The discovery of DNA ligase provides some classic examples of the development of enzyme assays (see this chapter’s Moment of Discovery). In the paper first reporting their detection of a DNA-joining activity, Lehman and colleagues described the use of partially purified enzyme extracts from E. coli that had this joining activity. For a DNA substrate, they used polydeoxyadenosine, poly(dA), along with polydeoxythymidine, poly(dT), the latter digested with micrococcal nuclease (which degrades DNA strands at random locations) until the average length of the poly(dT) strands was about 250 nucleotides. These DNA strands were then labeled at their 5′ ends with a ³²P-labeled phosphate group. When the labeled DNA was precipitated with HCl and filtered, the label remained with the precipitated DNA on the filter. The labeled DNA was then treated with an enzyme called alkaline phosphatase, which removes terminal phosphates from DNA; with this treatment, the label is freed from the DNA and washes through the filter.

In their experiments, Olivera and Lehman found that the poly(dT) strands were linked together, because the label became resistant to the alkaline phosphatase treatment. Some of their results are shown below, for a reaction containing 1 μmol (micromole) of labeled DNA ends.

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