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    • agomelatine Being a facultative intracellular pathogen C pse

    2022-05-13

    Being a facultative intracellular pathogen, C. pseudotuberculosis is exposed to oxygen and nitrogen species (ROS and RNS, respectively) reactive environment within macrophages (Nathan and Shiloh, 2000), apart from species endogenously generated by metabolic processes. These molecules interact with DNA, ultimately causing different types of damage, including modified bases, abasic sites (AP) and strand breaks, with cytotoxic or mutagenic effects on the cell (Barnes and Lindahl, 2004, Dizdaroglu and Jaruga, 2012). One of the most stable mutagenic products of oxidative damage to DNA is the 7,8-dihydro-8-oxo-dGuanine (8-oxoguanine) lesion, which can pair with cytosine or agomelatine during replication, producing GC→TA transversions (Drsata et al., 2013, Patra et al., 2014, Wang et al., 2015). To prevent genomic instability caused by 8-oxoguanine, as well as other DNA lesions, specialized enzymes that operate in specific pathways of DNA recognition and repair were naturally selected in different organisms. The base excision repair (BER) is the principal pathway for cellular protection against oxidative damage to DNA (David et al., 2007, Scott et al., 2014, Storr et al., 2013, Veen and Tang, 2015). The four key enzymes involved in this repair mechanism (DNA glycosylases, AP endonuclease (or AP lyase), DNA polymerase, and DNA ligase) operate in a stepwise manner, to excise the damaged base and replace it with a correct base (Robertson et al., 2009). Repair of 8-oxoguanine lesion has been clearly demonstrated in Escherichia coli and is being described for other organisms, given the high mutagenic potential of this lesion (Furtado et al., 2012, Huang et al., 2006, Robles et al., 2011, Sanders et al., 2009). Three enzymes MutM (formamidopyrimidine/5-formyluracil/5-hydroxymethyluracil DNA glycosylase), MutT (dGTP-preferring nucleoside triphosphate pyrophosphohydrolase), and MutY (adenine DNA glycosylase) are involved in this repair, cooperating to prevent the deleterious effects of 8-oxoguanine, and do so through the pathway known as the GO system (Fowler et al., 2003, Michaels and Miller, 1992, Nagorska et al., 2012). The importance of this system in preventing mutations was evidenced by the high mutation rates found in strains deficient in anyone of the genes involved in this process (mutT−, mutY−, and mutM−), as well as in double mutants (mutY) (Nagorska et al., 2012, Robles et al., 2011). MutT is a nucleoside triphosphatase that hydrolyzes 8-oxo-dGTP into 8-oxo-dGMP in the nucleotide pool, preventing its incorporation into DNA (Aguiar et al., 2013, Maki and Sekiguchi, 1992, Setoyama et al., 2011), while MutM and MutY are DNA glycosylases that excise cytosine from 8-oxoG:C and adenine from 8-oxoG:A mismatches, respectively (Eberle et al., 2015, Li, 2010, Markkanen et al., 2013). Some studies showed that E. coli MutY is a monofunctional enzyme; however, the change of one amino acid residue in its active site, S120K, is capable of introducing an additional AP lyase activity to this enzyme (Oliveira et al., 2014, Williams and David, 2000). An unique feature of the MutY protein family is the [4Fe–4S] cluster that interlinks two protein subdomains and some studies have shown the importance of this organometallic arrangement in C. pseudotuberculosis MutY stability, and the action of metal ions, pH and temperature on its folding and function (Eberle et al., 2015). Despite scarce evidences, repair mechanisms in C. pseudotuberculosis remain poorly understood and research in this area can contribute to narrow the search for molecular targets against lymphadenitis. In silico analyses of DNA repair genes in 15 Corynebacterium species, including C. pseudotuberculosis, showed that genes involved with oxidative damage repair, such as fpg/mutM (formamidopyrimidine-DNA glycosylase), nth/mutY (endonuclease III), nei/endoVIII (endonuclease VIII), and xht/exoIII (exonuclease III), are preserved, being present in all species investigated (Resende et al., 2011). This degree of conservation suggests that BER is an important protective mechanism against oxidative DNA damage in this organism. Considering these aspects and the need for genetic information on C. pseudotuberculosis, so as to expand the current knowledge on processes involved with DNA repair, the purpose of this study was to characterize the MutY protein from C. pseudotuberculosis (CpMutY) and evaluate its involvement with DNA repair. CpMutY was characterized through computational analyses, an in vivo functional complementation assay was performed under oxidative stress to investigate if CpMutY is capable to prevent mutations and an in vitro assay was carried out to verify if it presents an A/G-specific adenine glycosylase/AP lyase activity.