Enzymatic Breakage and Joining of Deoxyribonucleic Acid: IX. SYNTHESIS AND PROPERTIES OF THE DEOXYRIBONUCLEIC ACID ADENYLATE IN THE PHAGE T4 LIGASE REACTION

Abstract

Abstract Polynucleotide ligase purified from Escherichia coli infected with bacteriophage T4 reacts with ATP to form a ligase-AMP complex which can be isolated by gel filtration. When this ligase-AMP complex is incubated with DNA containing single strand breaks (nicks), there is a transfer of the AMP from the enzyme to the 5'-phosphoryl terminus of the nick to form a pyrophosphate bond (AppDNA). If the reaction is carried out at 0° and at pH 5.6, AppDNA is accumulated. The AppDNA has been isolated and the character of the linkage of AMP to DNA has been elucidated by a number of physical and enzymatic studies. The chemical properties of this enzymatically synthesized AppDNA are identical with those of AppT(pT)n in which AMP has been chemically linked by a pyrophosphate bond to the 5'-terminal phosphate of an oligomer of thymidylic acid. App[poly(N)], whether synthesized by enzymatic or chemical means, serves as an intermediate in the reaction which is catalyzed by the T4 ligase, and in which phosphodiester bond formation is accompanied by stoichiometric release of adenosine 5'-phosphate. The reaction occurs in the absence of ATP and is specific for nicks in duplex polynucleotides. If the enzyme is first incubated with ATP, the reaction is strongly inhibited. A temperature-sensitive polynucleotide ligase purified from E. coli cells infected with a mutant of T4 bacteriophage is unable to carry out either this reaction or the over-all joining reaction at elevated temperatures. When dAMP is substituted for AMP in the chemically synthesized polynucleotide intermediate, joining occurs at 8% the normal rate. No joining is detected if GMP is used as the activating species. The rates of joining of d(pT)7, d(pT)12, AppT(pT)6, and AppT(pT)11 in the presence of poly d(A) have been determined at various temperatures, and provide a sensitive method for the analysis of secondary structure of polynucleotides.