5692–5705 Nucleic Acids Research, 2010, Vol. 38, No. 17 doi:10.1093/nar/gkq350 Published online 10 May 2010 A novel single-stranded DNA-specific 30–50 exonuclease, Thermus thermophilus exonuclease I, is involved in several DNA repair pathways Atsuhiro Shimada 1 , Ryoji Masui1,2, Noriko Nakagawa1,2, Yoshio Takahata1, Kwang Kim1, Seiki Kuramitsu1,2 and Kenji Fukui2,* 1Department of Biological Sciences, Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka, Osaka 560-0043 and 2RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan Received January 12, 2010; Revised April 21, 2010; Accepted April 22, 2010 ABSTRACT Single-stranded DNA (ssDNA)-specific exonucle- ases (ssExos) are expected to be involved in a variety of DNA repair pathways corresponding to their cleavage polarities; however, the relationship between the cleavage polarity and the respective DNA repair pathways is only partially understood. To understand the cellular function of ssExos in DNA repair better, genes encoding ssExos were dis- rupted in Thermus thermophilus HB8 that seems to have only a single set of 50–30 and 30–50 ssExos unlike other model organisms. Disruption of the tthb178 gene, which was expected to encode a 30–50 ssExo, resulted in significant increase in the sensitivity to H2O2 and frequency of the spontaneous mutation rate, but scarcely affected the sensitivity to ultravio- let (UV) irradiation. In contrast, disruption of the recJ gene, which encodes a 50–30 ssExo, showed little effect on the sensitivity to H2O2, but caused increased sensitivity to UV irradiation. In vitro char- acterization revealed that TTHB178 possessed 30–50 ssExo activity that degraded ssDNAs containing deaminated and methylated bases, but not those containing oxidized bases or abasic sites. Consequently, we concluded that TTHB178 is a novel 30–50 ssExo that functions in various DNA repair systems in cooperation with or independently of RecJ. We named TTHB178 as T. thermophilus exonuclease I. INTRODUCTION Single-stranded DNA (ssDNA)- and double-stranded DNA (dsDNA)-speciﬁc exonucleases are essential for DNA replication, repair and recombination. Functional defects of exonucleases are known to have a profound impact on human diseases, such as Aicardi–Goutieres syndrome, familial chilblain lupus and ataxia telangiectasia-like disorder (1–3). The ssDNA-speciﬁc exo- nucleases (ssExos) are categorized by their cleavage polarity; from 30 to 50 (30–50) and from 50 to 30 (50–30). A variety of DNA repair pathways require ssExos to process the intermediate DNA structures generated during the re- actions (4–6). As the diverse intermediate DNA structures are yielded depending on the repair pathway, living cells are considered to require several kinds of ssExos with dif- ferent polarities. For example, a 50–30 ssExo is required for the early stage of double-strand break (DSB) repair. DSB is a potentially lethal lesion that is spontaneously generated in normal cells and also generated by external factors including ultraviolet (UV)-C (100–280 nm) (7). Bacterial DSB repair mainly occurs through homologous recombination (8). In this mechanism, an ssExo with 50–30 polarity processes the termini of a dsDNA to a 30-overhanging structure, generating an entry point for downstream enzymes. However, the type of ssExo polarity required for other DNA repair pathways is unclear. First, in DNA mismatch repair (MMR), which corrects a mismatched base generated during DNA replication, an ssExo is required for the excision of the ssDNA region containing the mis- matched base. Most organisms, except Escherichia coli and its closely related species, are thought to adopt the system in which the MutS/MutL complex (or its counter- part) nicks the 30- and 50-sides of the mismatched base (9–15). The ssExo polarity responsible for the removal of the error-containing ssDNA region in bacteria is unknown. Second, it also remains to be established how cells process intermediates in the repair of deaminated bases, such as xanthine, hypoxanthine and uracil. Endonuclease V is known to hydrolyse the phosphodiester bond at the 30-side of the deaminated lesion (16,17); however, the downstream reaction is unclear. ssExos *To whom correspondence should be addressed. Tel: +81-791-58-2891; Fax: +81-791-58-2892; Email: k.fukui@spring8.or.jp The Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.