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J. Biol. Chem., Vol. 279, Issue 26, 26876-26884, June 25, 2004

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The Escherichia coli 3-Methyladenine DNA Glycosylase AlkA Has a Remarkably Versatile Active Site^*

Patrick J. O'Brien and Tom Ellenberger

From the Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115

3-Methyladenine DNA glycosylase II (AlkA) from Escherichia coli is induced in response to DNA alkylation, and it protects cells from alkylated nucleobases by catalyzing their excision. In contrast to the highly specific 3-methyladenine DNA glycosylase I (E. coli TAG) that catalyzes the excision of 3-methyl adducts of adenosine and guanosine from DNA, AlkA catalyzes the excision of a wide variety of alkylated bases including N-3 and N-7 adducts of adenosine and guanosine and O² adducts of thymidine and cytidine. We have investigated how AlkA can recognize a diverse set of damaged bases by characterizing its discrimination between oligonucleotide substrates in vitro. Similar rate enhancements are observed for the excision of a structurally diverse set of substituted purine bases and of the normal purines adenine and guanine. These results are consistent with a remarkably indiscriminate active site and suggest that the rate of AlkA-catalyzed excision is dictated not by the catalytic recognition of a specific substrate but instead by the reactivity of the N-glycosidic bond of each substrate. Damaged bases with altered base pairing have a modest advantage, as mismatches are processed up to 400-fold faster than stable Watson-Crick base pairs. Nevertheless, AlkA does not effectively exclude undamaged DNA from its active site. The resulting deleterious excision of normal bases is expected to have a substantial cost associated with the expression of AlkA.

Received for publication, April 7, 2004 , and in revised form, May 3, 2004.

^* This work was supported by National Research Service Award Fellowship F32 GM65043 (to P. J. O.) and National Institutes of Health Grant R01 GM52504 (to T. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S5.

The Hsien Wu and Daisy Yen Wu Professor of Biochemistry at Harvard Medical School. To whom correspondence should be addressed: Dept. of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115. Tel.: 617-432-0458; Fax: 617-432-3380; E-mail: tome{at}hms.harvard.edu.

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