but it is instructive to investigate how DNA replication proceeds to understand what is involved in this process in present-day biology.
In a cell, DNA replication begins at specific locations on the DNA, or origins of replication. In bacteria, there is a single origin of replication on their circular genome or chromosome, whereas in eukaryotes, that have longer linear chromosomes, replication is initiated at multiple origins. The unwinding of DNA at these locations and the synthesis of new strands result in a replication fork (Figure 5.14). Let's examine this process in more detail.
Figure 5.14 The replication of DNA. The figure shows some of the diversity of machinery involved in the process.
An enzyme called a helicase is used to separate the two strands of DNA, essentially “unzipping” the hydrogen bonds. As the helicase separates the DNA at the replication fork, the DNA ahead of it is forced to rotate. This process results in a build-up of twists in the DNA, and a resistance becomes established, which, if not dealt with, would eventually halt the progress of the replication fork. A topoisomerase is an enzyme that temporarily breaks the strands of DNA, relieving the tension caused by unwinding the two strands of the DNA helix.
The replication fork generates two single strands of DNA. Each of these strands (Figure 5.14) can then be used as a template to make the corresponding strand, resulting in two new double helices. This process is called semi-conservative replication because only one strand of the parent double helix is conserved in each new DNA molecule that is made.
The first important point to understand about this process of making two new double-stranded DNA molecules is that the process has directionality. The energy for making a new DNA strand is acquired by cleaving the 5′-triphosphate of the nucleotide that is added to the growing DNA chain. This means that DNA synthesis can only proceed from the 5′ to 3′ direction. On one of the strands, this means that the new complementary strand can be made by synthesizing DNA by following the replication fork as it moves forwards. This is called the “leading” strand. The synthesis of the new strand is accomplished by DNA polymerase. The DNA polymerase is an ancient multimeric enzyme that is responsible for assembling the nucleotides into the newly forming DNA strand.
However, on the other strand of DNA, the “lagging” strand, synthesis must always restart near the replication fork as the DNA unzips (Figure 5.14) and move back in the opposite direction to the replication fork because of the directionality of DNA synthesis. This results in the complication that the lagging strand must be synthesized in fragments. To accomplish this task, a small piece of RNA, an RNA primer, is synthesized and attached to the DNA by a primase enzyme (the primase enzyme is a type of RNA polymerase because it makes RNA). DNA synthesis then proceeds from the RNA primer, accomplished by DNA polymerase. The strands of DNA produced in this way are called “Okazaki” fragments. These fragments are finally stitched together with an enzyme called DNA ligase, generating the complete double strand.
There is one other problem during replication. Single-stranded DNA, produced after the helicase has separated the two DNA strands, tends to fold back on itself forming secondary structures. These structures would interfere with the movement of DNA polymerase along the strand. To prevent this, single-strand binding proteins bind to the DNA until the second strand is synthesized, preventing secondary structure formation.
Termination of DNA replication requires that the progress of the DNA replication fork be stopped. Termination involves the interaction between two components: a specific “termination site sequence” in the DNA, and a protein which binds to this sequence to physically stop DNA replication. In various bacterial species, this protein is named the DNA replication terminus site-binding protein, or Ter protein.
Because bacteria have circular chromosomes, termination of replication occurs when the two replication forks that started out from the origin meet each other on the opposite side of the chromosome.
5.6.6 Plasmids
Organisms carry DNA in forms other than their primary chromosomal DNA. A plasmid is a small DNA molecule that is separate and can replicate independently of the chromosomal DNA in a cell, although it is considered to be part of the total genome of an organism (Figure 5.15). Plasmids are most commonly found as small circular, double-stranded DNA molecules in bacteria, archaea, and eukaryotic organisms. They carry genes that can benefit the survival of the organism in the natural environment. Examples include genes for antibiotic resistance (which makes them of central significance in the transfer of antibiotic resistance between bacteria in hospitals) and resistance to heavy metals (which allows organisms to live in diverse environments with heavy metals, from volcanic hot springs to human-polluted industrial sites). Plasmids can also provide bacteria with the ability to fix nitrogen gas from the atmosphere into useful nitrogen compounds or to degrade certain organic compounds that provide an advantage when nutrients are scarce. Plasmids vary in size from about a thousand to over a million base pairs, and the number of identical plasmids in a single cell can range from one to several thousand depending upon the environment and the species.
Figure 5.15 Plasmids are small circular pieces of DNA. They can be introduced into microorganisms by genetic engineers to produce industrially important products. The plasmid shown here has an origin of replication, which is the sequence that allows it to be copied. It contains a “coding sequence,” which could be a DNA sequence producing an important drug. The “promoter” is a DNA sequence that allows the coding region to be read. The transcription termination sequence tells the genetic machinery when to stop transcribing. It also contains a gene for antibiotic resistance which allows for bacteria successfully carrying the plasmid to be selected by researchers if they want to make sure that only the drug-producing bacteria are growing. The other sites marked on this diagram (e.g. SpeI, kpnI) are locations where specific enzymes (restriction enzymes) cut the DNA and allow the researcher to insert or remove bits of DNA.
Artificial plasmids are widely used in molecular cloning, where they serve as “carriers” of foreign genes that the researcher might wish to incorporate and have read inside a particular organism. For this reason, they are one of the most important tools in genetic engineering (Figure 5.15).
Plasmids are sometimes also called replicons, capable of replicating autonomously within a suitable host. However, plasmids, like viruses, are not considered to be a form of life. You might like to consider whether you agree with this view, and why, in the light of discussions in Chapter 2.
Discussion Point: What Is the Minimum Size of a Cell?
The minimum size of a cell is an important measure, since it defines the size below which we would question whether a putative fossil or living cell was a self-replicating living organism. For a cell to grow and reproduce, it must have some minimal machinery to produce proteins needed for division. It must have other molecules and proteins to gather energy and carry out basic functions such as replicating genetic information. Consider the following statement from a United States National Research Council report on the “Size Limits of Very Small Microorganisms”: “Free-living organisms require a minimum of 250–450 proteins along with the genes and ribosomes necessary for their synthesis. A sphere capable of holding this minimal molecular complement would be 250–300 nm in diameter, including its bounding membrane. Given the uncertainties
