dna repair 7 ( 2 0 0 8 ) 1962–1972 available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/dnarepair Repair of deaminated base damage by Schizosaccharomyces pombe thymine DNA glycosylase Liang Dong, Rongjuan Mi, Robert A. Glass, John N. Barry, Weiguo Cao * Department of Genetics and Biochemistry, South Carolina Experiment Station, Clemson University, Room 219 Biosystems Research Complex, 51 New Cherry Street, Clemson, SC 29634, United States article info Article history: Received 12 June 2008 Received in revised form 14 August 2008 Accepted 14 August 2008 Published on line 25 September 2008 Keywords: Nitrosative stress DNA damage DNA repair Deamination Thymine DNA glycosylase Fission yeast abstract Thymine DNA glycosylases (TDG) in eukaryotic organisms are known for their double- stranded glycosylase activity on guanine/uracil (G/U) base pairs. Schizosaccharomyces pombe (Spo) TDG is a member of the MUG/TDG family that belongs to a uracil DNA glycosylase superfamily. This work investigates the DNA repair activity of Spo TDG on all four deam- inated bases: xanthine (X) and oxanine (O) from guanine, hypoxanthine (I) from adenine, and uracil from cytosine. Unexpectedly, Spo TDG exhibits glycosylase activity on all deam- inated bases in both double-stranded and single-stranded DNA in the descending order of X>I>U ≫ O. In comparison, human TDG only excises deaminated bases from G/U and, to a much lower extent, A/U and G/I base pairs. Amino acid substitutions in motifs 1 and 2 of Spo TDG show a significant impact on deaminated base repair activity. The overall mutational effects are characterized by a loss of glycosylase activity on oxanine in all five mutants. L157I in motif 1 and G288M in motif 2 retain xanthine DNA glycosylase (XDG) activity but reduce excision of hypoxanthine and uracil, in particular in C/I, single-stranded hypoxanthine (ss- I), A/U, and single-stranded uracil (ss-U). A proline substitution at I289 in motif 2 causes a significant reduction in XDG activity and a loss of activity on C/I, ss-I, A/U, C/U, G/U, and ss-U. S291G only retains reduced activity on T/I and G/I base pairs. S163A can still excise hypoxanthine and uracil in mismatched base pairs but loses XDG activity, making it the clos- est mutant, functionally, to human TDG. The relationship among amino acid substitutions, binding affinity and base recognition is discussed. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Among the four DNA bases, three of them (adenine (A), cyto- sine (C), and guanine (G)) are subject to deamination caused by endogenous and environmental agents [1–7]. Deamination of adenine or cytosine produces hypoxanthine (I) or uracil (U), respectively [1,5]. Xanthine (X) and oxanine (O), on the other hand, are the corresponding deamination products derived from guanine [8]. Oxidative deamination of guanine produces a diazoate intermediate, which goes through two different ∗ Corresponding author. Tel.: +1 864 656 4176; fax: +1 864 656 0393. E-mail address: wgc@clemson.edu (W. Cao). pyrimidine ring opening and closing steps to form xanthine or oxanine [9]. Both xanthine and oxanine are stable lesions in DNA under physiological conditions [10–12]. The content of xanthine in DNA increases when it is exposed to reactive nitrogen species such as nitric oxide [5,13–15]. According to in vitro DNA polymerase studies or in vivo analysis, all four types of deaminated base damage may result in mutations if not removed by DNA repair systems [11,16–22]. In addition to the natural DNA bases, 5-methylcytosine is also subject to deam- ination, yielding a thymine base paired with guanine [1]. It 1568-7864/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.dnarep.2008.08.006