RESUMO
8-oxoguanine DNA glycosylase 1 (OGG1) is a DNA repair enzyme that excises 7,8-dihydro-8-oxoguanine (8oxoG) from DNA. Since 8oxoG is a highly mispairing lesion, decreased OGG1 expression level could lead to a higher background mutation frequency and could possibly increase the cancer risk of an individual under oxidative stress. In order to analyse the natural variation of OGG1, we measured the DNA repair activity in human lymphocytes of healthy individuals by means of an 8oxoG-containing oligonucleotide assay. The data obtained revealed a two fold interindividual variation of OGG1 activity in lymphocytes. There was no difference in OGG1 activity due to gender and smoking behaviour. Transcriptional analyses of OGG1 showed the expression of two isoforms, 1a and b, in lymphocytes. Structural analysis of the human OGG1 (hOGG1) gene revealed a Ser326/Cys326 polymorphism in the Caucasian population with allele frequencies of 75% for Ser326 and 25% for Cys326. This polymorphism was not associated with altered OGG1 activity. The described routine test system for measuring OGG1 activity in cryopreserved lymphocytes provided highly reproducible results and is a useful tool for risk assessment associated with alterations in the repair of oxidative DNA damage.
Assuntos
Reparo do DNA , Linfócitos/metabolismo , N-Glicosil Hidrolases/genética , N-Glicosil Hidrolases/metabolismo , Alelos , Sequência de Aminoácidos , Sequência de Bases , Dano ao DNA , Primers do DNA/genética , Primers do DNA/metabolismo , DNA-Formamidopirimidina Glicosilase , Frequência do Gene , Humanos , Técnicas In Vitro , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Polimorfismo Genético , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
In methanogenic archaea the transfer of the methyl group of N5-methyltetrahydromethanopterin to coenzyme M is coupled with energy conservation. The reaction is catalyzed by a membrane associated multienzyme complex composed of eight different subunits MtrA-H. The 23 kDa subunit MtrA harbors a corrinoid prosthetic group which is methylated and demethylated in the catalytic cycle. We report here that the 34 kDa subunit MtrH catalyzes the methylation reaction. MtrH was purified and shown to exhibit methyltetrahydromethanopterin:cob(I)alamin methyltransferase activity. Sequence comparison revealed similarity of MtrH with MetH from Escherichia coli and AcsE from Clostridium thermoaceticum: both enzymes exhibit methyltetrahydrofolate:cob(I)alamin methyltransferase activity.
Assuntos
Methanobacterium/enzimologia , Metiltransferases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Metabolismo Energético , Proteínas Ferro-Enxofre/metabolismo , Metiltransferases/química , Metiltransferases/genética , Dados de Sequência Molecular , Pterinas/metabolismo , Homologia de Sequência de Aminoácidos , Tetra-Hidrofolatos/metabolismo , Vitamina B 12/análogos & derivados , Vitamina B 12/metabolismoRESUMO
The insertion mutant HF231 of Alcaligenes eutrophus H16 failed to grow anaerobically on nitrate and nitrite. When grown under oxygen limitation, mutant HF231 specifically excreted coproporphyrin III, an intermediate of heme biosynthesis. With the help of a Tn5-labeled fragment, we identified and cloned the corresponding wild-type fragment. Sequence analysis of the mutant locus revealed an open reading frame consisting of 1,473 bp, predicting a protein of 491 amino acids that corresponds to a size of 54.2 kDa. In the non-coding upstream region, consensus elements that are indicative for binding sites of the anaerobic transcriptional regulator Fnr were identified. The deduced polypeptide showed extensive sequence similarity with various bacterial oxygen-independent coproporphyrinogen III oxidases designated HemN. HemN catalyzes the oxidative decarboxylation of coproporphyrinogen III to yield protoporphyrinogen IX. Anaerobic growth on nitrate and nitrite of mutant HF231 was restored by introducing the hemN gene of A. eutrophus or of Pseudomonas aeruginosa on a broad-host-range vector. Likewise, the A. eutrophus hemN complemented heme biosynthesis of a Salmonella typhimurium hemF/hemN double mutant during anaerobic and aerobic growth. Analysis of a transcriptional lacZ gene fusion showed that expression of hemN in A. eutrophus is nitrate-independent and repressed by oxygen.
Assuntos
Alcaligenes/enzimologia , Proteínas de Bactérias/metabolismo , Coproporfirinogênio Oxidase/metabolismo , Heme/biossíntese , Alcaligenes/genética , Alcaligenes/crescimento & desenvolvimento , Sequência de Aminoácidos , Anaerobiose , Proteínas de Bactérias/genética , Clonagem Molecular , Coproporfirinogênio Oxidase/genética , Coproporfirinas/biossíntese , Genes Bacterianos/genética , Teste de Complementação Genética , Dados de Sequência Molecular , Mutação , Nitratos/metabolismo , Oxigênio/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de Fusão , Mapeamento por Restrição , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
A recently cloned Bacillus subtilis open reading frame (hemN) upstream of the dnaK operon was identified as encoding a protein involved in oxygen-independent coproporphyrinogen III decarboxylation. B. subtilis hemN functionally complemented two Salmonella typhimurium hemF hemN double mutants under aerobic and anaerobic conditions. A B. subtilis hemN mutant accumulated coproporphyrinogen III only under anaerobic conditions. Interestingly, growth experiments using the B. subtilis hemN mutant revealed normal aerobic and anaerobic growth, indicating the presence of an alternative oxygen-independent enzymatic system. Northern blot experiments identified hemN mRNA as part of an approximately 7-kb pentacistronic transcript consisting of lepA, hemN, hrcA, grpE, and dnaK. One potential start site for aerobic and anaerobic transcription was located 37 bp upstream of the translational start codon of lepA. Comparable amounts of hemN transcript were observed under aerobic and anaerobic growth conditions. No experimental evidence for the presence of hemF in B. subtilis was obtained. Moreover, B. subtilis hemY did not substitute for hemF hemN deficiency in S. typhimurium. These results indicate the absence of hemF and suggest the presence of a second hemN-like gene in B. subtilis.
