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Science 29 November 1974:
Vol. 186. no. 4166, pp. 790 - 797
DOI: 10.1126/science.186.4166.790
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Articles

DNA Ligase: Structure, Mechanism, and Function

I. R. Lehnman 1

1 Department of Biochemistry at the Stanford University School of Medicine. Stanford, California 94305

DNA ligase of E. coli is a polypeptide of molecular weight 75,000. The comparable T4-induced enzyme is somewhat smaller (63,000 to 68,000). Both enzymes catalyze the synthesis of phosphodiester bonds between adjacent 5'-phosphoryl and 3'-hydroxyl groups in nicked duplex DNA, coupled to the cleavage of the pyrophosphate bond of DPN (E. coli) or ATP (T4). Phosphodiester bond synthesis catalyzed by both enzymes occurs in a series of these discrete steps and involves the participation of two covalent intermediates (Fig. 1). A steady state kinetic analysis of the reaction-catalyzed E. coli ligase supports this mechanism, and further demonstrates that enzyme-adenylate and DNA-adenylate are kinetically significant intermediates on the direct path of phosphodiester bond synthesis.

A strain of E. coli with a mutation in the structural gene for DNA ligase which results in the synthesis of an abnormally thermolabile enzyme is inviable at 42°C. Although able to grow at 30°C, the mutant is still defective at this temperature in its ability to repair damage to its DNA caused by ultraviolet irradiation and by alkylating agents. At 42°C, all the newly replicated DNA is in the form of short 10S "Okazaki fragments," an indication that the reason for the mutant's failure to survive under these conditions is its inability to sustain the ligation step that is essential for the discontinuous synthesis of the E. coli chromosome. DNA ligase is therefore an essential enzyme required for normal DNA replication and repair in E. coli. Purified DNA ligases have proved to be useful reagents in the construction in vitro of recombinant DNA molecules.


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