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 Citations (Scopus)

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
Pages (from-to)2491-2497
Number of pages7
JournalEuropean Journal of Biochemistry
Volume269
Issue number10
DOIs
StatePublished - Jun 1 2002
Externally publishedYes

Fingerprint

Methyltransferases
DNA
Type I DNA Topoisomerase
Cytosine
2-Hydroxy-5-nitrobenzyl Bromide
Amino Acid Sequence
Amino Acids
Bacterial DNA
Proteolysis
Peptide Fragments
DNA Ligases
Recombinant proteins
DNA Methylation
m(5)C rRNA methyltransferase
Bromides
Recombinant Proteins
Alanine
Molecular mass
Catalytic Domain
Proteins

Keywords

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

ASJC Scopus subject areas

  • Biochemistry

Cite this

The N-terminus of m5C-DNA methyltransferase MspI is involved in its topoisomerase activity. / Bhattacharya, Sanjoy K; Dubey, Ashok K.

In: European Journal of Biochemistry, Vol. 269, No. 10, 01.06.2002, p. 2491-2497.

Research output: Contribution to journalArticle

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AB - 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.

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