meningtidis (Mc) recombinant Fpg protein. (A) 1 ng of purified Mc Fpg or 0.032 Units of E. coli Fpg was incubated with 10–50 fmol of a 24 bp duplex oligodeoxyribonucleotide containing a single 8oxoG residue opposite A, T C or G. Base excision and strand cleavage were analysed by 20% PAGE and phosphorimaging. The arrow indicates the cleaved DNA substrate. * denotes 32P-labelled strand. 4SC-202 cost S; substrate. (B) Quantification of strand cleavage activity by Mc Fpg. The results represent the average of three independent experiments and
error bars indicate the standard deviation of the mean. Table 3 DNA glycosylase activity of N. meningitidis (Mc) recombinant Fpg protein. Substrate Released bases (fmol) Average (St. dev.)c N. meningitidis Fpga 75 (± 30) E. coli Fpgb 64 (± 44) No enzyme 12 (± 4) a 500 ng of protein was employed in each reaction b 160 Units of protein was employed in each reaction c standard deviation
of the mean Removal of formamidopyrimidine (faPy) from [3H]-methyl-faPy-poly(dG·dC) DNA by recombinant Mc and E. coli Fpg. The results Geneticin are given as the average of five independent measurements. Mc is a bacterium that seemingly spontaneously produces a plethora of variants upon which selection can act, instead of sensing the environment and changing accordingly . One of the major processes governing genetic changes in Neisseria sp. is phase variation. Phase variation is mediated by unstable polynucleotide tracts allowing the gene expression to be switched on or off . Recently, click here several genome maintenance genes have been shown to modulate phase variation frequencies, including the mismatch repair components mutS and mutL, the nucleotide excision repair
gene uvrD and the translesion DNA polymerase dinB [38–41]. Since Mc Fpg is able to remove oxidized Parvulin guanines, although in an error-free manner, we wanted to investigate a potential contribution of Mc fpg on phase variation of polyG tracts. Mc strains NmZ1099_UROS (Control), NmZ1099_UROSΔfpg (Δfpg) and NmZ1099_UROSΔmutS (ΔmutS) were constructed and examined by S12 ribosomal gene switching in a spectinomycin-selection assay (Figure 3). Phase variation was, as previously reported [38–41], significantly increased in the ΔmutS (30-fold) background compared to the wild-type level (***p < 0.001). However, the Mc fpg mutant exhibited only moderate increase (2-fold) compared to the wild-type level (***p < 0.001), and thus MutS exerts a more profound effect on the stability of Mc polyG tracts than Fpg. Likewise, the Mc fpg mutant was recently shown to generate only a weak mutator phenotype when assessed for its spontaneous mutation frequency in a rifampicin assay . In conclusion, Fpg is not a major player in modulating Mc mutation frequencies. Figure 3 Assessment of meningococcal (Mc) phase variation. Phase variation frequency for Mc strains NmZ1099_UROS (Control), NmZ1099_UROSΔfpg (Δfpg) and NmZ1099_UROSΔmutS (ΔmutS) as examined by a spectinomycin assay.