DNA III: _{The replication of DNA}

The synthesis of DNA

As mentioned in our DNA II module, the moment James Watson and Francis Crick first gazed upon their newly built model of DNA, they could see clues about one of the major properties that they knew DNA must somehow exhibit: self-replication. The mystery of self-replication had confused scientists for many years. But one thing was certain: Every cell, whether a yeast, a bacterium, or a human cell, must be able to copy all of its genes, all of its DNA. This is because when a cell divides in two, both resulting cells are genetically identical to each other and to the original parent cell. The sheer number of times that the DNA in your body has been replicated (and accurately) is astounding.

You began life as a single cell, a zygote, the result of the fusion of a sperm and an egg. Since then, you have developed into an organism with somewhere between 10 and 100 trillion cells (>10,000,000,000,000). And, with certain rare exceptions, every single one of your trillions of cells has the same DNA sequence as the one cell did when you were just a zygote. How does all of this copying of DNA take place?

As mentioned, the structure of the double-stranded DNA molecule gave powerful hints as to how DNA might be accurately copied. Specifically, the complementary base-pairing of DNA follows a strict pattern that allows us to accurately predict what one strand of DNA looks like just by looking at the other, complementary strand. Put another way, if someone took a regular DNA molecule, pulled the two strands apart, and showed us only one strand, we could accurately list the series of nucleotides of the missing strand.

Watson and Crick saw this possibility when they ended their paper saying, "It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material." This possible copying mechanism is called semi-conservative DNA replication, because if a cell would duplicate its DNA in this manner, the DNA helix would split and half of both of the new double helices would retain DNA from the original strand (Figure 1). While this scheme makes good sense, it was just a logical guess at first. It wasn't until the late 1950s that Matthew Meselson and Franklin Stahl performed the scientific experiment that showed that the replication of DNA was indeed semi-conservative. (See our Meselson and Stahl: Models of DNA Replication.)

In the 1950s, Meselson and Stahl, Watson and Crick, and many other scientists explored the properties of DNA using the intestinal bacterium Escherichia coli. Because a few rare strains of E. coli have been found to cause gastrointestinal illness, E. coli is frequently associated with outbreaks of food poisoning. But actually, most strains of E. coli are harmless and our large intestines are filled with this bacterium. E. coli was among the first routinely used "model organisms," a species that is chosen for extensive study in the laboratory because it offers certain practical advantages that make research easier. E. coli, in particular, is among the fastest growing organisms on Earth, with a generation time of under 20 minutes in ideal conditions. Since long before they knew what DNA was, scientists had noticed that the amount of DNA in an E. coli cell (and any other cell for that matter) doubles prior to cell division. The pool of DNA in the cell is then split equally between the two "daughter cells" that result, so that both have the same amount of DNA that the original bacterium had before replication. Because all of this happens in E. coli in about 20 minutes, it was the logical organism for early molecular biologists to select.

In vitro DNA replication

While Meselson and Stahl and others were testing the possible hypothetical models of DNA replication, other scientists set out to understand its molecular mechanism by re-creating it in a test tube. This process is called in vitro reconstitution and is often used in the field of biochemistry as a way of simplifying a complex cellular event so that it happens in isolation and can thus be observed and manipulated at will. The scientists who were first able to reconstitute DNA replication in a test tube were Arthur Kornberg and his wife Sylvy and the research team that they led. They achieved this incredible feat through a painstaking process of successive chemical purification of different proteins and other components from large batches of E. coli bacteria. By separating and purifying individual components, the Kornberg research team made several important discoveries about how DNA replication occurs.

These discoveries all began with the development of a critically important technique – the DNA synthesis assay. An assay is a quantitative laboratory measurement of a certain biological or chemical process, usually in a test tube (in vitro). The DNA synthesis assay is a technique for measuring the synthesis of new DNA molecules. The Kornberg laboratory was the first to develop this assay, and the assay itself is quite simple. First, DNA polymers are easily separated from free nucleotides because DNA is not soluble in solutions that contain trichloroacetic acid (TCA), while free nucleotides are. If a scientist adds TCA to a liquid mixture of DNA and free nucleotides, the DNA will precipitate out, while the nucleotides will remain dissolved in the liquid. The DNA precipitate can then be easily separated from the liquid by centrifugation.

The second important feature of the DNA synthesis assay is its use of radioactively labeled nucleotides. A scientist can add radioactive nucleotides when preparing a DNA synthesis assay, and then later, if DNA synthesis has occurred, some of the radioactive label will be incorporated into the TCA-insoluble DNA. This provides evidence that some of the labeled nucleotides were polymerized into a new DNA molecule. This DNA synthesis assay is very simple to execute and also very quantitative, which means that it gives very reliable and reproducible numerical values that can be used to calculate how much DNA was made and how fast the synthesis took place.

Armed with this assay, the Kornberg laboratory was the first to report the synthesis of DNA outside of a living cell. The popular press of the time announced that Arthur Kornberg had "created life in a test tube."

