Principles of Biochemistry Volume 3 - A. Lehninger 1985

Molecular mechanisms of genetic information transfer
DNA replication and transcription
Circular DNA replicates bidirectionally

We have already seen that the DNA of Bacteria and many Introduction/6.html">DNA-containing Viruses is a circular double helix. As soon as this discovery was made, the question arose as to how Circular DNA is replicated. Does the circular DNA first split into a linear molecule prior to Replication, or is it able to replicate as a circle? Landmark experiments performed by John Cairns demonstrated that DNA in intact E. coli Cells replicates while maintaining its circular conformation. Cairns grew E. coli in a medium containing thymidine labeled with the radioactive hydrogen isotope tritium (³H), which rendered the cellular DNA radioactive. When this DNA was gently isolated in a relaxed form and placed on a photographic emulsion, the radioactive thymidine residues produced tracks of silver grains on the exposed plate, outlining the DNA molecule. Based on these images, Cairns concluded that the intact chromosome is a giant circle—a finding consistent with the previously established genetic circular map of E. coli. However, the radioactive DNA isolated from cells undergoing replication revealed an additional radioactive loop (Fig. 28-3). Cairns proposed that this DNA loop arises from The formation of two radioactive daughter strands, complementary to the parental strands, as the Replication fork moves around the parental DNA circle.

It was initially believed that replication begins at a fixed point on the parental DNA—known as THE ORIGIN OF replication—and that a single replication fork moves around the circular DNA molecule in one direction (Fig. 28-3). However, subsequent experiments with E. coli and viral Chromosomes showed that replication is typically bidirectional, meaning that two replication forks are involved. Both forks originate at the same point and travel away from it simultaneously in opposite directions until they meet (Fig. 28-3). At this point, the two fully synthesized daughter double-stranded circles separate, with each containing one old and one newly synthesized strand.

Class="center">

Fig. 28-3. Replication of the E. coli chromosome. A. Schematic representation of a tritium-labeled E. coli chromosome during replication. B. Interpretation of the replication process (newly synthesized daughter strands are shown in red). According to one model, only a single replication fork moves away from the origin of replication. According to the other model, two replication forks originate at the origin and move in opposite directions until they meet. The chromosomes of E. coli and other bacteria, as well as many DNA-containing viruses, replicate according to the second model.

The origin of replication is a nucleotide sequence approximately 100–200 Base Pairs in length, without which DNA cannot replicate. This sequence is recognized by specific cellular Proteins that initiate the replication cycle at this site. It is precisely this initiation step of replication that is subject to cellular regulation.

Based on The rate of movement of the E. coli replication fork, it can be concluded that at 37°C, new DNA is synthesized at a rate exceeding 45,000 nucleotide residues per minute per fork. Given that there are ~ 10 base pairs per complete turn of The Double Helix (Section 27.6), the rate at which parental DNA unwinds at the replication fork in E. coli cells exceeds 4,500 rpm—faster than the engine shaft speed of a car traveling at 110 km/h. Such rapid unwinding presumably poses significant mechanical challenges for replication due to the double-helical nature of Native DNA molecules. As we will see later, The Cell employs specific mechanisms to overcome these problems.