An Interview with Manuel F. Varela and Ann F. Varela: Reiji Okazaki—DNA Replication

Dec 27, 2020 by

My father showed me the process of bacterial cells dying due to penicillin used for treatment. I naturally became interested in organisms and bioscience.”

—Tsuneko Okazaki

Never give up the research…

—Arthur Kornberg, to newly widowed Tsuneko Okazaki

Michael F. Shaughnessy

1) Reiji Okazaki—where was he born, when was he born—and where did he start his long and winding road to his many discoveries?

Dr. Reiji Okazaki is well-known for discovering the so-called discontinuous DNA synthesis mechanism on lagging strands using the famous Okazaki fragments. Reiji Okazaki (岡崎 令治) was born in Hiroshima, Japan, on October 8, 1930. He graduated from Nagoya University in 1953 with a Ph.D. in genetics. Okazaki was a postdoctoral fellow in the laboratory of Arthur Kornberg at Stanford University in California, the U.S. In 1963, Okazaki moved back to Nagoya University, where he became a professor.

2) The old saying—behind every man is a woman—who was he married to, and what was her involvement?

Okazaki’s wife and research partner was Dr. Tsuneko Okazaki. See Figure 71. In 1933, Tsuneko Okazaki was born in the Aichi Prefecture of Japan. During her undergraduate education, she studied biology at Nagoya University School of Science. Tsuneko Okazaki graduated with her Ph.D. from Nagoya University School of Science in 1956. She met Reiji Okazaki that same year. Shortly after they married, they collaborated with their research efforts in the laboratory.

Tsuneko and Reiji Okazaki began their research at Nagoya University in 1963. They decided to try to solve this paradox relating to the replication of DNA. They recognized that semiconservative replication could be rationalized if daughter strands of DNA were synthesized in vivo by an intermittent process.

File:Tsuneko Okazaki cropped 1 Tsuneko Okazaki 201511.jpg

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Figure 71. Tsuneko Okazaki, Emeritus Professor of Nagoya University, received the Person of Cultural Merit in November 2015.

Okazaki and his group looked at the replication point in detail. They speculated whether the daughter strand growing in the 3’ → 5’ direction (known as the “lagging” strand) might be synthesized as short fragments in the 5’ → 3’ order. DNA was hypothesized to grow in the direction opposite to the actual movement of extension for the newly synthesized daughter DNA. If this was the circumstance, the daughter strand could grow longer when these short fragments were linked together.

To corroborate their premise, the Okazaki group conducted meticulous experimentations and discovered that short fragments of DNA were being synthesized in replicating cells. These fragments contained 1,000 to 2,000 base pairs. The group also found that such short fragments accumulated upon impairment of the function of DNA ligase, the enzyme that links together pieces of DNA. In comparison, when DNA ligase existed, long strands of DNA were generated from short fragments linked together by the ligase.

The Okazaki group’s research findings culminated in the proposal of the discontinuous growth model of replicating strands of DNA. The DNA replication on the lagging strand occurs via the formation of short DNA fragments that are consequently linked to each other. Rollin Hotchkiss named these short fragments of DNA “Okazaki pieces” in 1968 while attending a Cold Spring Harbor Symposium.

Reiji Okazaki died in his prime in August 1975. His wife, Tsuneko, might have quit her research if it had not been for a letter of support she received from Arthur Kornberg. Tsuneko had worked with Kornberg as a Fulbright fellow at Stanford University. Kornberg told her, “Never give up the research; the world is waiting for the outcome of your research at Nagoya.” Providentially, a neighbor volunteered to help look after her son and daughter.

The theory of discontinuous growth of replicating DNA steadily won approval. However, there continued to be a need to identify primers required to initiate the replication process on the lagging strand.

Dr. Tsuneko Okazaki and her associates persisted in studying the problem and identified the RNA primer responsible for replication. This RNA primer was the final piece of the puzzle, providing evidence for the model initially proposed by Tsuneko and Reiji Okazaki.

3) Now, what exactly are these Okazaki fragments, and how did he research these things along with this wife?

