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The O6-methylguanine-DNA methyltransferase from the hyperthermophilic archaeon Pyrococcus sp. KOD1: a thermostable
M M Leclere1, M Nishioka, T Yuasa
1Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Japan.
Abstract:
The enzyme O6-methylguanine-DNA methyltransferase (MGMT) is the most common form of cellular defense against the biological effects of O6-methylguanine (O6-MeG) in DNA. Based on PCR amplification using primers derived from conserved amino acid sequences of MGMTs from 11 species, we isolated the DNA region coding for MGMT from the hyperthermophilic archaeon Pyrococcus sp. KOD1. The MGMT gene from KOD1 (mgtk) comprises 522 nucleotides, encoding 174 amino acid residues; its product shows considerable similarity to the corresponding mammalian, yeast and bacterial enzymes, especially around putative methyl acceptor sites. Phylogenetic analysis of MGMTs showed that archaeal MGMTs were grouped with their bacterial counterparts. The location of the MGMT gene on the KOD1 chromosome was also determined. The cloned KOD1 MGMT gene was overexpressed using the T7 RNA polymerase expression system, and the recombinant protein was purified by ammonium sulfate fractionation, heat treatment, ion-exchange chromatography and gel filtration chromatography. The purified recombinant protein was assayed for its enzyme activity by monitoring transfer of [3H]methyl groups from the substrate DNA to the MGMT protein; the activity was found to be stable at 90 degrees C for at least 30 min. When the mgtk gene was placed under the control of the lac promoter and expressed in the methyltransferase-deficient Escherichia coli strain KT233 (delta ada, delta ogt) cells, a MGMT was produced. The enzyme was functional in vivo and complemented the mutant phenotype, making the cells resistant to the cytotoxic properties of the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine.
Insights
The O6-methylguanine-DNA methyltransferase (MGMT) enzyme from Pyrococcus sp. KOD1 was cloned, expressed, and purified. This archaeal MGMT is heat-stable and functional in E. coli, demonstrating its role in DNA repair.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- O6-methylguanine-DNA methyltransferase (MGMT) is crucial for cellular defense against DNA damage from O6-methylguanine (O6-MeG).
- Understanding MGMT diversity and function across different life forms, including archaea, is important for DNA repair mechanisms.
Purpose of the Study:
- To isolate and characterize the MGMT gene from the hyperthermophilic archaeon Pyrococcus sp. KOD1.
- To investigate the enzymatic activity and stability of the recombinant archaeal MGMT.
- To assess the functional complementation of the gene in a bacterial system.
Main Methods:
- PCR amplification using conserved primers to isolate the MGMT gene (mgtk) from Pyrococcus sp. KOD1.
- Gene cloning, overexpression using the T7 RNA polymerase system, and protein purification via chromatography.
- Enzyme activity assays at high temperatures and complementation studies in an MGMT-deficient E. coli strain.
Main Results:
- The KOD1 MGMT gene (mgtk) encodes a 174-amino acid protein with similarity to other MGMTs.
- Phylogenetic analysis places archaeal MGMTs with bacterial counterparts.
- The purified recombinant enzyme is heat-stable up to 90°C and functionally complements E. coli mutants, conferring resistance to alkylating agents.
Conclusions:
- The study successfully cloned, expressed, and characterized a heat-stable MGMT from a hyperthermophilic archaeon.
- The findings highlight the conserved nature of MGMT across domains and its potential applications in DNA repair research.
- Archaeal MGMT exhibits functional similarity to its bacterial and eukaryotic homologs.