Recombinant alpha and beta subunits of M.AquI constitute an active DNA methyltransferase.
AquI DNA methyltransferase, M.AquI, catalyses the transfer of a methyl group from S-adenosyl-L-methionine to the C5 position of the outermost deoxycytidine base in the DNA sequence 5'CYCGRG3'. M.AquI is encoded by two overlapping ORFs (termed alpha and beta) instead of the single ORF that is customary for Class II methyltransferase genes. The structural organization of the M.AquI protein sequence is quite similar to that of other bacterial C5-DNA methyltransferases. Ten conserved motifs are also present in the correct order, but only on two polypeptides. We separately subcloned the genes that encode the alpha and beta subunits of M.AquI into expression vectors. The overexpressed His-fusion alpha and beta subunits of the enzyme were purified to homogeneity in a single step by Nickel-chelate affinity chromatography. The purified recombinant proteins were assayed for biological activity by an in vitro DNA tritium transfer assay. The alpha and beta subunits of M.AquI alone have no DNA methyltransferase activity, but when both subunits are included in the assay, an active enzyme that catalyses the transfer of the methyl group from S-adenosyl-Lmethionine to DNA is reconstituted. We also showed that the beta subunit alone contains all of the information that is required to generate recognition of specific DNA duplexes in the absence of the alpha subunit
Pinarbasi
Hatice
H
Krebs Institute, University of Sheffield, Department of Molecular Biology and Biotechnology, S10 2TN UK. hpinar@cumhuriyet.edu.tr
Pinarbasi
Ergun
E
Hornby
David
D
D001665:Binding Sites; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D015257:DNA-Cytosine Methylases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000378:metabolism
D017931:DNA Primers; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D006639:Histidine; D012436:S-Adenosylmethionine; D004247:DNA; C049759:DNA modification methylase M.AquI; D015257:DNA-Cytosine Methylases