Dihydrofolate reductase: Translational regulation and effects of mutations on binding and catalytic efficiency

One mechanism of antifolate resistance observed in-vivo experiments in tumors has been mutations in the dihydrofolate reductase (DHFR) at leucine 22 (Leu22) or at phenylalanine 31 (Phe-31). We investigated the enzyme kinetic and antifolate inhibitory properties of human DHFR enzyme by constructing site specific mutations at both positions. Leu22 was changed to methionine (Met), isoleucine (Ile), phenylalanine (Phe), tyrosine (Tyr) and arginine (Arg) to generate the various mutant enzymes. Furthermore, Phe31 was mutated to serine (Ser) in Phe22 and Tyr22 variants resulting in double substitutions. The overall catalytic efficiency (;A second goal of this thesis was to study the translational regulation of DHFR. Previous studies have shown that cellular DHFR protein levels increase within hours following MTX exposure. This rapid induction was originally attributed either to the stabilization of DHFR protein bound to MTX or to an increase in the translation of DHFR mRNA. The studies presented show that in in-vitro DHFR protein regulates its own translation by feedback inhibition of translation, and that addition of MTX relieves this inhibition. A Chinese hamster cell line lacking the DHFR gene transfected with four different constructs of human DHFR gene was used as an in-vivo model to investigate the binding site of DHFR mRNA to its own protein. The results of these studies show that the binding region of RNA for its cognate enzyme is in the coding region. UV-crosslinking experiments confirm that the binding site is in the coding region.