Myron Goodman and colleagues discovered the E. coli DNA polymerase V, the first example of a translesion DNA polymerase, using a reconstituted system including Pol III, the UmuD′ and UmuC proteins, and RecA protein. A control experiment was carried out in which Pol III was left out of the reaction mixture, and translesion DNA synthesis still occurred. This was the first suggestion that UmuC and UmuD′ might have DNA polymerase activity, but more work was needed to prove it.

Goodman and colleagues cultured E. coli cells that expressed Pol V, using a strain with mutations giving rise to both an inactivated Pol II and a temperature-sensitive Pol III.

The researchers partially purified Pol V and then carried out a gel filtration experiment to separate Pol III from Pol V. Their results are shown in Figure 1, a Western blot analysis of the fractions from the gel filtration column, using antibodies to the α subunit of Pol III and to UmuC.

Three fractions—50, 56, and 64—were chosen for further analysis, as shown in Figure 2. DNA polymerization activity was examined on a primer template (labeled P in the gel) in which the second position in the single-stranded template had an abasic site (X). The work was done at two temperatures, 37°C and 47°C.