Molecular cloning and characterization of the DNA mismatch repair gene class 2 from the Trypanosoma cruzi

Genes with homology to the bacterial mutS gene, which encodes a protein inv olved in post-replication DNA mismatch repair, are known in several organis ms. Using a degenerate PCR strategy, we cloned a Trypanosoma cruzi. genomic DNA fragment homologous to the mutS gene class two (MSH2). This fragment w as used as a probe to select the cot-responding cDNAs from a T. cruzi cDNA library. The complete sequence of the gene (3304 bp), denominated TcMSH2, w as obtained. The sequence contained an open reading frame of 2889 bp coding for a putative protein of 962 amino acids. Computational analyses of the a mino acid sequence showed 36% identity with MSH2 proteins from other eukary otes and revealed the presence of all functional domains of MutS proteins. Hybridization analyses indicated that the TcMSH2 gene is present as a singl e copy gene that is expressed in all forms of the T. cruzi life cycle. The role of the product of the TcMSH2 gene in mismatch repair was investigated by negative dominance phenotype analyses in Escherichia coli. When eukaryot ic muts genes are expressed in a prokaryotic system, they increase the bact erial mutation rate. The same phenomenon was observed with the TcMSH2 cDNA, indicating that T cruzi MSH2 interferes with the bacterial mismatch system . Phylogenetic analyses showed that the T. cruzi gene grouped with the MSH2 clade confirming the nature of the gene isolated in this work. (C) 2001 Pu blished by Elsevier Science B.V. All rights reserved.