Q.37 The schematic of a plasmid with a gap in one of the strands is shown below:
Which of the following enzyme(s) is/are required to fill the gap and generate a
covalently closed circular plasmid?
[P] DNA ligase
[Q] Alkaline phosphatase
[R] DNA polymerase
[S] Polynucletoide Kinase

Correct Answer

P (DNA Ligase) and R (DNA Polymerase) only

Introduction

In molecular biology, circular plasmids are widely used in cloning, DNA repair and recombinant technology.
When a gap occurs in one of the strands of a plasmid, specific enzymes are required to rebuild the missing section
and seal the strand to regenerate a covalently closed circular DNA molecule.

Problem Understanding

The plasmid shown contains:

• A single-stranded gap
• A free 3′-OH end
• A 5′-phosphate end

Two steps are needed for repair:

1. Filling the missing nucleotides
2. Sealing the phosphodiester bond to close the DNA strand

Explanation of Each Enzyme

[P] DNA Ligase

DNA ligase forms phosphodiester linkages and seals nicks in the DNA backbone.
Once DNA polymerase fills the gap, ligase completes the repair by joining the ends.

Conclusion: Required

[Q] Alkaline Phosphatase

This enzyme removes 5′ phosphate groups from DNA molecules.
Although useful in cloning to prevent self-ligation, removing the phosphate would prevent DNA ligase from working.

Conclusion: Not required

[R] DNA Polymerase

DNA polymerase fills the missing nucleotides using the 3′-OH end to extend the DNA strand.
Without this step, ligase cannot seal the gap.

Conclusion: Required

[S] Polynucleotide Kinase

Polynucleotide kinase adds phosphate to the 5′ end of DNA.
This is only needed when a phosphate is absent, such as when alkaline phosphatase has removed it.
Since the 5′ phosphate is already present in the plasmid, the enzyme is unnecessary.

Conclusion: Not required

Final Answer

Correct Option: P and R only

Conclusion

To produce a covalently closed circular plasmid from a DNA strand with a gap,
DNA polymerase fills the missing bases,
and DNA ligase seals the nick.
No additional enzymatic modification is necessary, making P and R the correct pair.