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

Authors
Publisher
Elsevier Inc.
Publication Date
Volume
449
Issue
1
Identifiers
DOI: 10.1016/j.bbrc.2014.04.162
Keywords
  • Ecop15I
  • Dna Restriction And Modification
  • 2-Aminopurine Fluorescence
  • Nucleotide Flipping
  • Time Correlated Single Photon Counting
Disciplines
  • Biology

Abstract

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.

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