Corynebacterium pseudotuberculosis is the etiological agent of caseous lymphadenitis, a disease that predominantly affects small ruminants, causing significant economic losses worldwide. As a facultative intracellular pathogen, this bacterium is exposed to an environment rich in reactive oxygen species (ROS) within macrophages. To ensure its genetic stability, C. pseudotuberculosis relies on efficient DNA repair pathways for excision of oxidative damage such as 8-oxoguanine, a highly mutagenic lesion. MutY is an adenine glycosylase involved in adenine excision from 8-oxoG:A mismatches avoiding genome mutation incorporation. The purpose of this study was to characterize MutY protein from C. pseudotuberculosis and determine its involvement with DNA repair. In vivo functional complementation assay employing mutY gene deficient Escherichia coli transformed with CpmutY showed a 13.5-fold reduction in the rate of spontaneous mutation, compared to cells transformed with empty vector. Also, under oxidative stress conditions, CpMutY protein favored the growth of mutY deficient E. coli, relative to the same strain in the absence of CpMutY. To demonstrate the involvement of this enzyme in recognition and excision of 8-oxoguanine lesion, an in vitro assay was performed. CpMutY protein was capable of recognizing and excising 8-oxoG:A but not 8-oxoG:C presenting evidences of glycosylase/AP lyase activity in vitro. In silico structural characterization revealed the presence of preserved motifs related to the MutY activity on DNA repair, such as catalytic residues involved in glycosylase/AP lyase activity and structural DNA-binding elements, such as the HhH motif and the [4Fe-4S] cluster. The three-dimensional structure of CpMutY, generated by comparative modeling, exhibits a catalytic domain very similar to that of E. coli MutY. Taken together, these results indicate that the CpmutY encodes a functional protein homologous to MutY from E. coli and is involved in the prevention of mutations and the repair of oxidative DNA lesions.
Keywords: 8-oxoguanine; Corynebacterium pseudotuberculosis; DNA repair; Functional assays; GO system; MutY.
Copyright © 2016 Elsevier B.V. All rights reserved.