Question

Function of DNA Ligase Some \(E\). coli mutants contain defective DNA ligase. When researchers expose these mutants to \({ }^{3} \mathrm{H}\)-labeled thymine and then sediment the DNA produced on an alkaline sucrose density gradient, two radioactive bands appear. One corresponds to a high molecular weight fraction, the other to a low molecular weight fraction. Explain.

Short answer

Defective DNA ligase in mutants leads to incomplete ligation, resulting in both high and low molecular weight DNA fragments in the density gradient.

Step-by-step solution

Understanding DNA Ligase Function

DNA ligase is an enzyme that facilitates the joining of DNA strands together by catalyzing the formation of a phosphodiester bond. It is crucial for completing the process of DNA replication by joining Okazaki fragments on the lagging strand and sealing nicks in DNA. In mutants with defective DNA ligase, these joining processes are impaired.

Sedimentation in Alkaline Sucrose Density Gradient

When DNA is exposed to an alkaline sucrose density gradient, it is separated based on molecular weight and sedimentation velocity. High molecular weight DNA strands sediment faster and form a distinct band, while lower molecular weight DNA forms a lighter band that sediments more slowly.

Analyzing the Appearance of Radioactive Bands

In the case of the described E. coli mutants, the two radioactive bands observed represent different fractions of DNA. The high molecular weight band correlates with complete DNA strands that include joined Okazaki fragments, despite some defects. The low molecular weight band corresponds to single, unjoined Okazaki fragments due to the mutants' inability to effectively ligate DNA strands together.

Interpretation of Results

The presence of a low molecular weight radioactive band indicates that, in the absence of functional DNA ligase, DNA replication is incomplete, resulting in the accumulation of unligated Okazaki fragments. This is why there are two distinct bands: one from the fragmented and another from the joined DNA pieces.

Key concepts

DNA replication

DNA replication is the fundamental process of copying a cell's DNA to ensure that each new cell has a complete set of genetic information. This is essential for cell division and growth. DNA replication takes place in several steps:

• Initiation: Starts at specific sequences called origins of replication.
• Elongation: New strands of DNA are synthesized by DNA polymerases.
• Termination: Process ends when the entire DNA molecule has been replicated.

Special enzyme activities ensure that replication is both accurate and efficient. DNA polymerases are essential as they add nucleotides to form new DNA strands. However, they can only add bases in one direction, which leads to the formation of Okazaki fragments on the lagging strand.

Okazaki fragments

Okazaki fragments are short sequences of DNA nucleotides synthesized discontinuously and later linked together during DNA replication. These fragments form on the lagging strand, which is synthesized in the opposite direction of the replication fork's movement. This backward directionality necessitates periodic priming and synthesis of new fragments:

• Each Okazaki fragment begins with an RNA primer synthesized by primase.
• DNA polymerase extends the fragment until it reaches the previous primer.
• After which, RNA primers are removed and replaced with DNA.

Finally, DNA ligase seals the gaps between adjacent fragments, forming a continuous DNA strand. Mutations affecting ligase can lead to incomplete or abnormal DNA replication.

Escherichia coli mutants

Escherichia coli, commonly known as E. coli, is a bacterium often used in scientific research due to its relative simplicity and prevalence. In experiments with E. coli mutants, especially those with defective enzymes such as DNA ligase, researchers can observe how important these proteins are for cellular processes. If the mutants contain defective DNA ligase, crucial steps in DNA replication can be disrupted.

• Inability to effectively join Okazaki fragments.
• Accumulation of gaps within the DNA sequence.
• Potential errors in DNA synthesis and repair processes.

These mutants can thus provide insight into both the normal function of DNA replication enzymes and the effects of their failure.

Phosphodiester bond formation

Phosphodiester bonds are the chemical linkages between the sugar-phosphate backbone of DNA and RNA strands. They are essential for maintaining the structural integrity of nucleic acid molecules. During DNA replication, DNA ligase catalyzes the formation of these bonds to link the 3' hydroxyl end of one nucleotide with the 5' phosphate end of another. This process is crucial for sealing nicks left after Okazaki fragments are synthesized.

• Without these bonds, DNA strands would not remain intact.
• They ensure continuity and stability of the DNA molecule.
• Defective phosphodiester bond formation can lead to unstable genetic material.

Thus, mutations that impair ligase function can result in incomplete DNA replication and a buildup of fragmented DNA, as observed in certain E. coli mutants.