Okazaki Fragments - Experiments

Experiments

The work of Kiwako Sakabe and Reiji Okazaki provided experimental evidence supporting the hypothesis that DNA replication is a discontinuous process. Previously, it was commonly accepted that replication was continuous in both the 3’ to 5’ and 5’ to 3’ directions. 3’ and 5’ are specifically numbered carbons on the deoxyribose ring in nucleic acids, and refer to the orientation or directionality of a strand. In 1967, the Okazakis and their colleagues suggested that there is no found mechanism that showed continuous replication in the 3’ to 5’ direction, only 5’ to 3’ using DNA polymerase, a replication enzyme. The team hypothesized that if discontinuous replication was used, short strands of DNA, synthesized at the replicating point, could be attached in the 5’ to 3’ direction to the older strand.

To distinguish the method of replication used by DNA experimentally, the team pulse-labeled newly replicated areas of Escherichia coli chromosomes, denatured, and extracted the DNA. A large amount of radioactive short units meant that the replication method was likely discontinuous. The hypothesis was further supported by the discovery of polynucleotide ligase, an enzyme that links short DNA strands together.

In 1968, the Okazakis gathered additional evidence of nascent DNA strands. They hypothesized that if discontinuous replication, involving short DNA chains linked together by polynucleotide ligase, is the mechanism used in DNA synthesis, then “newly synthesized short DNA chains would accumulate in the cell under conditions where the function of ligase is temporarily impaired.” E.coli were infected with Bacteriophage T4 that produce temperature-sensitive polynucleotide ligase. The cells infected with the T4 Phages accumulated a large amount of short, newly synthesized DNA chains, as predicted in the hypothesis, when exposed to high temperatures. This experiment further supported the Okazakis’ hypothesis of discontinuous replication and linkage by polynucleotide ligase. It disproved the notion that short chains were produced during the extraction process as well.

The Okazakis’ experiments provided extensive information on the replication process of DNA and the existence of short, newly synthesized DNA chains that later became known as Okazaki fragments.

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