Time-resolved fluorescence of 2-aminopurine in DNA duplexes in the presence of the EcoP15I Type III restriction-modification enzyme

Long Ma, Xiaohua Wu, Geoffrey G. Wilson, Anita C. Jones, David T.f. Dryden

Research output: Contribution to journalArticlepeer-review

Abstract

EcoP15I is a Type III DNA restriction and modification enzyme of Escherichia coli. We show that it contains two modification (Mod) subunits for sequence-specific methylation of DNA and one copy of a restriction endonuclease (Res) subunit for cleavage of DNA containing unmethylated target sequences. Previously the Mod2 dimer in the presence of cofactors was shown to use nucleotide flipping to gain access to the adenine base targeted for methylation (Reddy and Rao, J. Mol. Biol. 298 (2000) 597–610.). Surprisingly the Mod2 enzyme also appeared to flip a second adenine in the target sequence, one which was not subject to methylation. We show using fluorescence lifetime measurements of the adenine analogue, 2-aminopurine, that only the methylatable adenine undergoes flipping by the complete Res1Mod2 enzyme and that this occurs even in the absence of cofactors. We suggest that this is due to activation of the Mod2 core by the Res subunit.
Original languageEnglish
Pages (from-to)120-125
JournalBiochemical and Biophysical Research Communications
Volume449
Issue number1
Early online date9 May 2014
DOIs
Publication statusPublished - 1 Jun 2014

Keywords

  • EcoP15I
  • DNA restriction and modification
  • 2-Aminopurine fluorescence
  • Nucleotide flipping
  • Time correlated single photon counting

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