Published online 7 August 2007 Nucleic Acids Research, 2007, Vol. 35, No. 15 e100 doi:10.1093/nar/gkm575 Analysis of a nuclease activity of catalytic domain of Thermus thermophilus MutS2 by high-accuracy mass spectrometry Kenji Fukui1,2, Yoshio Takahata2, Noriko Nakagawa1,2, Seiki Kuramitsu1,2 and Ryoji Masui1,2,* 1RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148 and 2Department of Biological Sciences, Graduate School of Science, Osaka University, 1-1, Machikaneyama-cho, Toyonaka, Osaka 560-0043, Japan Received May 7, 2007; Revised July 12, 2007; Accepted July 13, 2007 ABSTRACT Electrospray ionization with Fourier-transform ion cyclotron resonance mass spectrometry (ESI–FT ICR MS) is a powerful tool for analyzing the precise structural features of biopolymers, including oligo- nucleotides. Here, we described the detailed char- acterization of a newly discovered nuclease activity of the C-terminal domain of Thermus thermophilus MutS2 (ttMutS2). Using this method, the length, nucleotide content and nature of the 5’- and 3’-termini of the product oligonucleotides were accurately identified. It is revealed that the C-terminal domain of ttMutS2 incised the phosphate backbone of oligodeoxynucleotides non-sequence- specifically at the 3’ side of the phosphates. The simultaneous identification of the innumerable frag- ments was achieved by the extremely high-accuracy of ESI–FT ICR MS. INTRODUCTION Nucleases play critical roles in DNA transactions including DNA replication, repair and recombination events (1–4). Detailed analysis of the enzymatic properties of a newly discovered nuclease activity is required in order to help identify cellular function. Nuclease digestion patterns are generally studied using radiolabeled oligonucleotides as substrate followed by electrophoretic analysis of the reaction mixture. However, this method only detects fragments containing the radiolabel. Multiple labeling of the substrate DNA is required in order to ascertain the location of all the cleaved sites. Furthermore, electrophor- esis does not reveal the precise nature of the 50 and 30-termini at the cleaved site. Alternatively, mass spectro- metry can detect all fragments produced by the nuclease without the need for using radioisotopes. Electrospray ionization (ESI) is a soft ionization technique which is suitable for large biopolymers, such as oligonucleotides, because the multiply charged ions lower the m/z (5–8). In addition, Fourier-transform ion cyclotron resonance mass spectrometry (FT ICR MS) is expected to achieve especially high mass accuracy in the analysis of mixtures of biopolymers and determination of the nature. In this study, we analyzed the degradation pattern of double-stranded DNA (dsDNA) by the catalytic domain of Thermus thermophilus MutS2 (ttMutS2) using ESI–FT ICR MS. To the best of our knowledge, this is the ﬁrst report that a nuclease activity was characterized by ESI–FT ICR MS. Bacterial MutS2 possesses domains homologous to the MutS family proteins (9–11) which are involved in DNA mismatch repair, DNA recombination and other DNA modiﬁcations. Recent studies showed that bacterial MutS2 is involved in suppression of homologous recombination (12–14) or/and protection from oxidative DNA damage (15). We previously revealed that ttMutS2 contains a nuclease activity (16,17), and the activity is conﬁned to the C-terminal domain whose sequence is not conserved in the other MutS homologs. The amino acid sequences homo- logous to the C-terminal domain of bacterial MutS2 distribute in variety of proteins other than MutS homolog (18,19). It is also reported that the C-terminal domain of human BCL-3-binding protein shows sequence similar- ity to that of bacterial MutS2 and possesses a nuclease activity (20), although there is no relationship between the biological function of human BCL-3-binding protein and that of bacterial MutS2. The precise characteristics of their activities had been unknown. MATERIALS AND METHODS Oligonucleotides The 50-hydroxylated 3, 7, 15, 21 and 37-mer single- stranded oligodeoxynucleotides were synthesized. *To whom correspondence should be addressed. Tel: +81 06 6850 5433; Fax: +81 06 6850 5442; Email: rmasui@bio.sci.osaka-u.ac.jp 2007 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.