This conservation was confirmed by in silico fusion of the crysta

This conservation was confirmed by in silico fusion of the crystal Baf-A1 chemical structure structure of Lactococcus lactis Fpg with Mc Fpg using the PDB (Figure 1B). Interestingly, the 11-mer DUS sequence encodes amino acids that are not identified as functional residues and is localized in an fpg region showing relatively low sequence homology across species boundaries (see additional file 1, Figures S1 and S2). Fpg has been extensively studied in

E. coli and is characterized in several other prokaryotes as well [32–34], displaying identical MM-102 price substrate specificities. In order to analyze the substrate specifiCity of Mc Fpg, the gene was over-expressed in E. coli and recombinant Mc Fpg protein purified to homogeneity (see additional file 1, Figure S4). Mc Fpg has an apparent size in SDS-PAGE of approximately 30 kDa, corresponding to the molecular selleck chemicals weight predicted from the genome deduced amino acid sequence and similar to Fpg of E. coli and L. lactis [32, 33]. The preferred substrates for recognized Fpg proteins are 8oxoG and faPy residues. The ability of recombinant Mc Fpg to remove these lesions was investigated, using E. coli Fpg as a positive control. Activity towards

C:faPy residues in a 3H-labeled poly(dG-dC) substrate was identified (Table 3). When assessing the 8oxoG excision, the Mc Fpg displayed both DNA glycosylase and AP lyase activity (Figure 2). Equivalent levels of base excision of 8oxoG opposite C, T and G and much lower activity toward 8oxoG when mispaired with A was demonstrated (Figure 2). No activity was dectected in the absence of 8oxoG residues (see additional file 1, Figure S5). This discrimination of the base opposite the lesion is in keeping with findings on E. coli Fpg [35], although the remaining activity against 8oxoG:A seen in Mc Fpg was not found in the original characterization of substrate specifiCity in E. coli.

8oxoG:C is probably the most important physiological substrate for Mc Fpg, despite the similar levels of nicking observed in 8oxoG:T and 8oxoG:G, as the former is by far the most common substrate in vivo in E. coli [4]. The removal of 8oxoG from the genome prevents G:C→T:A transversions in E. coli, but the mutation rates in single fpg mutants are too low in Mc to detect these lesions [9], despite this being the most likely event Dolutegravir when 8oxoG is preferentially mis-incorporated with adenine and left unrepaired. Recent studies in M. smegmatis have identified an alternative pattern of preferential incorporation of guanine opposite 8oxoG, creating G:C→C:G transversions or A:T→C:G transitions in the absence of Fpg [36]. 8oxoG:G and G:C→C:G transversions can also be found in E. coli and S. pombe, however, they are rare compared to 8oxoG:A events. In conclusion, these results demonstrate that the protein encoded by the Mc fpg gene excises base lesions that are typical substrates of other Fpg orthologues and are consistent with this protein being an Fpg DNA glycosylase.

Comments are closed.