Reiji and Tuneko Okazaki conducted a now classic experiment in 1968 in which they discovered a population of short fragments synthesized during DNA replication. They introduced a short pulse of ³H-thymidine into a culture of E. coli and extracted DNA from the cells at various intervals. In analyzing the DNA after centrifugation in denaturing gradients, they noticed that as the interval between the time of ³H-thymidine introduction and the time of centrifugation increased, the proportion of short strands decreased and more labeled DNA was found in larger strands. What would account for this observation?

Verified step by step guidance

1. Understand the context of the experiment: Reiji and Tuneko Okazaki were studying DNA replication in E. coli. They used ³H-thymidine, a radioactive form of thymidine, to label newly synthesized DNA strands. The goal was to observe the dynamics of DNA strand synthesis over time.
2. Recall the mechanism of DNA replication: DNA replication occurs in a semi-discontinuous manner. On the leading strand, synthesis is continuous, while on the lagging strand, synthesis occurs in short fragments called Okazaki fragments. These fragments are later joined together by DNA ligase.
3. Interpret the experimental observations: The short fragments observed immediately after the pulse of ³H-thymidine correspond to Okazaki fragments on the lagging strand. Over time, these fragments are joined into longer strands, which explains the decrease in the proportion of short fragments and the increase in labeled larger strands.
4. Explain the role of enzymes: DNA polymerase synthesizes the Okazaki fragments, and DNA ligase seals the gaps between them to form a continuous strand. The observed shift from short fragments to longer strands reflects the action of DNA ligase over time.
5. Conclude the significance of the experiment: The findings provided key evidence for the semi-discontinuous nature of DNA replication and the existence of Okazaki fragments, which are essential for lagging strand synthesis.

Key Concepts

DNA Replication

DNA replication is the biological process by which a cell duplicates its DNA before cell division. It involves the unwinding of the double helix and the synthesis of new strands using existing strands as templates. This process is crucial for genetic continuity and is facilitated by enzymes such as DNA polymerase, which adds nucleotides to form new strands.

Okazaki Fragments

Okazaki fragments are short sequences of DNA synthesized on the lagging strand during DNA replication. They are formed because DNA polymerase can only add nucleotides in a 5' to 3' direction, necessitating the synthesis of these fragments in short bursts as the replication fork opens. These fragments are later joined together by the enzyme DNA ligase to create a continuous strand.

Centrifugation and Denaturing Gradients

Centrifugation is a technique used to separate components of a mixture based on their density by spinning them at high speeds. In the context of DNA analysis, denaturing gradients can be employed to separate DNA fragments of different lengths. As DNA is subjected to varying conditions, shorter fragments migrate differently than longer ones, allowing researchers to analyze the size distribution of DNA synthesized during replication.