The Okazaki fragments are short pieces of single-stranded DNA molecules. These Okazaki segments are formed along the lagging DNA strand during its discontinuous DNA replication process. The Okazaki DNA fragments are about 1,000 to 2,000 nucleotides long in bacteria and about 100 to 200 bases in length within the eukaryotes. The Okazaki fragments are made during the initial stages of DNA synthesis, but only along the lagging strand. These DNA pieces participate in the so-called discontinuous or semi-continuous mode of DNA replication. The Okazaki fragments are quickly ligated to each other by a DNA ligase enzyme almost as soon as the DNA polymerase enzyme makes them. The new DNA strand formed by the joining of Okazaki fragments is called the lagging strand because of its relative slowness in being produced. In prokaryotes, the Okazaki fragments are made by DNA polymerase III. However, in eukaryotes, the Okazaki fragments are produced by DNA polymeraseδ (delta) along the lagging strand.

Tsuneko and Reiji Okazaki conducted their famous experiment as follows. First, they cultured Escherichia coli in radioactively labeled thymidine (T). The radioisotope was called tritium (hydrogen 3, denoted 3H). The [3H]-thymidine exposure to the bacteria was quick. Tsuneko and Reiji Okazaki briefly presented the radioactive thymidine for only 30 seconds. They had called this brief exposure a “pulse.” The radiation pulse permitted the bacteria to incorporate the labeled T nucleotide into newly synthesized DNA. The reason for the brief pulse-labeling stemmed from the fact that the Okazaki fragments would be caught at the initial stages of DNA replication before they were connected by DNA ligase.

After the pulse-labeling step occurred, Tsuneko and Reiji Okazaki transferred their radioactive bacteria to a culture medium lacking radioactive thymidine. The thymidine was unlabeled in the second stage. Tsuneko and Reiji Okazaki would refer to this new exposure as a “chase.” The chasing step permitted the Okazaki fragments to be ligated to each other and become part of the overall new DNA of the Escherichia coli genome. Together, the two stages were referred to as the “pulse-chase” experiment.

Then, Tsuneko and Reiji Okazaki prepared DNA from the pulse-chasing process, which was allowed to proceed at different time points. Hoping to catch the small Okazaki fragments, they used density gradient centrifugation. They found that the Escherichia coli had made a significant amount of short DNA fragments at initial time points. When allowed to proceed for a more extended time, the small pieces disappeared. Still, the radioactivity appeared in much larger amounts, indicating that the fragments had been connected.

File:DNA-Okazaki-Fragment-prelim.PNG

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Figure 72. Depiction of DNA replication with replication fork, strands, and Okazaki-fragments. a: template strands, b: leading strand, c: lagging strand, d: replication fork, e: primer, f: Okazaki fragment.

In Figure 72, the mechanism for DNA replication is shown. The diagram shows a replication fork because the leading and lagging strands look like the “tines” of a fork. In the top part, “c” denotes the lagging strand synthesis with the adjacent Okazaki fragments (“f”) undergoing the discontinuous mode of replication.

File:DNA replication en.svg

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Figure 73. DNA replication machinery to make a new DNA. This DNA synthesis system is paramount to all life as we know it.

Others have referred to the replication fork more as like a trombone-like system. Topoisomerase paves the way for the helicase to unwind the double-stranded DNA. The outcome is the formation of two single DNA strands. See Figure 73. The top part shows the lagging strand synthesis. The Okazaki fragments are made by DNA polymerase and connected by DNA ligase. As the replication fork unwinds further with the helicase enzyme’s help, the primase enzyme makes an RNA primer.

DNA polymerase is the primary enzyme that produces new DNA. Then single-stranded binding proteins stabilize the two strands. Next, the primase enzyme makes a short stretch of RNA, which serves as a primer. The RNA primer is needed to make DNA. Next, the DNA polymerase enzyme makes new DNA along both template strands, connecting the new DNA to the RNA primer. The strand of new DNA in the bottom of Figure 73 is the leading strand because it proceeds continuously along the template’s path.

Conversely, the new strand of DNA made along the template (top of Figure 73) is the lagging strand because it is produced somewhat more slowly. DNA polymerase will remove the RNA primers and fill in the gaps left behind. The DNA ligase will then covalently join the Okazaki fragments to produce an intact double-stranded molecule. Both leading and lagging strands will end up as complete double-stranded DNA molecules.

4) How did some of his discoveries enhance our understanding of the DNA replication process? Is this relevant today?

During the late 1950s and early 1960s, molecular biologists worked on the justification of daughter strand growth, in opposing directions, from a single replication point in vivo. The research by Tsuneko and Reiji Okazaki resulted in revolutionary discoveries in the field of molecular biology. Their perseverance has served as a model and inspiration for successive generations of investigators, who continue to test their hypotheses and seek the truth in many areas of scientific endeavor.

