Synthesis and reactivity of intermediates formed in the T4 RNA ligase reaction.

The intermediate adenylated donor derivatives A(5')pp(5')dTp and A(5')pp(5')GpGpGp have been prepared from suitable phosphorylating reagents activated by 1-hydroxybenzotriazole. Phosphodiester bond formation between donor and acceptor oligonucleotides as catalyzed by T4 RNA ligase is shown to be more efficient when the adenylated form of the donor molecule is used.     

A novel DNA joining activity catalyzed by T4 DNA ligase.

The use of T4 and E. coli DNA ligases in genetic engineering technology is usually associated with nick-closing activity in double stranded DNA or ligation of 'sticky-ends' to produce recombinant DNA molecules. We describe in this communication the ability of T4 DNA ligase to catalyze intramolecular loop formation between annealed oligodeoxyribonucleotides wherein Watson-Crick base pairing is absent on one side of the ligation site. Enzyme concentration, loop size, substrate specificity, and base composition were explored in an effort to maximize yield. Amounts of T4 DNA ligase in large molar excess to DNA template and ligated product are necessary to achieve high yields.     

A deoxyribonucleic acid ligase from nuclei of rat liver. Purification and properties.

A DNA ligase has been extensively purified from nuclei of rat livers. The ligase seals single strand nicks in DNA with any of the four usual bases on either the 3 or 5 sides. It requires ATP and a divalent cation (Mg-2plus or Mn-2plus) for activity. At low Mg-2plus concentrations the activity is greatly stimulated by a variety of monovalent cations. Relatively small excesses of either monovalent or divalent cation above the amounts which give maximal activity lead to inhibition of activity. Poly(G) and poly(I) inhibit ligase activity; several other polyribonucleotides are not inhibitory. Low concentrations of inorganic pyrophosphate are inhibitory. The molecular weight of the ligase is estimated from gel filtration to be about 10 times 10-4.