after a restriction enzyme cuts a DNA fragment we are left with two fragments of double stranded DNA which can be glued back together by a single molecule of T4 DNA ligase, right? in that case it will have to form two phosphodiester bonds , will it require the transfer of two phosphates? one for each bond ? and will the enzyme need to be adenylated twice for one reaction?
The phosphate remains on the DNA molecule after cleavage of the phosphodiester backbone by a restriction enzyme. DNA ligase reforms the phosphodiester bond, but the ATP does not contribute phosphate to the bond. The reaction has 3 steps.
1. DNA ligase reacts with ATP, forming a covalent adenylated-enzyme intermediate and releasing inorganic pyrophosphate (PPi).
2. The adenylate (AMP) is transferred to the phosphate group at the nick site on the DNA, resulting in adenylated DNA.
3. The phosphodiester bond is created, sealing the nick, and releasing the AMP.
In the case of a restriction site, there is a nick on each strand, so this reaction must occur twice.
Along with T4 DNA ligase, we use T4 DNA ligase buffer, which contains everything required for the ligation process. So, there is no need to add anything else.
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