Purification and characterization of rice DNA methyltransferase.
Epigenetic modification is essential for normal development and plays important roles in gene regulation in higher plants. Multiple factors interact to regulate the establishment and maintenance of DNA methylation in plant genome. We had previously cloned and characterized DNA methyltransferase (DNA MTase) gene homologues (OsMET1) from rice. In this present study, determination of DNA MTase activity in different cellular compartments showed that DNA MTase was enriched in nuclei and the activity was remarkably increased during imbibing dry seeds. We had optimized the purification technique for DNA MTase enzyme from shoots of 10-day-old rice seedlings using the three successive chromatographic columns. The Econo-Pac Q, the Hitrap-Heparin and the Superdex-200 columns yielded a protein fraction of a specific activity of 29, 298 and 800 purification folds, compared to the original nuclear extract, respectively. The purified protein preferred hemi-methylated DNA substrate, suggesting the maintenance activity of methylation. The native rice DNA MTase was approximately 160-170 kDa and exhibited a broad pH optimum in the range of 7.6 and 8.0. The enzyme kinetics and inhibitory effects by methyl donor analogs, base analogs, cations, and cationic amines on rice DNA MTase were examined. Global cytosine methylation status of rice genome during development and in various tissue culture systems were monitored and the results suggested that the cytosine methylation level is not directly correlated with the DNA MTase activity. The purification and characterization of rice DNA MTase enzyme are expected to enhance our understanding of this enzyme function and their possible contributions in Gramineae plant development.
Teerawanichpan
Prapapan
P
Department of Biotechnology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand.
Krittanai
Palika
P
Chauvatcharin
Nopmanee
N
Narangajavana
Jarunya
J
D002467:Cell Nucleus / Q000201:enzymology; D002845:Chromatography; D003596:Cytosine / Q000378:metabolism; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D018744:DNA, Plant; D044127:Epigenesis, Genetic; D018506:Gene Expression Regulation, Plant; D012275:Oryza / Q000737:chemistry / Q000196:embryology / Q000201:enzymology*; D036226:Seedlings / Q000201:enzymology; D012639:Seeds / Q000201:enzymology; D013379:Substrate Specificity
D018744:DNA, Plant; D003596:Cytosine; D015254:DNA Modification Methylases