by Xinyue Xu
Credits: Nagoya University
Okazaki Fragments are short lengths of DNA produced by the discontinuous replication of the lagging strand in DNA. Okazaki fragments form because DNA polymerase only works in the 5’-to-3’ direction, attaching the 5th carbon of the new nucleotide to the 3rd carbon of the last, so the two DNA strands, that run in opposite directions, are replicated in different directions. One strand - the leading strand - undergoes continuous replication as the template strand runs from the 3’-to-5’ direction. In contrast, the other undergoes discontinuous replication - the lagging strand-forming little fragments of DNA which are later joined together by DNA ligase.
Logically, this doesn’t make sense. Surely, it would be easier to assume that DNA polymerase works in both directions and if you were a scientist back in 1966, this assumption would be commonly accepted. Although at the time “all known DNA polymerases could elongate the DNA chain only in the 5′-to-3′ direction, the mechanism of the apparent 3′-to-5′ elongation of one of the two daughter chains was an enigma”.[1]
In the spring of 1963, married couple Reiji Okazaki and Tsuneko Okazaki returned to Japan after Reiji Okazaki had accepted an associate professor position at Nagoya University. Both of them had graduated from Nagoya University previously, with Tsunkeo Okazaki receiving a Ph.D. in Biology. They decided to investigate the “paradox of the directions of DNA chain elongation” [1] as part of their research. There two different models were formed: one, that the two chains were simultaneously replicated with one being in the 5’-to-3’ direction and the other being in the 3’-to-5’ direction or two, that the two stands were duplicated in the 5’-to-3’ direction, with one being replicated in fragments, rather than a long continuous strand. In 1967, Tsuneko Okazaki and Toru Owaga suggested that due to the lack of evidence for the existence of an enzyme that catalyzes DNA elongation in the 3’-to-5’ direction, hypothesised instead that there was a way in which the fragments synthesised could be attached to the 5’-to-3’ to the strand. [2]
Later on in the year, Reiji Okazaki, Tunekuo Okazaki, Kiwako Sakabe, Kazunari Sugimoto and Akio Sugino pulse-labelled with the radioactive compound 3H-thymidine newly replicated Escherichia coli chromosomes, in conditions that reduced the growth and replication of the E.coli cells. The E.coli cells were cultivated for generations, before being exposed to 3H-thymidine for 10 seconds, ensuring only the growing ends of the DNA were exposed to the 3H-thymidine. As 3H-thymidine is a radioactive compound, the newly synthesised DNA let out a “pulse” that allowed the team to monitor them. The majority of the newly replicated 3H-DNA first appeared as short discontinuous fragments, before becoming long stands of 3H-DNA. This discovery meant that the DNA replication process was most likely discontinuous. [3] The discovery of polynucleotide ligase, an enzyme which joins short DNA strands together into a continuous strand of DNA, in the same year further supported the Okazaki’s hypothesis.
The team continued doing experiments, further providing evidence of the DNA replication process and the existence of DNA fragments, later named Okazaki fragments. It’s thanks to Tunekuo Okazaki and her husband’s work that we have such an extensive knowledge of DNA replication as we do now.
Citations
[1] Okazaki T. Days weaving the lagging strand synthesis of DNA—A personal recollection of the discovery of Okazaki fragments and studies on discontinuous replication mechanism—. Proceedings of the Japan Academy, Series B. 2017 May 11;93(5):322-38.
[2]Ogawa T, Okazaki T. Discontinuous DNA replication. Annual review of biochemistry. 1980 Jul;49(1):421-57.
[3]Okazaki R, Okazaki T, Sakabe K, Sugimoto K, Sugino A. Mechanism of DNA chain growth. I. Possible discontinuity and unusual secondary structure of newly synthesized chains. Proceedings of the National Academy of Sciences. 1968 Feb;59(2):598-605.
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