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NCERT tells you that on one template strand replication is continuous, on the other it is "discontinuous", and that "the discontinuously synthesised fragments are later joined by the enzyme DNA ligase" (NCERT Class 12 Biology, Chapter 5). Those fragments carry a family name. This post covers the puzzle Reiji and Tsuneko Okazaki set out to solve, the experiment that found the fragments, and the hard years that followed.
What was the puzzle?
The two strands of DNA run in opposite directions, and both are copied at the same moving fork. But every known DNA polymerase could only extend a chain in the 5' to 3' direction. Arthur Kornberg's enzyme, covered in our post on Kornberg and DNA polymerase, was the one everyone knew. So how could one strand appear to grow 3' to 5'?
Tsuneko Okazaki tells the story in her 2017 recollection in the Proceedings of the Japan Academy. There were two options. Either an unknown enzyme built DNA 3' to 5', or that strand was made from short pieces, each built 5' to 3' in the direction opposite to the fork's movement, then joined. Studies that saw continuous growth had used methods that only detected long chains, so short pieces could have been missed.
Who were the Okazakis?
Tsuneko was born in Aichi Prefecture in 1933 and entered Nagoya University in 1952, among the first generation of Japanese women educated alongside men at university, she writes. In 1956 she married Reiji Okazaki (1930 to 1975). Nagoya's buildings had burned in the war, laboratories were in a barrack, and they bought a fraction collector with their own pocket money.
As Fulbright fellows they worked in the United States from 1960, including 15 months in Kornberg's laboratory at Stanford. There they saw that the test-tube enzyme could not explain replication in living cells. In 1963 Reiji became an associate professor at Nagoya, and they made the direction paradox one of their new laboratory's research themes.
How did they catch the fragments?
The trick was a very short pulse of radioactive thymidine, so only the newest DNA was labelled. Their own estimate showed that at 37 °C the pulse would need to be under 0.1 seconds, far too short to handle. So they cooled the bacteria to 20 °C or lower, which slowed replication enough to label for several seconds.
Reiji's first graduate student, Ms. Sakabe, ran the low-temperature experiment in E. coli. Unexpectedly, the label went into short DNA pieces only 1,000 to 2,000 nucleotides long. When the pulse was followed by a "chase" of unlabelled thymidine, the label moved into long DNA. Short pieces were made first and joined later. They had these results in 1966, after three years of effort.
The group published in PNAS in February 1968. At the Cold Spring Harbor Symposium in June 1968, where Reiji presented the model, the short pieces were given the name Okazaki fragments.
How did they show ligase joins the pieces?
Critics suggested the fragments were broken bits of fragile DNA. The team repeated the experiments with many systems and extraction methods. They then used a mutant virus, T4 phage, whose DNA ligase stopped working at high temperature. At 43 °C, fragments piled up. When the infected cells were moved back to 30 °C, where the ligase worked again, the fragments were quickly joined into long chains.
Worked example: counting fragments and joints
Use the paper's fragment length of 1,000 to 2,000 nucleotides. Imagine a 10,000-nucleotide stretch of lagging strand. This is a sample length for practice.
With 2,000-nucleotide fragments: 10,000 ÷ 2,000 = 5 fragments.
With 1,000-nucleotide fragments: 10,000 ÷ 1,000 = 10 fragments.
Joints needed: one fewer than fragments, so 4 to 9 ligase reactions.
Each fragment also starts on a short RNA primer. Tsuneko writes that the primer turned out to be fewer than 10 nucleotides long, far shorter than first expected.
Common mistakes:
Saying the lagging strand is built 3' to 5'. Each fragment is built 5' to 3'; only the overall strand appears to grow the other way.
Saying polymerase joins the fragments. DNA ligase makes the final joining bond.
Forgetting the primer. DNA polymerase cannot start a chain on its own; NCERT says so too.
What happened next?
While the group hunted for the RNA primer, Reiji developed leukaemia. He had been in Hiroshima when the atomic bomb fell, and Tsuneko writes that he felt lucky to have lived 30 more years. He died on 1 August 1975, aged 44, "without knowing the nature of the RNA primer". Tsuneko carried the work on, defended the fragments against new doubts, and later became a professor at Nagoya. She received the L'Oréal-UNESCO Award for Women in Science in 2000. For an earlier replication experiment, read our post on Meselson and Stahl.
What can a student take from their way of working?
Change the conditions to see more. Cooling the cells made an impossibly fast event measurable.
Test the joiner. A ligase mutant turned a model into evidence.
Keep going. They did serious science in a barrack laboratory, and Tsuneko finished what they started.
If the replication fork is hard to picture, the Fermi Simulation Lab offers interactive simulations for ideas easier to see than read. Our guide to DNA replication and transcription covers the exam detail.
Next step: draw a replication fork, label the leading and lagging strands, and mark the 5' and 3' ends of three Okazaki fragments.
