Novel DNA glycosylases from Mycobacterium tuberculosis

Oxidized bases are removed from DNA of Escherichia coli by enzymes formamidopyrimidine DNA glycosylase (Eco-Fpg) and endonuclease VIII (Eco-Nei) of the same structural family Fpg/Nei. New homologs of these enzymes not characterized earlier have been found in genomes of Actinobacteria. We have cloned and expressed two paralogs (Mtu-Nei2 and Mtu-Fpg2) from 36KAZ and KRA94 isolates of Mycobacterium tuberculosis and studied their ability to participate in DNA repair. Under heterologous expression in E. coli, Mtu-Nei2 decreased the rate of spontaneous mutagenesis in the rpoB gene, whereas Mtu-Fpg2 moderately increased it, possibly due to absence of residues crucially important for catalysis in this protein. Mtu-Nei2 was highly active toward double-stranded DNA substrates containing dihydrouracil residues and apurine-apyrimidine sites and was less efficient in cleavage of substrates containing 8-oxoguanine and uracil residues. These lesions, as well as 8-oxoadenine residues, were also recognized and removed by the enzyme from single-stranded DNA. Fpg and Nei homologs from M. tuberculosis can play an important role in protection of bacteria against genotoxic stress caused by oxidative burst in macrophages.