The N-terminus of m5C-DNA methyltransferase MspI is involved in its topoisomerase activity

Sanjoy K. Bhattacharya, Ashok K. Dubey

Research output: Contribution to journalArticle

5 Scopus citations

Abstract

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 ≈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.

Original languageEnglish (US)
Pages (from-to)2491-2497
Number of pages7
JournalEuropean Journal of Biochemistry
Volume269
Issue number10
DOIs
StatePublished - 2002

Keywords

  • DNA binding
  • Methyltransferase MspI
  • Proteolysis
  • Topoisomerase I.

ASJC Scopus subject areas

  • Biochemistry

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