rifampin and 8-hydroxyguanine

Oxidative damage to DNA results in the occurrence of 7,8-dihydro-8-oxoguanine (8-oxoG) in the genome. In eubacteria, repair of such damage is initiated by two major base-excision repair enzymes, MutM and MutY. We generated a MutY-deficient strain of Mycobacterium smegmatis to investigate the role of this enzyme in DNA repair. The MutY deficiency in M. smegmatis did not result in either a noteworthy susceptibility to oxidative stress or an increase in the mutation rate. However, rifampicin-resistant isolates of the MutY-deficient strain showed distinct mutations in the rifampicin-resistance-determining region of rpoB. Besides the expected C to A (or G to T) mutations, an increase in A to C (or T to G) mutations was also observed. Biochemical characterization of mycobacterial MutY (M. smegmatis and M. tuberculosis) revealed an expected excision of A opposite 8-oxoG in DNA. Additionally, excision of G and T opposite 8-oxoG was detected. MutY formed complexes with DNA containing 8-oxoG : A, 8-oxoG : G or 8-oxoG : T but not 8-oxoG : C pairs. Primer extension reactions in cell-free extracts of M. smegmatis suggested error-prone incorporation of nucleotides into the DNA. Based on these observations, we discuss the physiological role of MutY in specific mutation prevention in mycobacteria.

Topics: Animals; Bacterial Proteins; Cell Line; DNA Damage; DNA Glycosylases; DNA Repair; DNA, Bacterial; Guanine; Hydrogen Peroxide; Mice; Mutation; Mycobacterium smegmatis; Rifampin; Substrate Specificity

The human Ogg1 protein (hOgg1) is an antimutator DNA glycosylase/AP lyase that catalyzes the excision of 8-oxo-7,8-dihydroguanine (8-oxoG) and the incision of apurinic and apyrimidinic (AP) sites in DNA. In this study, we have investigated the functional role of H270, Q315 and F319, three amino acids that are located in the 8-oxoG-binding pocket of hOgg1. Wild-type and mutant hOgg1 proteins (H270A, H270R, H270L, Q315A and F319A) were purified to apparent homogeneity. The catalytic activities and the DNA-binding properties of the various hOgg1 mutants were compared to those of the wild-type. The results show that hOgg1 mutated at H270 (H270A and H270L) or F319 (F319A) exhibits greatly reduced (50- to 1000-fold) DNA glycosylase activity, whereas the AP lyase activity is only moderately affected (<4-fold). The affinity of the hOgg1 mutants (H270A, H270L and F319A) for 8-oxoG.C-containing DNA is also greatly reduced (>30-fold), whereas their affinity for THF.C-containing DNA is only moderately reduced (<7-fold). The results also show that hOgg1 mutated at Q315 (Q315A) exhibits catalytic and DNA-binding properties similar to those of the wild-type. Therefore, H270 and F319 are essential to form the functional 8-oxoG-binding pocket, whereas Q315 is less crucial. In contrast, H270, Q315 and F319 are not required for efficient binding of THF.C and cleavage of AP sites. Finally, hOgg1 mutant proteins with a substitution of H270A or F319A are members of a new type of hOgg1 that is deficient in DNA glycosylase but proficient in AP lyase.

Topics: Binding Sites; Catalysis; Circular Dichroism; DNA; DNA Glycosylases; DNA-(Apurinic or Apyrimidinic Site) Lyase; DNA-Binding Proteins; DNA-Formamidopyrimidine Glycosylase; Drug Resistance, Bacterial; Escherichia coli; Escherichia coli Proteins; Guanine; Humans; Models, Molecular; Mutation; Protein Binding; Rifampin; Structure-Activity Relationship

Although both bacillary and coccoid forms of Helicobacter pylori reside in human stomach, the pathophysiological significance of the two forms remains obscure. The present work describes the effect of oxygen tension on the transformation and reactive oxygen species (ROS) metabolism of this pathogen. Most H. pylori cultured under an optimum O2 concentration (7%) were the bacillary form, whereas about 80% of cells cultured under aerobic or anaerobic conditions were the coccoid form. The colony-forming unit of H. pylori decreased significantly under both aerobic and anaerobic culture conditions. The bacillary form of H. pylori generated predominantly superoxide radical, whereas the coccoid form generated preferentially hydroxyl radical. Specific activities of cellular respiration, urease, and superoxide dismatase decreased markedly after transformation of the bacillary form to the coccoid form, with concomitant generation of protein carbonyls and 8-hydroxyguanine. The frequency of mutation of cells increased significantly during culture under nonoptimum O2 conditions. These results indicate that ROS generated by H. pylori catalyze the oxidative modification of cellular DNA, thereby enhancing the transformation from the bacillary to the coccoid form. The enhanced generation of mutagenic hydroxyl radicals in the coccoid form might accelerate mutation and increase the genetic diversity of H. pylori.

Topics: Aerobiosis; Anaerobiosis; Bacterial Proteins; Ciprofloxacin; Cytochromes c; DNA; Drug Resistance; Guanine; Helicobacter pylori; Metronidazole; Mutation; Oxidative Stress; Oxygen; Reactive Oxygen Species; Rifampin; Superoxide Dismutase; Urease