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J. Biol. Chem., Vol. 282, Issue 38, 27578-27586, September 21, 2007

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Excision of 5-Halogenated Uracils by Human Thymine DNA Glycosylase

ROBUST ACTIVITY FOR DNA CONTEXTS OTHER THAN CpG^*

From the Department of Biochemistry and Molecular Biology, Marlene and Stewart Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, Maryland 21201

Thymine DNA glycosylase (TDG) excises thymine from G·T mispairs and removes a variety of damaged bases (X) with a preference for lesions in a CpG·X context. We recently reported that human TDG rapidly excises 5-halogenated uracils, exhibiting much greater activity for CpG·FU, CpG·ClU, and CpG·BrU than for CpG·T. Here we examine the effects of altering the CpG context on the excision activity for U, T, FU, ClU, and BrU. We show that the maximal activity (k_max) for G·X substrates depends significantly on the 5' base pair. For example, k_max decreases by 6-, 11-, and 82-fold for TpG·ClU, GpG·ClU, and ApG·ClU, respectively, as compared with CpG·ClU. For the other G·X substrates, the 5'-neighbor effects have a similar trend but vary in magnitude. The activity for G·FU, G·ClU, and G·BrU, with any 5'-flanking pair, meets and in most cases significantly exceeds the CpG·T activity. Strikingly, human TDG activity is reduced 10^2.3–10^4.3-fold for A·X relative to G·X pairs and reduced further for A·X pairs with a 5' pair other than C·G. The effect of altering the 5' pair and/or the opposing base (G·X versus A·X) is greater for substrates that are larger (bromodeoxyuridine, dT) or have a more stable N-glycosidic bond (such as dT). The largest CpG context effects are observed for the excision of thymine. The potential role played by human TDG in the cytotoxic effects of ClU and BrU incorporation into DNA, which can occur under inflammatory conditions and in the cytotoxicity of FU, a widely used anticancer agent, are discussed.

Received for publication, May 23, 2007 , and in revised form, June 29, 2007.

^* This work was supported by National Institutes of Health Grant R01-GM72711 (to A. C. D.) and the University of Maryland Marlene and Stewart Greenebaum Cancer Center. 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.

¹ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 N. Greene St., Baltimore MD 21201. Tel.: 410-706-8118; Fax: 410-706-8297; E-mail: adroh001{at}umaryland.edu.

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				A. Maiti, M. T. Morgan, E. Pozharski, and A. C. Drohat Crystal structure of human thymine DNA glycosylase bound to DNA elucidates sequence-specific mismatch recognition PNAS, July 1, 2008; 105(26): 8890 - 8895. [Abstract] [Full Text] [PDF]