Question

A geneticist has discovered a yeast cell, which encodes a DNA polymerase that may add nucleotides in both the \(5^{\prime}\) to \(3^{\prime}\) and \(3^{\prime}\) to \(5^{\prime}\) directions. Which of the following structures would this cell NOT likely generate during DNA replication? (A) RNA primers (B) Okazaki fragments (C) Replication fork (D) Nicked DNA by topoisomerases

Short answer

The yeast cell with a DNA polymerase that can add nucleotides in both the 5' to 3' and 3' to 5' directions would NOT likely generate (B) Okazaki fragments during DNA replication, as the bidirectional polymerase overcomes the directionality constraint that usually causes their synthesis on the lagging strand.

Step-by-step solution

Understand the process of DNA replication

During the normal process of DNA replication, the double-stranded DNA helix unwinds, and the two single strands serve as templates for synthesizing new complementary strands. DNA polymerases can only synthesize new strands in the 5' to 3' direction. In the leading strand, this occurs in a continuous manner, while in the lagging strand, it occurs in segments known as Okazaki fragments due to the 5' to 3' directionality constraint.

Explain the role of each structure in the options

(A) RNA primers: These are short RNA sequences that bind to the single-stranded DNA template and provide a free 3' end for the DNA polymerase to begin synthesizing the new strand. (B) Okazaki fragments: These are short DNA segments synthesized discontinuously on the lagging strand due to the directionality constraint of DNA polymerase. (C) Replication fork: This is the structure that forms when the double-stranded DNA helix unwinds and separates into single strands during replication. (D) Nicked DNA by topoisomerases: Topoisomerases are enzymes that temporarily cut one or both strands of the DNA helix to relieve tension generated during replication, resulting in nicked DNA.

Identify the structure that wouldn't form with bidirectional DNA polymerase

Since the DNA polymerase in this yeast cell is capable of adding nucleotides in both the 5' to 3' and 3' to 5' directions, it overcomes the directionality constraint that usually causes the synthesis of Okazaki fragments on the lagging strand. Therefore, if the DNA polymerase can synthesize in both directions, we would not observe the formation of Okazaki fragments during DNA replication. Thus, the correct answer is: (B) Okazaki fragments.

Key concepts

DNA polymerase

One of the key players in DNA replication is DNA polymerase. This is an enzyme responsible for assembling DNA molecules by synthesizing new strands based on a template strand. Normally, DNA polymerase works in the 5' to 3' direction, adding nucleotides to the growing DNA strand. This directionality is crucial because it ensures the new DNA strand is complementary to the template strand, maintaining the genetic code during cell division.

DNA polymerase is essential for both the leading and lagging strands. On the leading strand, it moves continuously towards the replication fork. However, on the lagging strand, due to the antiparallel structure of DNA, it works in short bursts, creating Okazaki fragments. This behavior is due to the traditional unidirectionality of DNA polymerase.

In this geneticist's observation, the yeast's unique DNA polymerase that synthesizes both 5' to 3' and 3' to 5' could technically eliminate the need for Okazaki fragments, simplifying replication on the lagging strand.

Okazaki fragments

During DNA replication, Okazaki fragments are short DNA segments synthesized discontinuously on the lagging strand. These fragments are named after the Japanese scientists Reiji and Tsuneko Okazaki, who first proposed their existence.

In typical DNA replication, DNA polymerase can only synthesize new DNA in the 5' to 3' direction. This restriction poses a challenge on the lagging strand, which runs in the opposite 3' to 5' direction relative to the replication fork. To overcome this, replication on the lagging strand occurs in small segments called Okazaki fragments. These fragments are later connected by the enzyme DNA ligase to create a continuous strand.

However, in the yeast cell described in the exercise, a DNA polymerase that operates in both directions would potentially replicate both strands continuously. This capability would negate the necessity for Okazaki fragments.

Replication fork

The replication fork is a crucial structure in the process of DNA replication. It is formed when the double-stranded DNA helix unwinds and separates into two single strands. This area is where active synthesis of new DNA strands occurs.

At the replication fork, multiple enzymes perform their essential functions:

• Helicase unwinds the DNA.
• Single-strand binding proteins stabilize the unwound DNA.
• Primase synthesizes RNA primers necessary for DNA polymerase to start replication.

The fork is dynamic, moving as replication progresses, with the leading strand being synthesized continuously and the lagging strand in fragments.

Understanding the role of the replication fork helps in appreciating the coordinated effort required by different enzymes, including DNA polymerase and supporting proteins, to ensure efficient and accurate DNA replication.

Topoisomerase

Topoisomerases are enzymes that play a critical role during DNA replication by managing DNA supercoiling and tension, which result from unwinding the DNA double helix.

As the replication fork advances, DNA ahead of the fork can become overwound, creating tension in the DNA helix. Topoisomerase enzymes mitigate this by creating temporary breaks in the DNA strands:

• Type I topoisomerases cleave one strand of DNA, allowing it to unwind and relieve tension.
• Type II topoisomerases break both strands and manage more complex topological issues.

These nicks or breaks are essential for unhindered replication process and they are quickly repaired to maintain DNA integrity.

Without topoisomerases, the replication machinery would become stalled, and replication could result in genomic instability. Therefore, they are indispensable during the replication process, ensuring the DNA structure is properly maintained and replicated faithfully.