536–544 Nucleic Acids Research, 2011, Vol. 39, No. 2 doi:10.1093/nar/gkq791 Published online 17 September 2010 Dissecting endonuclease and exonuclease activities in endonuclease V from Thermotoga maritima Rongjuan Mi, Anne K. Abole and 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, USA Received June 3, 2010; Revised August 1, 2010; Accepted August 2, 2010 ABSTRACT Endonuclease V is an enzyme that initiates a conserved DNA repair pathway by making an endonucleolytic incision at the 30-side 1 nt from a deaminated base lesion. DNA cleavage analysis using mutants defective in DNA binding and Mn2+ as a metal cofactor reveals a novel 30-exonuclease activity in endonuclease V [Feng,H., Dong,L., Klutz,A.M., Aghaebrahim,N. and Cao,W. (2005) Defining amino acid residues involved in DNA- protein interactions and revelation of 30- exonuclease activity in endonuclease V. Biochemistry, 44, 11486–11495.]. This study defines the enzymatic nature of the endonuclease and exo- nuclease activity in endonuclease V from Thermotoga maritima. In addition to its well-known inosine-dependent endonuclease, Tma endonucle- ase V also exhibits inosine-dependent 30-exonuclease activity. The dependence on an inosine site and the exonuclease nature of the 30-exonuclease activity was demonstrated using 50-labeled and internally-labeled inosine-containing DNA and a H214D mutant that is defective in non-specific nuclease activity. Detailed kinetic analysis using 30-labeled DNA indicates that Tma endonuclease V also possesses non-specific 50-exonuclease activity. The multiplicity of the endo- nuclease and exonuclease activity is discussed with respect to deaminated base repair. INTRODUCTION Endonuclease V (endo V) is a DNA repair enzyme which hydrolyzes the second phosphodiester bond 30 from a deaminated base lesion (1–4). Inosine (I) derived from ad- enosine deamination, xanthosine and oxanosine from guanosine deamination, and uridine from cytidine de- amination are all substrates in vitro for bacterial endo V (1–3,5,6). The endonuclease activity towards inosine and its role in repair of deaminated inosine damage in vivo have been studied intensely. Biochemical and kinetic analysis reveals that endo V remains bound to inosine-containing DNA after strand cleavage (1,3,7,8). It was proposed that this unique property may act as a sensor to recruit other proteins for downstream repair process (3). Genetic analysis using endo V (encoded by nﬁ gene) deletion mutants indicates that endo V is a primary repair enzyme for the repair of inosine lesions produced under nitrosative stress conditions (9–11). After the 30-nicking downstream of the inosine site by endo V, a repair patch needs to be created to remove the lesion. Taking advantage of an oligonucleotide-mediated transformation system, a genetic study demonstrates that repair of inosine in chromosomes of Escherichia coli creates an 5 nt repair patch, which is deﬁned by removal of 2 nt from the 50-side and 3 nt from the 30-side (12). The structure and function relationship of this repair enzyme has been investigated intensely using thermostable endonuclease V from the thermophilic bacterium Thermotoga maritima (Tma) as a model system. A system- atic site-directed mutagenesis analysis on all seven conserved motifs deﬁnes the role of a series of residues in motifs I, III, IV, V, VI, VII in interactions with inosine in DNA (Figure 1A) (13). Biochemical and struc- tural studies reveal that D43, E89, D110 and H214 are part of the active site that coordinates metal binding and catalytic function (14,15). Several conserved amino acid residues in motifs III and IV play an important role in protein–DNA interactions and recognition of deaminated DNA bases. In addition to the endonuclease activity nicking the inosine site, Tma endo V also contains non-speciﬁc nuclease activities (3,4,16–18). An important ﬁnding from previous systematic site-directed mutagenesis analysis is the revelation of 30-exonuclease activity from Tma endo V (13). Previous biochemical characterization of the nuclease activity from Tma endo V primarily focused on endonuclease activity on deaminated lesions *To whom correspondence should be addressed. Tel: +1 864 656 4176; Fax: +1 864 656 0393; Email: wgc@clemson.edu 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.