The work of Tsuneko and Reiji Okazaki solved a significant problem of their day. The trouble was that DNA polymerase worked only in a so-called 5’ → 3’ (pronounced 5-prime to a 3-prime) direction. After a lengthy search, no enzyme known to humankind had ever been discovered that worked in a 3’ → 5’ manner. This direction meant that for the lagging strand, the replication fork had to be opened further for DNA polymerase to move into the fork’s aperture. The DNA polymerase had to work its way along the template to make DNA in the required 5’ → 3’ direction.

Reiji Okazaki reasoned that the bacteria required time to unwind, move DNA polymerase into the fork’s crevice, make a short fragment, and start the process over again. The steps had to occur in a repeated manner until the replication was complete on the lagging strand. The result would be a collection of short fragments that had to be joined.

The work of the Okazaki’s solved the problem of the stubborn requirement for a 5’ → 3’ DNA polymerase. Thus, the new findings of the Okazaki’s meant that no longer was there a futile need to search for the elusive 3’ → 5’ DNA polymerase. Even today, no such 3’ → 5’ DNA polymerase has yet been found on Earth. Their work also made it possible to exploit the new mechanism for its presence in all other types of living organisms. Further, using DNA replication machinery, DNA-based chemotherapies could be invoked to help regulate desired gene expression systems.

Knowledge of a new method for lagging strand synthesis emerged—the discovery by the Okazaki’s permitted investigators to evaluate the molecular and cellular DNA-making machinery. The new work let molecular biologists study the systems for making DNA in prokaryotes, eukaryotes, and viruses.

The Okazaki work facilitated new developments for DNA sequencing and comparative analyses of new sequences. Molecular cloning of genes would be accelerated by new knowledge of DNA replication mechanisms. The replication work would become necessary for making mutations in various organisms to study the molecular functions of new proteins. In modern times, the genome-editing technology will effectively take advantage of Okazaki fragments when making newly edited DNA. It is anticipated that the mechanism of DNA replication using Okazaki fragments will continue to permit new advances in all aspects of molecular biology for millennia.

5) In closing, we have to echo the words of Viktor Frankl in one of his books, “We must be alert—alert in a twofold sense—since Dachau and Auschwitz, we know what man is capable of—and since Nagasaki and Hiroshima—we know what is at stake.” Sadly, Okazaki died while very young—apparently, he was exposed to radiation outside of Hiroshima during World War II. When exactly did he die, and what did he die of?

Okazaki was severely irradiated in Hiroshima when the first atomic bomb was dropped and, as a most likely result, contracted leukemia and died at 44 on August 1, 1975. While it is not clear what caused Okazaki’s cancer, it is widely known that DNA mutation can play an essential role in its development. Sadly, because many of the Hiroshima and Nagasaki atomic bombing survivors later suffered significantly higher rates of cancers, such as leukemia, the likelihood that the etiology of Okazaki’s cancer being attributable to radiation exposure cannot be definitively discounted. It is ironic that the machinery that Okazaki studied, the DNA replication machinery of the living cell, was the same type of machinery that could have been the target for radioactive exposure.

Radioactive agents, especially ionizing radiation, have been conclusively demonstrated to mutate DNA. If that DNA damage occurs at specific sites along an individual’s genome, such as in a protooncogene, or a cell growth checkpoint gene, then the likelihood for carcinogenesis is tantamount. We, human Earthlings, live in a constant background of radiation, about 50 to 100 individual counts of radioactive particles per minute. We have active DNA repair machinery, the same type that Okazaki manipulated in his laboratory, to fix most of the DNA damage. Sometimes a “hit” or two is just enough to set off a transformation process to convert a healthy cell into a tumorogenic one. In relatively rare cases, the tumorous tissue changes from a benign one and can become malignant to produce a carcinogenic situation.

In modern times, cancer remains a significant health problem. The prime causes of such malignancies range from infection, carcinogens, and heredity. Because the human body is a complicated structural cellular, tissue, and organ system, various cancer treatments are likewise complicated with highly diverse networks and molecular mechanisms. A monumental amount of time, energy, and money is expended in the scientific investigation of cancer. It will be many years before investigators get a handle on the perplexing challenge of cancer.

For further information on the famous Okazaki experiment, visit:

DNA replication video:

List of Okazaki publications:

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