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  • To verify DNA glycosylase AP lyase activity we

    2021-09-16

    To verify DNA glycosylase/AP lyase activity we have employed a simple and efficient method to investigate based on fluorescein-labeled duplex oligonucleotides read on an automated sequencer (Allgayer et al., 2013, Lopes et al., 2007, Lopes et al., 2008). This approach using purified CpMutY proteins revealed that this enzyme was capable of recognizing and excising 8-oxoguanine paired with adenine, but not cytosine (Fig. 4). Wang et al. (2015)through crystallographic studies using Geobacillus stearothermophilus MutY protein revealed the strong interaction between MutY active site and the BMH-21 base. Additionally they showed that the MutY protein can discriminate cytosine from adenine, since cytosine was unable to enter the active site pocket of the enzyme due to imbalanced forces. MutY protein has kept cytosine in a region far from its active while recognizing the 8-oxoG:C lesion, thus evidencing a control strategy of its activity. This discrimination ability composes the structural basis for avoidance of promutagenic DNA repair BMH-21 by MutY adenine DNA glycosylase (Reynisson and Steenken, 2005, Wang et al., 2015). MutY proteins of Mycobacterium semegmatis and Mycobacterium tuberculosis from the CNMR group (Corynebacterium, Mycobacterium, Nocardia and Rhodococcus) shared the same characteristics: the ability to recognize and excise 8-oxoG:A but do not have activity on 8-oxoG:C lesions. Furthermore, in these organisms, the MutY protein was still able to repair other lesions as 8-oxoG:T and 8-oxoG:G (Dorella et al., 2006, Kurthkoti et al., 2010). Future studies will be performed to evaluate the CpMutY activity on different substrates. Currently MutY protein classification is quite controversial. Some authors have classified it as monofunctional by observing just the glycosylase activity (Michaels et al., 1992), though other studies have classified it as bifunctional due glycosylase/AP lyase activity in vitro (Lu et al., 1995, Lu et al., 1996). In in vitro assay the purified CpMutY showed evidences of AP lyase activity, cleaving the duplex oligonucleotide in the damaged residue, even in the absence of NaOH as observed in the EcMutY (Lu et al., 1995, Lu et al., 1996). Moreover, similar to EcMutY we also have verified that CpMutY presented low AP lyase activity in vitro (Michaels et al., 1992, Williams and David, 2000). As pointed by Williams and David (2000) several factors can influence in MutY bifunctional behavior, as the purification process that could contain contaminants which promoting the AP lyase activity. As suggested by these authors, other complementary purification assays have to be used to purification improvement. Furthermore, another important aspect in in vitro MutY studies is related to reaction buffers used in experiments because mono/bifunctional behavior could be influenced by different salts constitution. It is known that Lys 120 endonuclease III from E. coli is the residue responsible for AP lyase activity performing covalent Shiff base intermediate with DNA (Thayer et al., 1995). Endo3c functional domain is preserved in CpMutY and EcMutY, though Lys 120 is not maintained in both of them, which have Tyr 138 and Ser 120, respectively (Fig. 5). Some hypotheses for this bifunctional profile observed in in vitro assays were proposed, such as the existence of other residues that could be involved with this activity. Even as Lys 120, Lys 157 and Lys 158 residues, both preserved in CpMutY and EcMutY, could be involved with Shiff base intermediate (Williams and David, 2000). Zarkov and Grollman (1998) showed that Lys 142 residue in EcMutY has ability to bind covalently to DNA suggesting its involvement in AP lyase activity. In the same position CpMutY protein has Arg 160, another positive residue that could replace it and maintain the activity (Supplementary Fig. 1). Until now the mechanism proposed to MutY behavior is uncertain. Some studies have demonstrated that EcMutY protein with S120K modification showed well-defined bifunctional activity. However, in vivo assays using this model did not show repair the DNA damage efficiently, being classified as a monofunctional enzyme (Oliveira et al., 2014, Williams and David, 2000).