DNA ligase is the enzyme that forms covalent phosphodiester bonds between adjacent nucleotides, joining restriction fragments or Okazaki fragments. In recombinant DNA technology, after restriction endonucleases cut DNA and the insert is placed into the vector, DNA ligase seals the nicks by connecting the 3′-OH of one fragment to the 5′-phosphate of the adjacent fragment, using ATP or NAD⁺ as a cofactor. Without ligase, the recombinant DNA molecule would not be covalently sealed.