DNA Repair 2 (2003) 455–470 Endonuclease IV enhances base excision repair of endonuclease III from Methanobacterium thermoautotrophicum Jung Ho Back a,b , Ji Hyung Chung c , Young In Park b , Key-Sun Kim a , Ye Sun Han a,∗ a Structural Biology Research Center, Korea Institute of Science and Technology, P. O. Box 131, Cheongryang, Seoul, South Korea b The Graduate School of Biotechnology, Korea University, Seoul, South Korea c Yonsei Research Institute of Aging Science, and Cardiovascular Genome Center, Yonsei University, Seoul, 120-749, South Korea Accepted 4 December 2002 Abstract Damaged DNA strands are repaired by base excision (BER) in organisms, a process initiated by repair enzymes, which include DNA glycosylases and endonucleases. We expressed and characterized two putative endonuclease genes from Methanobacterium thermoautotrophicum, Mt0764 and Mt1010, encoding homologues of endonuclease III (endo III) and endonuclease IV (endo IV) of Escherichia coli. The Mt0764 and Mt1010 proteins showed endo III activity by removing thymine glycol from DNA strand and AP endonuclease activity, respectively. The Mt0764 protein not only cleaved the oligonu- cleotide duplex, containing a thymine glycol/adenine pair efficiently, but also showed activity on the 8-oxoguanine-containing oligonucleotide duplex. In this study, we report upon the stimulation of endo III activity by endo IV using two recombinant proteins (Mt1010 and Mt0764) from M. thermoautotrophicum. Mt1010 stimulated the DNA glycosylase activity of Mt0764 for DNA substrates containing 8-oxoguanine residues and increasing the formation of the Mt0764 protein–DNA complex. The interaction between Mt1010 and Mt0764 was observed by using an in vitro binding assay. These results suggest that association between endo III and endo IV may occur in vivo, and this contributes to efficient base excision repair for the oxidative damage of DNA. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Methanobacterium thermoautotrophicum; Endonuclease III; Endonuclease IV; DNA glycosylase/AP lyase 1. Introduction The oxidative DNA lesions are generated by DNA modification via reactive oxygen species (ROS) or by the misincorporation of damaged nucleotides during DNA synthesis. These lesions are mainly repaired via the base excision repair (BER) pathway, which is initiated by removal of the damaged base, and are catalyzed by DNA glycosylases [1–3]. There are two ∗ Corresponding author. Tel.: +82-2-958-5933; fax: +82-2-958-5939. E-mail address: yshan2@kist.re.kr (Y.S. Han). classes of DNA glycosylases, and these have dis- tinct substrate specificities, namely, monofunctional simple glycosylases and glycosylases with associated apurine/apyrimidine (AP) lyase activity. All oxidized base lesions are removed from DNA strand by DNA glycosylase/AP lyases, which not only catalyze the removal of the damaged bases, but which also cause strand cleavage by -elimination. Endonuclease III (endo III or Nth), the product of the nth gene from E. coli, is a monomeric pro- tein of 23 kDa, which contains a Fe–S cluster [4,5]. The enzyme possesses glycosylase activity and 3 ′ - apurinic/apyrimidinic (AP) endonuclease activity; 1568-7864/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. doi:10.1016/S1568-7864(02)00243-4