ACTIVE-SITE-DIRECTED DOUBLE MUTANTS OF DIHYDROFOLATE-REDUCTASE

Variants of dihydrofolate reductase (DHFR), which confer resistance to antifolates, are used as dominant selectable markers in vitro and in vivo and may be useful in the context of gene therapy, To identify imp roved mutant human DHFRs with increased catalytic efficiency and decre ased binding to methotrexate, we constructed by site-directed mutagene sis four variants with substitutions at both Leu(22) and Phe(31) (i.e. , Phe(22)-Ser(31), Tyr(22)-Ser(31), Phe(22)-Gly(31), and Tyr(22)-Gly(3 1)). Antifolate resistance has been observed previously when individua l changes are made at these active-site residues, Substrate and antifo late binding properties of these ''double'' mutants revealed that each have greatly diminished affinity for antifolates (>10,000-fold) yet o nly slightly reduced substrate affinity, Comparison of in vitro measur ed properties with those of single-residue variants indicates that dou ble mutants are indeed significantly superior, This was verified for o ne of the double mutants that provided high-level methotrexate resista nce following retrovirus-mediated gene transfer in NIH3T3 cells.