Question

Suppose you are studying a DNA repair system, such as the nucleotide excision repair in vitro. By mistake, you add DNA ligase from a tube that has already expired. What would be the result?

Short answer

Answer: Using expired DNA ligase in a nucleotide excision repair system in vitro could lead to incomplete or erroneous ligation of the repaired DNA segment. This may result in further DNA damage, interference with normal cellular processes like replication or transcription, and potentially harmful consequences for the cell.

Step-by-step solution

Understand nucleotide excision repair

Nucleotide excision repair (NER) is a DNA repair system that identifies and repairs damaged DNA by removing the damaged piece of DNA and then synthesizing a new, correct piece of DNA that replaces it. The process consists of several steps, including damage recognition, strand opening, damaged strand removal, DNA synthesis, and ligation.

Understand the role of DNA ligase

DNA ligase is an enzyme that plays a crucial role in the ligation step of the NER process, where it facilitates the joining of DNA strands (in this case, the repair patch) by forming a phosphodiester bond between the 3’-hydroxyl end of one nucleotide and the 5’-phosphate end of another nucleotide. Thus, DNA ligase ensures the continuity and stability of the newly synthesized DNA.

Analyze the effect of using expired DNA ligase

If the DNA ligase used in the nucleotide excision repair system in vitro is expired, it might lose its enzymatic activity or functions due to denaturation or degradation. This could potentially lead to incomplete or erroneous ligation of the repaired DNA strand.

Determine the consequences of incomplete or erroneous ligation

The consequences of an incomplete or erroneous ligation of the repaired DNA segment may lead to the DNA strand remaining broken or having errors in the repaired region. This might result in further DNA damage, which could propagate as mutations or interfere with normal cellular processes like replication or transcription, ultimately causing the repaired DNA sequence to be nonfunctional or potentially harmful to the cell.

Conclusion

In conclusion, adding expired DNA ligase in the nucleotide excision repair in vitro system would likely lead to incomplete or erroneous ligation of the repaired DNA segment. This can cause further DNA damage, affect normal cellular processes, and potentially have harmful consequences for the cell.

Key concepts

Nucleotide Excision Repair

Nucleotide excision repair (NER) is a vital DNA repair mechanism found in cells. Its primary function is to identify and correct lesions or abnormalities in DNA that can affect genetic stability. This repair process is initiated when specific proteins detect damaged DNA, often caused by UV light or chemical exposure. Once damage is identified, the DNA double helix is unwound to create a single-strand gap. Enzymes then remove the segment of DNA containing the error, creating a template for repair. This complex process ensures that inaccuracies can be fixed to maintain healthy cellular function.

NER involves multiple steps, each executed by specialized proteins:

• Damage Recognition: Proteins scan and identify irregularities in the DNA.
• Strand Opening: Enzymatic activities separate the DNA strands, creating space for repair.
• Excision: The damaged section is cut and removed.
• DNA Synthesis: A fresh DNA segment is synthesized using the remaining strand as a template.
• Ligation: The newly synthesized DNA is joined to the existing strand, restoring DNA integrity.

This repair system is highly conserved across species, highlighting its importance in preserving genetic information.

DNA Ligase

DNA ligase is a crucial enzyme in the process of DNA repair and replication. It specializes in joining DNA strands by forming phosphodiester bonds between nucleotides. This bonding is essential in overcoming breaks or nicks in the DNA backbone, ensuring that the integrity of the DNA double helix is maintained.

In the context of nucleotide excision repair, DNA ligase is responsible for sealing the newly synthesized DNA patch onto the older DNA strand after the damaged section has been removed and replaced. Without the activity of DNA ligase, the newly repaired DNA strand would remain incomplete or unstable, thus leaving the cell susceptible to further genetic errors.

If DNA ligase is inactive or improperly functioning, such as when using an expired sample, it might lead to:

• Incomplete DNA repair, resulting in persistent DNA nicks.
• Increased vulnerability to genetic mutations.
• Disruption of critical cellular processes like DNA replication and transcription.

Ensuring active DNA ligase is pivotal for effective DNA repair and cellular health.

DNA Synthesis

DNA synthesis is a key step in the DNA repair process. It occurs after the damaged DNA segment is excised and before the ligation step. During synthesis, DNA polymerase enzymes create a new DNA strand that complements the existing template strand. This newly synthesized DNA precisely replaces the erroneous or damaged sections removed during the excision step.

In nucleotide excision repair, DNA synthesis ensures that the genetic information remains unaltered and correctly restored. This step is highly dependent on the accuracy of the DNA polymerases involved, whose job is to match nucleotides accurately according to the base-pairing rules: Adenine (A) pairs with Thymine (T), and Cytosine (C) with Guanine (G).

Effective DNA synthesis is crucial because:

• It restores the genetic code with precision.
• Prevents mutations from propagating through cellular divisions.
• Maintains genetic stability across generations of cells.

Ensuring complete and accurate DNA synthesis is essential to preserve the fidelity of genetic information.

Enzyme Activity

Enzyme activity is fundamental to the functionality of DNA repair systems like nucleotide excision repair. Enzymes are biological catalysts that speed up chemical reactions. They are crucial in various steps of DNA repair, including damage recognition, excision, synthesis, and ligation.

In NER, each step relies on specific enzyme activities:

• Endonucleases act to excise damaged DNA sections.
• DNA polymerases facilitate the synthesis of new DNA strands.
• DNA ligase seals the newly synthesized DNA to the existing strand.

Each of these enzymes must be in optimal condition to effectively function in the repair process. If an enzyme, like DNA ligase, loses activity due to being expired or denatured, the entire repair procedure can be compromised.

This can have critical implications:

• Incomplete repair can lead to cellular damage and mutation.
• Proper enzyme activity is essential to maintaining genome stability.
• Monitoring enzyme conditions can prevent faults in the DNA repair mechanism.

In summary, enzyme activity is integral to every aspect of DNA repair, ensuring cellular resilience and genetic fidelity.