Question

A biologist systematically removes each of the proteins involved in DNA replication to determine the effect each has on the process. In one experiment, after separating the strands of DNA, she sees many short DNA/RNA fragments as well as some long DNA pieces. Which of the following is most likely missing? (A) Helicase (B) DNA polymerase (C) DNA ligase (D) RNA primase

Short answer

The most likely missing protein is (C) DNA Ligase, as its absence would result in many short DNA fragments due to the inability to connect Okazaki fragments on the lagging strand, while still allowing for some long DNA pieces to form on the leading strand.

Step-by-step solution

Understanding the role of Helicase

Helicase is an enzyme that unwinds and separates the two strands of DNA during replication. If helicase was missing, the replication process would not even begin because the strands remain joined together, and no replication would occur.

Understanding the role of DNA Polymerase

DNA Polymerase is an enzyme that adds nucleotides to the growing DNA chain during replication. It starts at the RNA primer and synthesizes the new DNA strand in the 5' to 3' direction. This process ensures that the new strand is complementary to the original DNA strand. If DNA polymerase were missing, no new DNA strands would be synthesized, and no replication would occur.

Understanding the role of DNA Ligase

DNA Ligase is an enzyme that joins together the short DNA fragments, called Okazaki fragments, formed on the lagging strand during DNA replication. If DNA ligase were missing, the Okazaki fragments would not connect, and we would see many short DNA fragments.

Understanding the role of RNA Primase

RNA Primase is an enzyme that adds a short sequence of RNA nucleotides, called a primer, to the single-stranded DNA template. This primer allows DNA Polymerase to bind and start replicating the new DNA strand. If RNA Primase were missing, we would have many short DNA/RNA fragments but no long DNA pieces, as DNA polymerase would not have a starting point to begin replication.

Determine which protein is missing based on the observation

Given the observation of many short DNA/RNA fragments and some long DNA pieces, the most likely missing protein would be DNA ligase, as described in Step 3. The missing DNA ligase would prevent the Okazaki fragments from connecting on the lagging strand, resulting in many short DNA fragments but still allowing for some long DNA pieces to form on the leading strand. Therefore, the correct answer is: (C) DNA Ligase

Key concepts

DNA Ligase

DNA ligase plays a pivotal role in DNA replication. It is the molecular 'glue' that stitches together the Okazaki fragments on the lagging strand, forming a continuous DNA molecule. These short fragments are a consequence of the replication mechanism, which can only add nucleotides in one direction (5' to 3'). As such, while the leading strand is synthesized continuously, the lagging strand is generated in pieces.

During replication, DNA ligase acts by sealing the nicks in the sugar-phosphate backbone of the DNA. This enzyme catalyzes the formation of a phosphodiester bond between the 3' hydroxyl end of one nucleotide and the 5' phosphate end of another. Without DNA ligase, cells would accumulate broken strands of DNA, a scenario detrimental to cell survival and function. Its absence, as seen in the exercise, results in many short DNA fragments since the Okazaki fragments cannot be joined to form a complete strand.

RNA Primase

RNA primase is the 'initiator' in the DNA replication process. It synthesizes a short RNA primer, which serves as a starting point for DNA synthesis. The primer is necessary because DNA polymerases, the enzymes that construct the DNA strand, can only add nucleotides to an existing chain; they cannot start from scratch.

The RNA primer is made up of a few ribonucleotides, which are similar to DNA nucleotides but contain ribose sugar instead of deoxyribose and uracil base instead of thymine. After the primer is in place, DNA polymerase can attach and begin adding DNA nucleotides. This process is essential on both the leading and lagging strands, although it occurs only once at the start of the leading strand and multiple times at various points along the lagging strand to initiate each Okazaki fragment.

DNA Polymerase

DNA polymerase is the chief builder during DNA replication. This crucial enzyme has several functions, but its primary role is to add nucleotides to a growing DNA strand. It matches each incoming nucleotide with the complementary base on the template strand, synthesizing the new strand in a 5' to 3' direction, ensuring accuracy through a proofreading mechanism.

Different types of DNA polymerases are involved in DNA replication and repair, each playing specific functions. For instance, in eukaryotic cells, DNA polymerase alpha helps initiate DNA synthesis by adding a few nucleotide bases to the primer. DNA polymerase delta then takes over to elongate the new DNA strand on the leading and lagging strands.

Helicase

Helicase is often referred to as the 'unzipper' of the DNA helix. It is an essential enzyme that unwinds the double-stranded DNA, creating two single strands that serve as templates for replication. Helicase breaks the hydrogen bonds between base pairs, resulting in the formation of a replication fork - a Y-shaped region where new DNA strands will be synthesized.

In addition to its role in DNA replication, helicase is also involved in DNA repair and transcription, the process by which RNA is made from a DNA template. The unwinding action of helicase is a critical first step; without it, the entire DNA replication process would be stalled from the beginning, as DNA polymerase requires single-stranded DNA to initiate synthesis.