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Bacillus subtilis alkA gene encoding inducible 3-methyladenine DNA glycosylase is adjacent to the ada operon.

ABSTRACT: In Bacillus subtilis, the adaptive response to DNA alkylation depends on the ada operon, which consists of the adaA and adaB genes, which encode methylphosphotriester DNA methyltransferase (AdaA protein) and O6-methylguanine DNA methyltransferase (AdaB protein), respectively. A structural gene (alkA) that encodes 3-methyladenine DNA glycosylase was found upstream of the ada operon, but in the opposite orientation. This cluster of genes was mapped at about 235 kb from the SfiI recognition site near the origin of replication in the physical map of the B. subtilis chromosome. Disruption of the alkA gene sensitized cells to N-propyl-N'-nitro-N-nitrosoguanidine, while its overproduction rendered cells highly resistant to N-propyl-N'-nitro-N-nitrosoguanidine, indicating that lethal DNA damage produced by bulky alkylating agents was effectively counteracted by AlkA glycosylase. Transcription of the alkA gene was induced by treating adaA+ cells with methylating agents concurrent with transcription of the ada operon. This was accomplished by using methylated AdaA protein bound to a 30-bp segment in the middle of the 100-bp sequence between the transcriptional start sites of the alkA gene and ada operon. Thus, in this organism, the adaptive response to DNA alkylation is achieved by autologous activation of a divergent regulon composed of the genes for a DNA glycosylase and two species of DNA alkyltransferase.

SUBMITTER: Morohoshi F

PROVIDER: S-EPMC206683 | biostudies-other | 1993 Sep

REPOSITORIES: biostudies-other

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Bacillus subtilis alkA gene encoding inducible 3-methyladenine DNA glycosylase is adjacent to the ada operon.

Morohoshi F F Hayashi K K Munkata N N

Journal of bacteriology 19930901 18

In Bacillus subtilis, the adaptive response to DNA alkylation depends on the ada operon, which consists of the adaA and adaB genes, which encode methylphosphotriester DNA methyltransferase (AdaA protein) and O6-methylguanine DNA methyltransferase (AdaB protein), respectively. A structural gene (alkA) that encodes 3-methyladenine DNA glycosylase was found upstream of the ada operon, but in the opposite orientation. This cluster of genes was mapped at about 235 kb from the SfiI recognition site ne ...[more]

PMID: 8376346

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Project description:BACKGROUND:The emergence of artemisinin-resistant malaria parasites highlights the need for novel drugs and their targets. Alkylation of purine bases can hinder DNA replication and if unresolved would eventually result in cell death. DNA-3-methyladenine glycosylase (MAG) is responsible for the repair of those alkylated bases. Plasmodium falciparum (Pf) MAG was characterized for its potential for development as an anti-malarial candidate. METHODS:Native PfMAG from crude extract of chloroquine- and pyrimethamine-resistant P. falciparum K1 strain was partially purified using three chromatographic procedures. From bio-informatics analysis, primers were designed for amplification, insertion into pBAD202/D-TOPO and heterologous expression in Escherichia coli of recombinant PfMAG. Functional and biochemical properties of the recombinant enzyme were characterized. RESULTS:PfMAG activity was most prominent in parasite schizont stages, with a specific activity of 147 U/mg (partially purified) protein. K1 PfMAG contained an insertion of AAT (coding for asparagine) compared to 3D7 strain and 16% similarity to the human enzyme. Recombinant PfMAG (74 kDa) was twice as large as the human enzyme, preferred double-stranded DNA substrate, and demonstrated glycosylase activity over a pH range of 4-9, optimal salt concentration of 100-200 mM NaCl but reduced activity at 250 mM NaCl, no requirement for divalent cations, which were inhibitory in a dose-dependent manner. CONCLUSION:PfMAG activity increased with parasite development being highest in the schizont stages. K1 PfMAG contained an indel AAT (asparagine) not present in 3D7 strain and the recombinant enzyme was twice as large as the human enzyme. Recombinant PfMAG had a wide range of optimal pH activity, and was inhibited at high (250 mM) NaCl concentration as well as by divalent cations. The properties of PfMAG provide basic data that should be of assistance in developing anti-malarials against this potential parasite target.

Dataset Information

Bacillus subtilis alkA gene encoding inducible 3-methyladenine DNA glycosylase is adjacent to the ada operon.

Publications

Bacillus subtilis alkA gene encoding inducible 3-methyladenine DNA glycosylase is adjacent to the ada operon.

Similar Datasets

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