Figure 4.
Increase in proofreading activity of Tne DNA polymerase Arg722Lys mutant. (A) The substrate used in the assay. The sequence recognized by BglII is underlined and the symbol * indicates the 32P-label at the 5′-end of the primer. The arrow indicates the cleavage position of BglII in the template. (B) An autoradiograph of the reaction products. In this experiment, mutants D137A and D137A/R722K were used. The amounts of DNA polymerase used in the reactions were 2.0 U for lanes 1, 4, 7 and 10, 1.0 U for lanes 2, 5, 8 and 11 and 0.5 U for lanes 3, 6, 9 and 12, respectively. Ten units of BglII were added to the reaction (lanes 4–6 and 10–12). EP and CP indicate the extended primer and cleavage products, respectively. The unextended primer is shown in lane 0. Because the Tne DNA polymerase was not removed from the extension reactions before incubating with BglII, the cleaved products were extended by 4 nt (indicated by CP) by the DNA polymerases at 37°C.