A crude soluble enzyme system capable of replicating oriC plasmids in a DnaA- and oriC-dependent manner was developed in 1980. In addition to DNA polymerase III (the replicative enzyme), the following proteins are also required to replicate an oriC plasmid:
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| Name |
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| DnaA | DnaA binds to oriC and initially opens an AT-rich sequence. DnaA then helps to assemble the DnaB (helicase), in the form of a DnaB-DnaC complex, around each strand. |
| DnaB (helicase) | DnaB (helicase) unwinds DNA strands using ATP energy. It moves processively along DNA (i. e., does not leave the DNA until replication is finished; it encircles the DNA strand). DnaA together with the use of ATP energy is required to load DnaB (helicase) onto DNA in the form of a DnaB-DnaC complex. After loading DnaB onto the replication fork, DnaC is released from the DnaB-DnaC complex and leaves the DNA. DnaB helicase activity is greatly stimulated by making contact with DNA polymerase III holoenzyme. |
| DnaC | DnaC forms a complex with DnaB. It is required for loading DnaB onto DNA. |
| DnaG (primase) | All known DNA polymerases require a pre-exisitng "primer" from which to begin replication. E. coli uses an RNA primer which is synthesized by an enzyme called DnaG (primase). DnaG makes RNA primers (about 10 nucleotides long) that are used by DNA pol III holoenzyme to start DNA synthesis. DnaG acts distributively (does not remain associated with DNA). It drops off DNA after primer synthesis, then reloads onto DNA a second or so later by protein-protein interactions with DnaB (helicase) to synthesize the next primer on the lagging strand. There are many places along the template where RNA primers can be made by primase. |
| SSB | SSB (single-strand binding protein) does not itself unwind DNA, but binds to and stabilizes unwound single-stranded DNA. |
| Gyrase (topoisomerase II) | Double-stranded DNA is unwound by DnaB helicase so that each strand can be replicated into new daughter double-stranded molecules. However, the unwinding activity of helicases at one position along a DNA molecule causes the "downstream" portion of the same DNA molecule to become OVERWOUND (remember class demonstration). Unless this overwinding is relieved by an enzyme called gyrase (topoisomerase II), DNA replication will soon stop. Gyrase uses ATP energy to introduce negative supercoiling into the DNA. The negative supercoiling compensates for the positive supercoiling generated during DNA replication. Gyrase is not limited to the site of DNA replication, but can act at any position on the DNA molecule. Gyrase can be considered as the SWIVEL for replicating molecules. |
| DNA polymerase I | Required to remove RNA primers by simultaneous action of 5'-to3' exonuclease and DNA polymerase (nick translation). |
| DNA ligase | Required to join Okazaki fragments together, uses NAD+ as energy cofactor. |
The initiation of DNA replication at oriC by DnaA protein is shown in this FIGURE.
The leading strand is synthesized continuously as shown in this FIGURE.
The lagging strand is synthesized discontinuously as Okazaki fragments. The primase-to-polymerase switch at the replication fork is shown in this FIGURE. In panel (A) DnaB helicase encircles the lagging strand, and the two core polymerases are held to the two strands of DNA by beta clamps. The tau dimer binds two cores, one gamma complex clamp loader, and contacts the DnaB helicase (this contact between tau and DnaB stimulates helicase activity). The requirement for primase to contact DnaB for activity localizes primers near the replication fork. Primase remains at the primed site through interaction with SSB. In panel (B) the gamma complex opens the beta ring in response to binding ATP. Upon binding to SSB through the chi subunit of the gamma complex, the primase-to-SSB contact becomes destabilized and dissociates from DNA. Panel (C) illustrates that the released primase can recycle to prime a new Okazaki fragment, consistent with the distributive action of primase in a replication fork system. The mechanism by which DNA polymerase acts on the lagging strand is also shown in the figure. Due to the antiparallel structure of duplex DNA, the polymerase on the lagging strand extends DNA in a direction opposite the polymerase on the leading strand. However, the lagging strand polymerase is connected to the leading strand polymerase and thus moves with the replisome. This results in a DNA loop on the lagging strand that grows larger as the lagging core polymerase draws DNA up through it until it finishes an Okazaki fragment, whereby it "bumps" into the fragment it synthesized previously. This is shown in panel (A). Upon completion of an Okazaki fragment, the lagging strand polymerase releases from DNA by severing its tie to the beta clamp, leaving the beta ring behind on the finished fragment as in panel (B). The DNA polymerase then reassociates with a new beta clamp assembled onto an upstream primer by the gamma complex (panel C).
When the synthesis of the new Okazaki fragment terminates by colliding with the downstream Okazaki fragment (FIGURE), the RNA primer that was used to initiate the downstream Okazaki fragment is removed by the 5'-to-3' exonuclease activity of DNA polymerase I with simultaneous filling of the gap with deoxyribonucleotides (nick translation). Finally, adjacent Okazaki fragments are joined together by DNA ligase using NAD+ as the energy cofactor.
The terminus (ter) of DNA replication is opposite the origin of replication on the circular E. coli chromosome. Ter is a "trap" (FIGURE): replication forks enter, but do NOT leave, this region spanning 450 kb (1 kb = 1,000 bp). There are six ter sites in this region. A protein called Tus binds to the ter sites, and this binding stops DnaB (helicase). Leading strand synthesis terminates one nucleotide away from bound Tus. Ter sites only work in one direction. For each round of replication, only one termination site is usually used. The fork that meets a termination site in its active orientation first is stalled and waits for the other fork to reach the same site. A replication fork halted at a properly oriented termination complex therefore appears to form a barrier to the opposing replication fork, ensuring that each segment of the chromosome is replicated exactly once. Interestingly, E. coli mutants lacking the ter region or having mutated Tus are viable, but segregate non-viable cells at high frequency.
At the conclusion of E. coli DNA replication the two circular daughter molecules remain linked together (like links in a chain). Segregation (separation) of circular daughter DNA molecules requires the action of topoisomerase IV (Click here to see Table of E. coli topoisomerases).