Antimutator role of DNA glycosylase MutY in pathogenic Neisseria species.

Genome alterations due to horizontal gene transfer and stress constantly generate strain on the gene pool of Neisseria meningitidis, the causative agent of meningococcal (MC) disease. The DNA glycosylase MutY of the base excision repair pathway is involved in the protection against oxidative stress. MC MutY expressed in Escherichia coli exhibited base excision activity towards DNA substrates containing A:7,8-dihydro-8-oxo-2'-deoxyguanosine and A:C mismatches. Expression in E. coli fully suppressed the elevated spontaneous mutation rate found in the E. coli mutY mutant. An assessment of MutY activity in lysates of neisserial wild-type and mutY mutant strains showed that both MC and gonococcal (GC) MutY is expressed and active in vivo. Strikingly, MC and GC mutY mutants exhibited 60- to 140-fold and 20-fold increases in mutation rates, respectively, compared to the wild-type strains. Moreover, the differences in transitions and transversions in rpoB conferring rifampin resistance observed with the wild type and mutants demonstrated that the neisserial MutY enzyme works in preventing GC-->AT transversions. These findings are important in the context of models linking mutator phenotypes of disease isolates to microbial fitness.

Growth inhibition of granulocyte-macrophage colony-forming cells by human cytidine deaminase requires the catalytic function of the protein.

Previous studies have indicated that cytidine deaminase (CDD) is a potent growth inhibitor of granulocyte-macrophage colony-forming cells (GM-CFC). In this study, we have undertaken molecular cloning and purification of recombinant human CDD to elucidate the growth regulatory potential and mechanism behind the growth suppressive effect. The purified protein had a specific activity of 1.35 x 10(5) U/mg and a Km value of 30 micromol/L. In the GM-CFC assay, the recombinant protein was shown to reduce colony formation to 50% at 16 pmol/L concentration. Similarly, as was observed with CDD derived from granulocyte extract, the effect depended on the presence of thymidine (>/= 4 x 10(-5) mol/L). These results imply that CDD is an extremely potent inhibitor of GM-CFC and that no additional factor from the granulocyte extract is required for the growth inhibitory effect. Modification of CDD by truncation from the C-terminal end, or by amino acid substitution of an active site glutamate residue, eliminated both the enzyme activity and the growth regulatory potential of CDD. Furthermore, CDD from Escherichia coli was found to be even more effective than human CDD in growth suppression of GM-CFC, with 10-fold higher inhibitory activity corresponding to a 10-fold higher enzymatic activity. Taken together, these results show that the catalytic nucleoside deaminating function of the protein is essential for the growth suppressive effect of CDD. Most probably, CDD exerts growth inhibition by depleting the cytidine and deoxycytidine pool required for DNA synthesis, as addition of deoxycytidine monophosphate, which is not a substrate for CDD, neutralizes the inhibiting effect.