The N-terminus of m5C-DNA methyltransferase MspI is involved in its topoisomerase activity.
DNA cytosine methyltransferase MspI (M.MspI) must require a different type of interaction of protein with DNA from other bacterial DNA cytosine methyltransferases (m5C-MTases) to evoke the topoisomerase activity that it possesses in addition to DNA-methylation ability. This may require a different structural organization in the solution phase from the reported consensus structural arrangement for m5C-MTases. Limited proteolysis of M.MspI, however, generates two peptide fragments, a large one (p26) and a small one (p18), consistent with reported m5C-MTase structures. Examination of the amino-acid sequence of M.MspI revealed similarity to human topoisomerase I at the N-terminus. Alignment of the amino-acid sequence of M.MspI also uncovered similarity (residues 245-287) to the active site of human DNA ligase I. To evaluate the role of the N-terminus of M.MspI, 2-hydroxy-5-nitrobenzyl bromide (HNBB) was used to truncate M.MspI between residues 34 and 35. The purified HNBB-truncated protein has a molecular mass of approximately equal 45 kDa, retains DNA binding and methyltransferase activity, but does not possess topoisomerase activity. These findings were substantiated using a purified recombinant MspI protein with the N-terminal 34 amino acids deleted. Changing the N-terminal residues Trp34 and Tyr74 to alanine results in abolition of the topoisomerase I activity while the methyltransferase activity remains intact.
Bhattacharya
Sanjoy K
SK
Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology-Delhi, New Delhi, India. bhattas@ccf.org
Dubey
Ashok K
AK
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D011088:DNA Ligases / Q000235:genetics; D026942:DNA Topoisomerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism; D015257:DNA-Cytosine Methylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli; D008969:Molecular Sequence Data; D010446:Peptide Fragments; D016415:Sequence Alignment
D004269:DNA, Bacterial; D010446:Peptide Fragments; C029641:DNA modification methylase HpaII; D015257:DNA-Cytosine Methylases; D026942:DNA Topoisomerases; D011088:DNA Ligases