Assuntos
Bacillus subtilis/enzimologia , DNA Nucleotidiltransferases/isolamento & purificação , Isoenzimas/isolamento & purificação , Cloromercurobenzoatos/farmacologia , Cromatografia DEAE-Celulose , Cromatografia por Troca Iônica , Citarabina/farmacologia , DNA Nucleotidiltransferases/metabolismo , DNA de Cadeia Simples , Estabilidade de Medicamentos , Etilmaleimida/farmacologia , Estudos de Avaliação como Assunto , Congelamento , Genes , Temperatura Alta , Isoenzimas/metabolismo , Métodos , Polietilenoglicóis , Moldes Genéticos , Nucleotídeos de Timina , Fatores de Tempo , Trítio , UltrassomAssuntos
Bacillus subtilis/enzimologia , DNA Nucleotidiltransferases/metabolismo , Genes , Sulfato de Amônio , Bacillus subtilis/crescimento & desenvolvimento , Bacteriófagos , Cromatografia DEAE-Celulose , Cromatografia por Troca Iônica , Mapeamento Cromossômico , Meios de Cultura , DNA Nucleotidiltransferases/antagonistas & inibidores , DNA Nucleotidiltransferases/isolamento & purificação , Reparo do DNA , Replicação do DNA , Estabilidade de Medicamentos , Endonucleases/metabolismo , Exonucleases/metabolismo , Temperatura Alta , Mutação , Radioisótopos de Fósforo , Nucleotídeos de Timina/metabolismo , Transdução Genética , Transformação Genética , Trítio , VibraçãoRESUMO
The antimicrobial agent, 6-(p-hydroxyphenylazo)-uracil, specifically inhibits DNA polymerase III of Bacillus subtilis with a K(i) of less than 1 muM. The inhibition requires prior reduction of the drug, is reversible, and is competitive with dGTP. High amounts of dATP prevent inhibition by the closely related drug, 6-(p-hydroxyphenylazo)-isocytosine. A model is presented in which the inhibitors base-pair with the template while part of a ternary complex with the enzyme. These results imply that DNA polymerase III of B. subtilis is necessary for chromosomal replication.
Assuntos
Bacillus subtilis/enzimologia , DNA Nucleotidiltransferases/antagonistas & inibidores , Uracila/farmacologia , Trifosfato de Adenosina/farmacologia , Compostos Azo/antagonistas & inibidores , Compostos Azo/farmacologia , DNA Nucleotidiltransferases/metabolismo , DNA Bacteriano/metabolismo , Ditiotreitol/farmacologia , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/farmacologia , Cinética , Fenóis/antagonistas & inibidores , Fenóis/farmacologia , Polinucleotídeos/antagonistas & inibidores , Polinucleotídeos/metabolismo , Moldes Genéticos , Nucleotídeos de Timina/metabolismo , Trítio , Uracila/antagonistas & inibidoresRESUMO
Of six deoxyribonucleic acid repair mutants of Bacillus subtilis assayed for deoxyribonucleic acid polymerase, only the methyl methanesulfonate-sensitive and ultraviolet light-sensitive mutant JB1-49(59) has impaired polymerase activity. Extracts prepared by sonic treatment or gentle lysis had about 10% of the wild-type activity with poly d(A-T), an alternating copolymer of deoxyadenylate and deoxythymidylate, used as template. The sensitivity to methyl methanesulfonate and ultraviolet light and the low level of polymerase activity transformed and reverted together, indicating that the two characteristics are a pleiotropic manifestation of a single mutation. Mixed extract and kinetic experiments mitigated against an altered nuclease activity as the enzymatic consequence of the mutation. Also, the mutant and wild type activities were stimulated equally by Escherichia coli exonuclease III. The residual activity in the mutant showed several differences from the wild-type activity: it purified differently, was more sensitive to sulfhydryl reagents, and displayed a different template specificity. We tentatively conclude that either the mutation in JB1-49(59) has introduced a qualitative as well as a quantitative change in the polymerase or the wild type contains two distinct polymerases, one of which is missing in the mutant.