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Active Site-directed Double Mutants of Dihydrofolate Reductase¹

Programs of Molecular Pharmacology and Therapeutics [E. A. E-A., S. M., S. N., M. C. W., D. B., J. R. B.] and Human Genetics [M. S.], Memorial Sloan-Kettering Cancer Center and Graduate School of Medical Sciences, Cornell University [Y. T.], New York, New York 10021

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 improved mutant human DHFRs with increased catalytic efficiency and decreased binding to methotrexate, we constructed by site-directed mutagenesis four variants with substitutions at both Leu²² and Phe³¹ (i.e., Phe²²-Ser³¹, Tyr²²-Ser³¹, Phe²²-Gly³¹, and Tyr²²-Gly³¹). Antifolate resistance has been observed previously when individual changes are made at these active-site residues. Substrate and antifolate binding properties of these "double" mutants revealed that each have greatly diminished affinity for antifolates (>10,000-fold) yet only slightly reduced substrate affinity. Comparison of in vitro measured properties with those of single-residue variants indicates that double mutants are indeed significantly superior. This was verified for one of the double mutants that provided high-level methotrexate resistance following retrovirus-mediated gene transfer in NIH3T3 cells.

¹ This work was supported in part by USPHS Grants CA59350 and P30 CA 08748-31.

² To whom requests for reprints should be addressed, at Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, Box 78, New York, NY 10021. E-mail: J-bertino@mskcc.org.

Received 6/ 3/96. Accepted 7/26/96.

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