Recombinant DNA technology: Which enzyme catalyzes formation of covalent phosphodiester bonds to join compatible restriction fragments?

Introduction:After restriction enzymes generate compatible DNA ends, the fragments must be covalently joined to create a continuous backbone. The enzyme responsible for sealing the nick between adjacent nucleotides is a mainstay of cloning workflows. This question asks you to identify that enzyme—DNA ligase.

Given Data / Assumptions:

• Restriction digestion produces either sticky (cohesive) or blunt ends.
• Annealing of sticky ends relies on base pairing but remains a nicked backbone until sealed.
• Cloning requires stable covalent joins for propagation in cells.

Concept / Approach:DNA ligase catalyzes formation of a phosphodiester bond between adjacent 3′-OH and 5′-phosphate termini. T4 DNA ligase uses ATP, whereas E. coli DNA ligase uses NAD+. Ligation can join both cohesive and blunt ends (the latter typically at lower efficiency), enabling insert–vector assembly in plasmids and other constructs.

Step-by-Step Solution:

Verification / Alternative check:Successful ligation yields transformants with intact plasmids; control reactions lacking ligase fail to produce stable constructs, demonstrating the necessity of covalent sealing.

Why Other Options Are Wrong:

• Primase synthesizes RNA primers; helicase unwinds duplexes; polymerase extends DNA but does not seal nicks; topoisomerase alters supercoiling and can transiently break/rejoin strands but is not used for cloning ligation.

Common Pitfalls:Assuming annealed sticky ends are permanently joined without ligase; base pairing alone is insufficient for stable propagation.

Final Answer:DNA ligase