Of course, this was hardly the case, but the new ability to synthesize DNA in vitro captured the attention of the general population and is recognized as one of the crucial successes paving the way for the emergence of genetic engineering in the 1970s and 80s. Initially, the laboratory synthesis of DNA was extremely slow (much slower than it occurs in a cell), and it occurred only when crude extracts of E. coli were added to the test tubes. Crude extracts contain all the contents of the cells – all proteins, nucleotides, DNA, RNA, lipids, carbohydrates, etc. Nevertheless, the DNA synthesis assay was a good starting point in which Kornberg and others could begin to dissect the process of DNA replication in detail.

The first discovery and arguably the most important occurred in 1955: Kornberg's research team purified the enzyme from the crude extract that is chiefly responsible for the synthesis of DNA – DNA polymerase. When purified DNA polymerase is added to the DNA synthesis assay, the synthesis of DNA occurs hundreds of times more rapidly than when it is not added. However, the in vitro synthesis of DNA still required the addition of small amounts of crude cell extract. This is because DNA polymerase does not make DNA all by itself – there are many other factors required and not all of these were known at the time. The Kornberg lab and others around the world worked to purify other important components from the crude extract, in the hopes that one day they could make DNA using only the necessary factors and no crude extract.

Some of these required components were obvious, while others were unexpected. For example, it was very quickly discovered that nucleotides were required for the synthesis of DNA, which isn't very surprising because it was well known, even in the 1950s, that nucleotides are the building blocks of DNA. However, only nucleotides in the tri-phosphate form could be used as DNA building blocks (Figure 2). Later studies demonstrated why this is so - the breaking of the high-energy terminal phosphate bond of each new nucleotide added to a growing DNA molecule provides the energy for the polymerization reaction.

Another important point that the Kornberg laboratory noted was that the test tube DNA synthesis reactions required the presence of an intact copy-template DNA in order for DNA polymerase to make more DNA. In other words, even in a test tube, DNA polymerase cannot build "random" DNA molecules through the willy-nilly polymerization of nucleotides. It can only make copies of DNA molecules that already exist. Think of it this way - DNA polymerase is like a copy machine, NOT like a computer with new sentences can be created. A copy machine cannot print anything unless it has a template to work with. So when Kornberg added purified intact DNA molecules to the DNA synthesis assay, once again the speed of DNA polymerase increased dramatically. (Prior to this discovery, DNA synthesis was occurring only because tiny amounts of DNA template were present in the crude extract that is added to the assay mixture.)

How DNA is pulled apart

In addition to the hunt for more of the individual factors involved in DNA replication, the DNA synthesis assay allowed researchers to study the properties of DNA synthesis. As scientists around the globe began to study DNA polymerase and DNA replication, they knew that the semi-conservative model of DNA replication, as proven by Meselson and Stahl, requires that the two original template strands of DNA are pulled apart in order to be copied separately. However, it was not known how this happens. Scientists had observed that the two strands of DNA are held very tightly together by the hydrogen bonds between complementary nucleotide base-pairs of the two strands. In the laboratory, the only way the two strands could be separated was by heating the DNA to near-boiling temperatures. Obviously, it is not likely that living cells generate high heat in order to pry apart the two strands of DNA, so the question remained, "Inside a living cell, what pulls apart the two original strands of DNA so that they may be copied?"

Because double-stranded DNA is very stable, scientists suspected that there must be an elaborate mechanism for pulling the two strands apart. Two research groups, including Arthur Kornberg's, discovered the answer in the late 1970s: an enzyme they named DNA helicase. This enzyme is capable of prying the two strands of DNA apart so that the two individual strands can then serve as templates for DNA polymerase, according to the semi-conservative model.

It turns out, however, that when helicase first pries apart a section of DNA, it does not start at the end of the molecule in the case of linear DNA, nor does it select a place at random. The initial "melting" of DNA occurs at specific locations, called origins of DNA replication. Each of these creates a bulge in the DNA double helix that is visible by electron microscopy. These bulges are called replication bubbles and represent sites of DNA synthesis (Figure 3).

When a replication bubble opens up and DNA synthesis begins, replication proceeds in both directions, away from the origin. A DNA helicase enzyme leads the way, unzipping the parental DNA as replication proceeds in its wake. Both of these mobile regions of DNA synthesis are referred to as replication forks, which are the sites at which the replication of DNA is executed (Figure 4).

DNA primers and unidirectional synthesis

Once scientists began to focus on the events that occur at replication forks, they made several interesting observations that helped them to realize that DNA synthesis was much more complicated than they first imagined. The first such intriguing discovery was made by a young Japanese scientist named Tsuneko Okazaki, while working as a postdoctoral fellow with Kornberg at Stanford. Okazaki noticed that DNA polymerase cannot simply begin copying a template once it is pried apart from its complementary strand. Something more is needed to "kick-start" the copying of DNA before DNA polymerase can jump into action. Okazaki then discovered that she could coax DNA polymerase into performing DNA replication if she added a short piece of DNA that was complementary to part of the DNA template (Figure 5). Because this short DNA molecule served to get DNA synthesis started, Kornberg named them primers.

