Binding of Specific DNA Base-pair Mismatches by N-Methylpurine-DNA Glycosylase and Its Implication in Initial Damage Recognition Tapan Biswas 1 , Lawrence J. Clos II 2 , John SantaLucia Jr 2 Sankar Mitra 1 and Rabindra Roy 1 * 1 Sealy Center for Molecular Science and Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch Galveston TX 77555-1079, USA 2 Department of Chemistry Wayne State University Detroit, MI 48202, USA Most DNA glycosylases including N-methylpurine-DNA glycosylase (MPG), which initiate DNA base excision repair, have a wide substrate range of damaged or altered bases in duplex DNA. In contrast, uracil- DNA glycosylase (UDG) is specific for uracil and excises it from both single-stranded and duplex DNAs. Here we show by DNA footprinting analysis that MPG, but not UDG, bound to base-pair mismatches especially to less stable pyrimidine–pyrimidine pairs, without catalyzing detectable base cleavage. Thermal denaturation studies of these normal and damaged (e.g. 1,N 6 -ethenoadenine, 1A) base mispairs indicate that duplex instability rather than exact fit of the flipped out damaged base in the catalytic pocket is a major determinant in the initial recognition of damage by MPG. Finally, based on our determination of binding affinity and catalytic efficiency we conclude that the initial recognition of sub- strate base lesions by MPG is dependent on the ease of flipping of the base from unstable pairs to a flexible catalytic pocket. q 2002 Elsevier Science Ltd. All rights reserved Keywords: N-methylpurine-DNA glycosylase; mismatch; DNA instability; substrate recognition; catalytic efficiency *Corresponding author Introduction Repair of DNA containing small DNA adducts, as well as altered and abnormal bases, occurs pri- marily via the base excision repair (BER) pathway, whose first step is cleavage of the base by a DNA glycosylase in all organisms. 1–2 Accumulated evidence on the properties of many DNA glycosyl- ases indicates the presence of two classes of DNA glycosylases on the basis of their substrate specificity. 2–3 UDG from both Escherichia coli and mammals, which is the most extensively studied glycosylase and is often used as the paradigm for the DNA glycosylase reaction, constitutes a distinct class that acts nearly exclusively on uracil in DNA. 4–5 It excises U from G·U mismatch or A·U equally well, is active on both single and double- stranded DNA substrates, and in fact prefers single-stranded DNA. 6–7 In contrast, other DNA glycosylases such as mammalian MPG, E. coli endonuclease III (Nth), and formamido pyrimi- dine-DNA glycosylase 3 (Fpg or MutM), which have a broad specificity for substrates with widely different structures, constitute the second class. The hallmark of this second group is low turnover, possibly several orders of magnitude lower than that of UDG, 7–10 and extremely low activity if any with single-stranded DNA substrates. How this second group of enzymes recognizes their cognate substrate lesions in DNA is an important question because in spite of their broad substrate range, the enzymes do not act indiscriminately on any DNA lesion. The mammalian MPG is known to excise at least 17 structurally diverse modified bases from DNA. 11 These lesions include 3-alkylpurines, 7-alkyl- guanine, 1,N 6 -ethenoadenine, N 2 ,3-ethenoguanine, and hypoxanthine (Hx), all of which are purine derivatives. 8,12 – 16 Moreover, the base alterations are located in both the major and minor grooves 0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved Present addresses: T. Biswas, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA; R. Roy, Division of Carcinogenesis and Molecular Epidemiology, American Health Foundation, Valhalla, NY 10595, USA. E-mail address of the corresponding author: rroy@ahf.org Abbreviations used: MPG, N-methylpurine-DNA glycosylase; UDG, uracil-DNA glycosylase; 1A, 1,N 6 -ethenoadenine; Hx, hypoxanthine. doi:10.1016/S0022-2836(02)00519-3 available online at http://www.idealibrary.com on B w J. Mol. Biol. (2002) 320, 503–513