Mismatch repair in Trypanosoma brucei: Heterologous expression of MSH2 from Trypanosoma cruzi provides new insights into the response to oxidative damage Alice Machado-Silva a,b , Santuza M.R. Teixeira a , Glória R. Franco a , Andréa M. Macedo a , Sérgio D.J. Pena a , Richard McCulloch b , Carlos Renato Machado a, ⁎ a Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Departamento de Bioquímica e Imunologia, Belo Horizonte, Brazil b Wellcome Centre for Molecular Parasitology, University of Glasgow, Glasgow, UK Received 4 September 2007; received in revised form 14 December 2007; accepted 17 December 2007 Received by F.G. Alvarez-Valin Available online 2 January 2008 Abstract Trypanosomes are unicellular eukaryotes that cause disease in humans and other mammals. Trypanosoma cruzi and Trypanosoma brucei are the causative agents, respectively, of Chagas disease in the Americas and sleeping sickness in sub-Saharan Africa. To better comprehend the interaction of these parasites with their hosts, understanding the mechanisms involved in the generation of genetic variability is critical. One such mechanism is mismatch repair (MMR), which has a crucial, evolutionarily conserved role in maintaining the fidelity of DNA replication, as well as acting in other cellular processes, such as DNA recombination. Here we have attempted to complement T. brucei MMR through the expression of MSH2 from T. cruzi. Our results show that T. brucei MSH2-null mutants are more sensitive to hydrogen peroxide (H 2 O 2 ) than wild type cells, suggesting the involvement of MSH2 in the response to oxidative stress in this parasite. This phenotype is reverted by the expression of either the T. cruzi or the T. brucei MSH2 protein in the MSH2-null mutants. In contrast, MMR complementation, as assessed by resistance to N-methyl-N′- nitro-N-nitrosoguanidine (MNNG) and microsatellite instability, was not achieved by the heterologous expression of T. cruzi MSH2. This finding, associated to the demonstration that mutation of MLH1, another component of the MMR system, did not affect sensitivity of T. brucei cells to H 2 O 2 , suggests an additional role of MSH2 in dealing with oxidative damage in these parasites, which may occur independently of MMR. © 2008 Elsevier B.V. All rights reserved. Keywords: DNA repair; Hydrogen peroxide; Trypanosomatids; Genetic stability 1. Introduction Trypanosomes cause potentially lethal maladies and collec- tively affect millions of people. Distinct strategies are used by these protozoans to survive in the face of the mammalian immune response. T. brucei is an exclusively extracellular parasite and possesses a large repertoire of surface proteins, known as VSGs (Variant Surface Glycoproteins), whose expression changes continually in a process termed antigenic variation (Barry et al., 2005). T. cruzi, on the other hand, is a predominantly intracellular pathogen and relies on an enormous heterogeneity of surface proteins among the population to survive in its different hosts (Buscaglia et al., 2006). Never- theless, both parasites take advantage of genetic diversity for a successful infection of their hosts. Several mechanisms may affect the levels of genetic diversity within a given population. Among these controlling processes is the mismatch repair (MMR) pathway, which plays a central role in the maintenance of genetic stability by enhancing the fidelity of Available online at www.sciencedirect.com Gene 411 (2008) 19 – 26 www.elsevier.com/locate/gene Abbreviations: MNNG, N-methyl-N′-nitro-N-nitrosoguanidine; MMR, mis- match repair; H 2 O 2 , hydrogen peroxide; 8oxoG, 7,8-dihydro-8-oxoguanine; RT- PCR, reverse trancriptase PCR; MTT, methylthiazoldiphenyltetrazolium bromide. ⁎ Corresponding author. Departamento de Bioquímica e Imunologia, ICB, UFMG. Av Antônio Carlos 6627, Caixa Postal 486, Belo Horizonte, MG, Brazil. Tel.: +55 31 34992628; fax: +55 31 34992984. E-mail address: crmachad@icb.ufmg.br (C.R. Machado). 0378-1119/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2007.12.021