Distribution of mutations in human thymidylate synthase yielding resistance to 5-fluorodeoxyuridine.
Thymidylate synthase (TS) catalyzes methylation of dUMP to dTMP and is the target of cancer chemotherapeutic agents (e.g. 5-fluorouracil). Here, we used error-prone PCR to mutagenize the full-length human TS cDNA and then selected mutants resistant to 5-fluorodeoxyuridine in a bacterial complementation system. We found that resistant mutants contained 1-5 amino acid substitutions and that these substitutions were located along the entire length of the polypeptide. Mutations were frequent near the active site Cys(195) and in the catalytically important Arg(50) loop; however, many mutations were also distributed throughout the remainder of the cDNA. Mutants containing a single amino acid replacement identified the following 14 residues as unreported sites of resistance: Glu(23), Thr(51), Thr(53), Val(84), Lys(93), Asp(110), Asp(116), Pro(194), Ser(206), Met(219), His(250), Asp(254), Tyr(258), and Lys(284). Many of these residues are distant from the active site and/or have no documented function in catalysis or resistance. We conclude that mutations distributed throughout the linear sequence and three-dimensional structure of human TS can confer resistance to 5-fluorodeoxyuridine. Our findings imply that long range interactions within proteins affect catalysis at the active site and that mutations at a distance can yield variant proteins with desired properties.
Kawate
Hisaya
H
The Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology, University of Washington, Seattle, Washington 98195-7705, USA.
Landis
Daniel M
DM
Loeb
Lawrence A
LA
D000595:Amino Acid Sequence; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D001120:Arginine / Q000737:chemistry; D001224:Aspartic Acid / Q000378:metabolism; D001665:Binding Sites; D002460:Cell Line; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm; D004926:Escherichia coli / Q000378:metabolism; D005467:Floxuridine / Q000494:pharmacology*; D015723:Gene Library; D006801:Humans; D007700:Kinetics; D008957:Models, Genetic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D013940:Thymidylate Synthase / Q000235:genetics*
D000964:Antimetabolites, Antineoplastic; D018076:DNA, Complementary; D005467:Floxuridine; D001224:Aspartic Acid; D001120:Arginine; D013940:Thymidylate Synthase; D003545:Cysteine