Fig. 3.
Expression of hTOP2βK600T, hTOP2βK646E, and hTOP2βR757W is not viable in rad52-deficient yeast strains. (A-B) The hTOP2β expression vector pKN28 was generated to facilitate introduction of point mutations into constructs that express hTOP2β from the TPI1 promoter. pKN28 with wild-type hTOP2β and pKN28 expressing either hTOP2βK600T or hTOP2βR757W were transformed into JN362a (RAD52+) or JN394 (rad52−) strains. While transformation efficiencies were similar in RAD52+ strains, a greatly reduced number of colonies in the rad52− strain were seen with pKN28 hTOP2β wild type, and no colonies were seen with pKN28 hTOP2βK600T or hTOP2βR757W. (C, D) A similar analysis was carried out with pKN27 encoding hTOP2βK646E and hTOP2βR757W, demonstrating that these alleles also could not be introduced into rad52− strains. All transformation plates were incubated at 30 °C for 3 d. The plates were then photographed by conventional photography.