The Nâterminus of m5CâDNA methyltransferase MspI is involved in its topoisomerase activity
2002
Bhattacharya, Sanjoy K. | Dubey, Ashok K.
DNA cytosine methyltransferase MspI (M.MspI) must require a different type of interaction of protein with DNA from other bacterial DNA cytosine methyltransferases (m5CâMTases) to evoke the topoisomerase activity that it possesses in addition to DNAâmethylation ability. This may require a different structural organization in the solution phase from the reported consensus structural arrangement for m5CâMTases. Limited proteolysis of M.MspI, however, generates two peptide fragments, a large one (p26) and a small one (p18), consistent with reported m5CâMTase structures. Examination of the aminoâacid sequence of M.MspI revealed similarity to human topoisomeraseâI at the Nâterminus. Alignment of the aminoâacid sequence of M.MspI also uncovered similarity (residues 245–287) to the active site of human DNA ligaseâI. To evaluate the role of the Nâterminus of M.MspI, 2âhydroxyâ5ânitrobenzyl bromide (HNBB) was used to truncate M.MspI between residues 34 and 35. The purified HNBBâtruncated protein has a molecular mass of ≈â45âkDa, retains DNA binding and methyltransferase activity, but does not possess topoisomerase activity. These findings were substantiated using a purified recombinant MspI protein with the Nâterminal 34 amino acids deleted. Changing the Nâterminal residues Trp34 and Tyr74 to alanine results in abolition of the topoisomeraseâI activity while the methyltransferase activity remains intact.
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