A trojan horse approach for silencing thymidylate synthase

Biochemistry
Deborah K WestFrank Maley

Abstract

In this paper we present a new and possibly more effective way of inhibiting thymidylate synthase (TS) in cells than through the use of substrate analogue inhibitors. An inactive double mutant of TS (DM), Arg(126)Glu/Cys(146)Trp, is shown to progressively impair the reactivation of native Escherichia coli TS when the two are denatured together in vitro. The individual single mutant proteins Arg(126)Glu and Cys(146)Trp showed little or no inhibition. When the DM is introduced into E. coli and induced from an expression plasmid, the mutant subunits act as a decoy in deceiving newly formed native TS subunits to fold with them to yield inactive heterodimers. As a consequence of the depletion of TS, the cells die a "thymineless" death when grown in medium devoid of thymine. Addition of thymine to the medium enables the cells to grow normally, although only very low levels of TS activity could be detected in those cells containing induced DM. The individual single-site mutations of the DM, Arg(126)Glu and Cys(146)Trp, did not inhibit growth, as might be expected from the in vitro studies. However, when a nontoxic level of 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) is added to growing DM-transformed cells, the combination is le...Continue Reading

Citations

Dec 1, 1988·Molecular and Cellular Endocrinology·R E Kramer
Dec 3, 1990·Molecular and Cellular Endocrinology·R E Kramer
Feb 3, 2007·Journal of Theoretical Biology·Marcio S de Queiroz, Grover L Waldrop
Jun 26, 2007·Protein Science : a Publication of the Protein Society·Ruth L SaxlFrank Maley

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