which is available only at the 3` end of the molecule. This leads to the important principle that DNA replication always proceeds from the 5` end to the 3` end, the 5`-phosphate of the incoming nucleotide being attached to the 3`-hydroxyl of the previously added nucleotide.
Enzymes that catalyze the addition of deoxynucleotides are called DNA polymerases. Several such enzymes exist, each with a specific function. There are five different DNA polymerases in Escherichia coli, called DNA polymerases I, II, III, IV, and V. DNA polymerase III (Pol III) is the primary enzyme for replicating chromosomal DNA. DNA polymerase I (Pol I) is also involved in chromosomal replication, though to a lesser extent. The other DNA polymerases help repair damaged DNA.
All known DNA polymerases synthesize DNA in the 5` to 3` direction. However, no known DNA polymerase can initiate a new chain; all of these enzymes can only add a nucleotide onto a pre-existing 3`-OH group. To start a new chain, a primer, (referring a nucleic acid molecule to which DNA polymerase can attach the first nucleotide) is required. In most cases this primer is a short stretch of RNA. When the double helix is opened at the beginning of replication, an RNA-polymerizing enzyme makes the RNA primer. This enzyme, called primase, synthesizes a short stretch of RNA of around 11–12 nucleotides that is complementary in base pairing to the template DNA. At the growing end of this RNA primer is a 3`-OH group to which DNA polymerase can add the first deoxyribonucleotide. Continued extension of the molecule thus occurs as DNA rather than RNA. The primer will eventually be removed and replaced with DNA.
Before DNA polymerase can synthesize new DNA, the double helix must be unwound to expose the template strands. The zone of unwound DNA where replication occurs is known as the replication fork. The enzyme DNA helicase unwinds the double helix, using energy from ATP, and exposes a short single-stranded region. Helicase moves along the DNA and separates the strands just in advance of the replication fork. The single-stranded region is covered by single strand binding protein. This stabilizes the single-stranded DNA and prevents the double helix from re-forming. Unwinding of the double helix by helicase generates positive supercoils ahead of the advancing replication fork. To counteract this, DNA gyrase travels along the DNA ahead of the replication fork and inserts negative supercoils to cancel out the positive supercoiling.
Bacteria possess a single location on the chromosome where DNA synthesis is initiated, the origin of replication (oriC). This consists of a specific DNA sequence of about 250 bases that is recognized by initiation proteins, in particular a protein called DnaA, which binds to this region and opens up the double helix. Next to assemble is the helicase (known as DnaB), which is helped onto the DNA by the helicase loader protein (DnaC). Two helicases are loaded, one onto each strand, facing in opposite directions. Next, two primase and then two DNA polymerase enzymes are loaded onto the DNA behind the helicases. Initiation of DNA replication then begins on the two single strands. As replication proceeds, the replication fork appears to move along the DNA.
An important distinction in replication between the two DNA strands due to the fact that replication always proceeds from 5` to 3` (always adding a new nucleotide to the 3`-OH of the growing chain). On the strand growing from the 5`-PO4 to the 3`-OH, called the leading strand, DNA synthesis occurs continuously because there is always a free 3`-OH at the replication fork to which a new nucleotide can be added. But on the opposite strand, called the lagging strand, DNA synthesis occurs discontinuously because there is no 3`-OH at the replication fork to which a new nucleotide can attach. Where is the 3`-OH on this strand? It is located at the opposite end, away fromthe replication fork. Therefore, on the lagging strand, RNA primers must be synthesized by primase multiple times to provide free 3`-OH groups. By contrast, the leading strand is primed only once, at the origin. As a result, the lagging strand is made in short segments, called Okazaki fragments, after their discoverer, Reiji Okazaki. These fragments are joined together later to give a continuous strand of DNA.
After synthesizing the RNA primer, primase is replaced by Pol III. This enzyme is a complex of several proteins, including the polymerase core enzyme itself. Each polymerase is held on the DNA by a sliding clamp, which encircles and slides along the single template strands of DNA. Consequently, the replication fork contains two polymerase core enzymes and two sliding clamps, one set for each strand. However, there is only a single clamp-loader complex. This is needed to assemble the sliding clamps onto the DNA. After assembly on the lagging strand, the elongation component of Pol III, DnaE, then adds deoxyribonucleotides until it reaches previously synthesized DNA. At this point, Pol III stops. The next enzyme to take part, Pol I, has more than one enzymatic activity. Besides synthesizing DNA, Pol I has a 5` to 3` exonuclease activity that removes the RNA primer preceding it. When the primer has been removed and replaced with DNA, Pol I is released. The last phosphodiester bond is made by an enzyme called DNA ligase. This enzyme seals nicks in DNAs that have an adjacent 5`-PO4 and 3`-OH (something that Pol III is unable to do), and along with Pol I, it also participates in DNA repair. DNA ligase is also important for sealing genetically manipulated DNA during molecular cloning.
The circular nature of the chromosome of Escherichia coli and most other prokaryotes creates an opportunity for speeding up replication. In E. coli, and probably in all prokaryotes with circular chromosomes, replication is bidirectional from the origin of replication. There are thus two replication forks on each chromosome moving in opposite directions. These are held together by the two Tau protein subunits. In circular DNA, bidirectional replication leads to the formation of characteristic shapes called theta structures (θ).
Bidirectional DNA synthesis around a circular chromosome allows DNA to replicate as rapidly as possible. Even taking this into account and considering that Pol III can add nucleotides to a growing DNA strand at the rate of about 1000 per second, chromosome replication in E. coli still takes about 40 min. Interestingly, under the best growth conditions, E. coli can grow with a doubling time of about 20 min. However, even under these conditions, chromosome replication still takes 40 min. The solution to this conundrum is that cells of E. coli growing at doubling times shorter than 40 min contain multiple DNA replication forks. That is, a new round of DNA replication begins before the last round has been completed. Only in this way can a generation time shorter than the chromosome replication time be maintained.
The DNA polymerization is different in leading and the lagging strands and also the enzymes involved. From such a simplified drawing it would appear that each replication fork contains a host of different proteins all working independently. Actually, this is not so. These proteins aggregate to form a large replication complex called the replisome. The lagging strand of DNA loops out to allow the replisome to move smoothly along both strands, and the replisome literally pulls the DNA template through it as replication occurs. Therefore, it is the DNA, rather than DNA polymerase, that moves during replication. Note also how helicase and primase form a sub-complex, called the primosome, which aids their working in close association during the replication process
In summary, in addition to Pol III, the replisome contains several key replication proteins: (1) DNA gyrase, which removes supercoils; (2) DNA helicase and primase (the primosome), which unwind and prime the DNA; and (3) single-strand binding protein, which prevents the separated template strands from reforming a double helix.
Proof reading
Proofreading activity occurs if an incorrect base has been inserted because this creates a mismatch in base pairing. Both Pol I and Pol III possess a 3` to 5` exonuclease activity that can remove such wrongly inserted nucleotides.
Termination of replication
Eventually the process of DNA replication is finished. How does the replisome know when to stop? On the opposite side of the circular chromosome from the origin is a site called the terminus of replication. Here the two replication forks collide as the new circles of DNA are completed. When replication of the circular chromosome is complete, the two circular molecules are linked together, much like the links of a chain. They are unlinked by another enzyme, topoisomerase IV. Obviously, it is critical that, after DNA replication, the DNA is partitioned so that each daughter cell receives a copy of the chromosome.
Youtube video about DNA replication
Bacterial DNA replication is bidirectional (Youtube)
Plasmids of bacteria perform different way of replication known as rolling circle DNA replication (Compare the difference between theta and rolling circle DNA replication)