Recognition and binding of mismatch repair proteins at an oncogenic hot spot

BACKGROUND: The current investigation was undertaken to determine key steps differentiating G:T and G:A repair at the H-ras oncogenic hot spot within the nuclear environment because of the large difference in repair efficiency of these two mismatches. RESULTS: Electrophoretic mobility shift (gel shift) experiments demonstrate that DNA containing mismatched bases are recognized and bound equally efficiently by hMutSα in both MMR proficient and MMR deficient (hMLH1-/-) nuclear extracts. Competition experiments demonstrate that while hMutSα predictably binds the G:T mismatch to a much greater extent than G:A, hMutSα demonstrates a surprisingly equal ratio of competitive inhibition for both G:T and G:A mismatch binding reactions at the H-ras hot spot of mutation. Further, mismatch repair assays reveal almost 2-fold higher efficiency of overall G:A repair (5'-nick directed correct MMR to G:C and incorrect repair to T:A), as compared to G:T overall repair. Conversely, correct MMR of G:T → G:C is significantly higher (96%) than that of G:A → G:C (60%). CONCLUSION: Combined, these results suggest that initiation of correct MMR requires the contribution of two separate steps; initial recognition by hMutSα followed by subsequent binding. The 'avidity' of the binding step determines the extent of MMR pathway activation, or the activation of a different cellular pathway. Thus, initial recognition by hMutSα in combination with subsequent decreased binding to the G:A mismatch (as compared to G:T) may contribute to the observed increased frequency of incorrect repair of G:A, resulting in the predominant GGC → GTC (Gly → Val) ras-activating mutation found in a high percentage of human tumors

Genetic microheterogeneity and phenotypic variation of Helicobacter pylori arginase in clinical isolates

BACKGROUND: Clinical isolates of the gastric pathogen Helicobacter pylori display a high level of genetic macro- and microheterogeneity, featuring a panmictic, rather than clonal structure. The ability of H. pylori to survive the stomach acid is due, in part, to the arginase-urease enzyme system. Arginase (RocF) hydrolyzes L-arginine to L-ornithine and urea, and urease hydrolyzes urea to carbon dioxide and ammonium, which can neutralize acid. RESULTS: The degree of variation in arginase was explored at the DNA sequence, enzyme activity and protein expression levels. To this end, arginase activity was measured from 73 minimally-passaged clinical isolates and six laboratory-adapted strains of H. pylori. The rocF gene from 21 of the strains was cloned into genetically stable E. coli and the enzyme activities measured. Arginase activity was found to substantially vary (>100-fold) in both different H. pylori strains and in the E. coli model. Western blot analysis revealed a positive correlation between activity and amount of protein expressed in most H. pylori strains. Several H. pylori strains featured altered arginase activity upon in vitro passage. Pairwise alignments of the 21 rocF genes plus strain J99 revealed extensive microheterogeneity in the promoter region and 3' end of the rocF coding region. Amino acid S232, which was I232 in the arginase-negative clinical strain A2, was critical for arginase activity. CONCLUSION: These studies demonstrated that H. pylori arginase exhibits extensive genotypic and phenotypic variation which may be used to understand mechanisms of microheterogeneity in H. pylori

Genetic Microheterogeneity and Phenotypic Variation of \u3ci\u3eHelicobacter pylori\u3c/i\u3e Arginase in Clinical Isolates

Background: Clinical isolates of the gastric pathogen Helicobacter pylori display a high level of genetic macro- and microheterogeneity, featuring a panmictic, rather than clonal structure. The ability of H. pylori to survive the stomach acid is due, in part, to the arginase-urease enzyme system. Arginase (RocF) hydrolyzes L-arginine to L-ornithine and urea, and urease hydrolyzes urea to carbon dioxide and ammonium, which can neutralize acid. Results: The degree of variation in arginase was explored at the DNA sequence, enzyme activity and protein expression levels. To this end, arginase activity was measured from 73 minimally-passaged clinical isolates and six laboratory-adapted strains of H. pylori. The rocF gene from 21 of the strains was cloned into genetically stable E. coli and the enzyme activities measured. Arginase activity was found to substantially vary (\u3e100-fold) in both different H. pylori strains and in the E. coli model. Western blot analysis revealed a positive correlation between activity and amount of protein expressed in most H. pylori strains. Several H. pylori strains featured altered arginase activity upon in vitro passage. Pairwise alignments of the 21 rocF genes plus strain J99 revealed extensive microheterogeneity in the promoter region and 3\u27 end of the rocF coding region. Amino acid S232, which was I232 in the arginase-negative clinical strain A2, was critical for arginase activity. Conclusion: These studies demonstrated that H. pylori arginase exhibits extensive genotypic and phenotypic variation which may be used to understand mechanisms of microheterogeneity in H. pylori

Recognition and binding of mismatch repair proteins at an oncogenic hot spot-3

<p><b>Copyright information:</b></p><p>Taken from "Recognition and binding of mismatch repair proteins at an oncogenic hot spot"</p><p>BMC Molecular Biology 2005;6():6-6.</p><p>Published online 14 Mar 2005</p><p>PMCID:PMC555755.</p><p></p>ir (differential) of DNA containing a G:T or a G:A mismatch at the H-codon 12 location. hMutSα recognizes and forms an initial recognition complex (IRC) with both mismatches equally, and then binds more strongly to G:T, perhaps by undergoing an additional conformational step to the ultimate recognition complex (URC), which does not occur with G:A [40]. This results in more accurate repair of G:T, but more frequent total repair of G:A. See text for further discussion

Genetic microheterogeneity and phenotypic variation of \u3cem\u3eHelicobacter pylori\u3c/em\u3e arginase in clinical isolates

Background: Clinical isolates of the gastric pathogen Helicobacter pylori display a high level of genetic macro- and microheterogeneity, featuring a panmictic, rather than clonal structure. The ability of H. pylori to survive the stomach acid is due, in part, to the arginase-urease enzyme system. Arginase (RocF) hydrolyzes L-arginine to L-ornithine and urea, and urease hydrolyzes urea to carbon dioxide and ammonium, which can neutralize acid. Results: The degree of variation in arginase was explored at the DNA sequence, enzyme activity and protein expression levels. To this end, arginase activity was measured from 73 minimally-passaged clinical isolates and six laboratory-adapted strains of H. pylori. The rocF gene from 21 of the strains was cloned into genetically stable E. coli and the enzyme activities measured. Arginase activity was found to substantially vary (\u3e100-fold) in both different H. pylori strains and in the E. coli model. Western blot analysis revealed a positive correlation between activity and amount of protein expressed in most H. pylori strains. Several H. pylori strains featured altered arginase activity upon in vitro passage. Pairwise alignments of the 21 rocF genes plus strain J99 revealed extensive microheterogeneity in the promoter region and 3\u27 end of the rocF coding region. Amino acid S232, which was I232 in the arginase-negative clinical strain A2, was critical for arginase activity. Conclusion: These studies demonstrated that H. pylori arginase exhibits extensive genotypic and phenotypic variation which may be used to understand mechanisms of microheterogeneity in H. pylori