ABSTRACT

The Escherichia coli Ada protein repairs methylphosphotriesters in DNA by direct, irreversible methyl transfer to one of its own cysteine residues. The methyl transfer process is autocatalyzed by coordination of the acceptor residue, Cys, to a tightly bound zinc ion. Kinetic data reveal a 4-fold reduction in the methylphosphotriester repair activity for the Cd(II) form of Ada versus the native Zn(II)-bound form, thus confirming a direct role for the metal in autocatalysis. Quantitative electrophoretic mobility shift assays reveal that the specific DNA affinity of the protein is increased 10-fold by transfer of a methyl group to Cys; the Cd(II) and the Zn(II) forms of the protein behave similarly in this respect. This methylation-sensitive stimulation of binding underlies the ability of Ada to activate inducibly the transcription of a methylation-dependent regulon. We conclude that the chemical properties of the bound metal influence the transition state for autocatalytic methyl transfer, but not the structure that ultimately results from this process.

FOOTNOTES

*

This work was supported by grants from the Chicago Community Trust (Searle Scholars Program) and the National Science Foundation (Presidential Young Investigator Award Program) (to G. L. V.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Howard Hughes Medical Institute Predoctoral Fellow.

¶

To whom correspondence and reprint requests should be addressed.

()

The abbreviations used are: MeP, methylphosphotriester; N-Ada10, Ada fragment containing residues 1-92; N-Ada20, Ada fragment containing residues 1-178; N-Ada17, Ada fragment containing residues 1-153; S-Me-CysN-Ada, Cys-methylated N terminus of Ada; FPLC, fast protein liquid chromatography.

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