The results obtained through in

The results obtained through in vitro, in vivo and in silico corroborated with those presented by other works and indicate that CpMutY is involved in oxidative damage repair in this organism. In this regard, MutY conservation in evolutionarily distant bacteria suggests that this protein plays an important role in cell defense against ROS detrimental to DNA in prokaryotic cells. Additional studies are necessary to investigate whether C. pseudotuberculosis mutY gene deficiency would affect this microorganism's pathogenicity and survival, compared to its wild-type counterpart, and could be used as a possible target for therapy against caseous lymphadenitis.
Conclusions The involvement of CpMutY in DNA repair and prevention of mutations was demonstrated here through functional assays. In in vivo assays the CpMutY protein was capable of suppressing the mutator phenotype of E. coli (BH980), reducing the rate of spontaneous mutation to levels similar to those of the wild strain. In addition, expression of CpMutY protein conferred advantage on growth under oxidative stress in mutY-gene-deficient Escherichia coli. In vitro assay suggested that CpMutY protein presented a specific glycosylase/AP lyase activity for 8-oxoG:A. Moreover computational analyses revealed that important features related to the activity of PF-00562271 glycosylase/AP lyase are preserved in the CpMutY protein, suggesting that its role in 8-oxoG:A mismatch repair is preserved. Taken together, the present results suggest that CpMutY protein presented evidences of functional enzyme homologous to MutY from E. coli and can performs a similar role in 8-oxoG:A mismatch repair, preventing GC→TA transversions and protecting against endogenous or exogenous oxidative damage to DNA. The following is the supplementary data related to this article.
Acknowledgments This investigation was supported by FAPEMIG (APQ00202-10).
Introduction Our genome is continually exposed to reactive oxygen species (ROS) produced by wide range of mechanisms, e.g., exogenous sources, ionizing radiations, ultraviolet radiations, chemotherapeutics, other harmful chemicals and endogenous sources such as by-products of aerobic metabolism, inflammation reactions, intermediates of metabolism and hormones (De Bont & van Larebeke, 2004). These ROS create oxidative stress which leads to several types (as many as 50,000) of lesions in DNA (Lomax et al., 2013). These lesions are genotoxic and/or mutagenic because they cause DNA strand break and base damage. Any of this damage can cause severe effect on genomic stability and can induce point or frameshift mutations. The ROS produced involve O2− (superoxide radical anion), OH (hydroxyl radical), singlet oxygen (1O2) and H2O2 (hydrogen peroxide) (De Bont & van Larebeke, 2004). OH is the most harmful (highly reactive) type of ROS which is also generated during inflammatory response (Griendling et al., 2000). Major lesions produced by oxidative stress are 5.6 dihydrouracil (DHU), 5-hydroxyuracil (5-OHU), 5, 6-thymine glycol (Tg), 5-(hydroxymethyl)-2′-deoxyuridine, 5-formyl-2′-deoxyuridine, 4.6-diamino-5-formamidopyrimidine (Fapy-A) and 2.6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy-G), 7.8-dihydro-8-oxoadenine (8-oxo-A), 8-dihydro-2′-deoxyguanosine (8-oxoG) and single-strand breaks (SSB) etc. (De Bont and van Larebeke, 2004, Lomax et al., 2013, Hegde et al., 2008, Jaruga et al., 2000). Only radiation produces around 850 pyrimidine lesions, 450 purine lesions, 1000 single-strand breaks (SSB) and 20–40 double-strand breaks (DSB)/cell/Gy (Cadet et al., 2008). These lesions generate spontaneous mutations in the genomic DNA, e.g., deamination of C to U or 5-OHU causes transition mutation (GC→AT) because U and 5-OHU both pair with adenine (A) and guanine (G) is oxidized to 8-oxoG which pairs with A that leads to GC→AT transversion mutation (Helbock et al., 1999, Shibutani et al., 1991). These mutations when produced spontaneously in genes essential for apoptosis, cell cycle, genome maintenance (DNA repair), tumor suppressor and oncogene can result in cancerogenesis or other diseases like rheumatoid arthritis, aging etc. (Lomax et al., 2013, Levine, 1997, Ames et al., 1993, Gotz et al., 1994, Lovell et al., 2000).