The mechanism behind the given reaction is the formation of the enzyme-AMP complex. The AMP supports this complex formation. The gel electrophoresis technique will help to determine whether the supercoiling in the DNA has in fact been relaxed.
a) In a three-step process, the ATP-dependent DNA ligases link single-stranded breaks (nicks) in the phosphodiester backbone of double-stranded DNA. The creation of a covalent enzyme-AMP complex (intermediate) is the initial stage in the ligation reaction.
A highly conserved lysine residue in the ligase's active region is covalently linked to AMP when the cofactor ATP is broken into pyrophosphate and AMP. The 5' phosphate of the nick receives the activated AMP residue, after which the nick is sealed by the formation of a phosphodiester bond and the removal of AMP.
b) The gel electrophoresis technique helps analyze the DNA. This technique uses an electric field to separate the DNA fragments based on size and charge. Supercoiled DNA moves faster than relaxed or open circular DNA in gel electrophoresis. And relaxed DNA migrates slowly and is found closer to the well. This way, we will find whether the DNA is relaxed or supercoiled.
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