The discovery of primers was a major advance because now the scientists knew all the crucial components that were needed to perform an efficient DNA synthesis reaction in vitro. They no longer had a need for crude cell extract. Furthermore, the discovery of primers led to another curious observation by scientists, including Tsuneko Okazaki and her husband Reiji Okazaki, both former trainees of Kornberg, who had returned to Japan and formed their own research group. The Okazakis noticed that when a DNA synthesis reaction is set up and a primer is added, DNA synthesis begins at the primer and proceeds in only one direction. Curiously, they did not observe replication of the DNA region on the other side of the primer.

Returning to the structural model of DNA built by Watson and Crick, the Okazaki research team realized that DNA polymerization was only occurring at one end of the primer, the 3' end, and continuing in that direction. This was not simply a peculiar artifact of in vitro DNA synthesis. DNA replication inside all living cells also proceeds only in one direction: 5' to 3' (Figure 6). This property is called unidirectional DNA synthesis.

Okazaki fragments

Once it was realized that DNA synthesis proceeds in only one direction, Okazaki, Kornberg, and the entire community of DNA scientists realized that this posed a serious problem for their understanding of the DNA replication fork. There was extensive evidence that DNA synthesis proceeds on both strands of the DNA template after the two strands are pulled apart, and they had seen how DNA polymerase enzymes follow behind DNA helicase, synthesizing the new DNA strands alongside both original template strands (the semi-conservative model of DNA replication). But how could this be if DNA synthesis can proceed only in one direction (Figure 7)?

It was Reiji Okazaki who first postulated the solution to this conundrum. He imagined that the only possible way that DNA replication can occur on both strands of a replication fork but still proceed only in the 5' to 3' direction was if DNA synthesis was continuous on one of the strands, trailing steadily behind the DNA helicase, but discontinuous on the other strand, proceeding in short stretches away from the replication fork. These short stretches are called Okazaki fragments in honor of Reiji Okazaki; however, it was the work of the whole Okazaki research team, the Kornberg research team, and several others that confirmed Okazaki's hypothesis regarding discontinuous replication of DNA (Figure 8). It was Kornberg who coined the terms leading strand for the strand in which DNA replication is continuous, and lagging strand for the strand in which DNA synthesis occurs in short discontinuous Okazaki fragments of ~300 nucleotides of DNA.

Tragically, Reiji Okazaki died seven years after his famous discovery of discontinuous DNA replication. A native of Hiroshima, he was 15 years old when the first atomic bomb was dropped and was heavily irradiated while searching for his parents amongst the rubble. He suffered the effects of radiation sickness, finally succumbing to leukemia at the age of 44. The Okazakis and Kornbergs were both great examples of husband-wife teams of scientists (Figure 9).

Following these major breakthroughs, scientists moved relatively quickly in mapping out the other major players of the replication fork (Figure 10). For example, it was discovered by the Kornberg lab that the primer that is necessary to initiate DNA synthesis inside cells is actually made of RNA, not DNA, and is put in place by an enzyme called DNA primase. This RNA is eventually replaced with DNA by a specialized version of DNA polymerase, called DNA polymerase I (DNA pol I), while the main workhorse of DNA polymerase is actually DNA polymerase III (DNA pol III).

Further, it was discovered that the individual Okazaki fragments of the lagging strand need to be covalently bonded together. The enzyme that seals the Okazaki fragments together is called DNA ligase (Figure 11). Because this enzyme "seals" two stretches of DNA together, DNA ligase would later prove to be an essential tool in genetic engineering, as DNA molecules from different sources were "cut and pasted" to make new combinations and new DNA sequences.

The Kornbergs win the Nobel prize

For all of the important discoveries that led to our understanding of the molecular events that take place at the DNA replication fork, Alfred Kornberg was honored with the Nobel Prize in Chemistry in 1959. Throughout his life, Dr. Kornberg mentored many young scientists who went on to great accomplishments of their own, including the Okazakis, whose pioneering work with discontinuous DNA replication led to the discovery of Okazaki fragments. Other famous students of the so-called "Kornberg school" include research leaders around the world in both academia and the biotechnology industry. In fact, it is no surprise that the biotech industry itself started mainly in the San Francisco bay area, because Kornberg spent most of his career at Stanford University, just 30 miles south of San Francisco. Among the most successful students of Arthur Kornberg is his son Roger Kornberg, who claims to have "grown up in the lab" watching his father make crucial discoveries about DNA synthesis. With his own research team, Roger painstakingly studied the processes of RNA synthesis, also called gene transcription, which has many parallels to DNA synthesis.

Just as Arthur Kornberg earned the Nobel Prize in 1959 for deciphering the events of DNA synthesis, his son Roger was awarded the Nobel Prize in 2006 for a lifetime of research on RNA synthesis. The success of this father-son duo demonstrates how the mentoring of the next generation of scientists is among the most important work that scientists perform, a reality further emphasized by the fact that the large majority of scientific research takes place in the academic setting and involves young scientists-in-training as the foot soldiers of discovery.