Time-resolved fluorescence of 2-aminopurine as a probe of base flipping in M.HhaI-DNA complexes

Robert K Neely, Dalia Daujotyte, Saulius Grazulis, Steven W Magennis, David T F Dryden, Saulius Klimasauskas, Anita C Jones

Research output: Contribution to journalArticlepeer-review

69 Citations (Scopus)


DNA base flipping is an important mechanism in molecular enzymology, but its study is limited by the lack of an accessible and reliable diagnostic technique. A series of crystalline complexes of a DNA methyltransferase, M.HhaI, and its cognate DNA, in which a fluorescent nucleobase analogue, 2-aminopurine (AP), occupies defined positions with respect the target flipped base, have been prepared and their structures determined at higher than 2 A resolution. From time-resolved fluorescence measurements of these single crystals, we have established that the fluorescence decay function of AP shows a pronounced, characteristic response to base flipping: the loss of the very short (approximately 100 ps) decay component and the large increase in the amplitude of the long (approximately 10 ns) component. When AP is positioned at sites other than the target site, this response is not seen. Most significantly, we have shown that the same clear response is apparent when M.HhaI complexes with DNA in solution, giving an unambiguous signal of base flipping. Analysis of the AP fluorescence decay function reveals conformational heterogeneity in the DNA-enzyme complexes that cannot be discerned from the present X-ray structures.

Original languageEnglish
Pages (from-to)6953-60
Number of pages8
JournalNucleic Acids Research
Issue number22
Publication statusPublished - 2005


  • 2-Aminopurine
  • Crystallography, X-Ray
  • DNA
  • DNA-Cytosine Methylases
  • Fluorescent Dyes
  • Hydrogen Bonding
  • Models, Molecular
  • Molecular Probes
  • Nucleic Acid Conformation
  • Spectrometry, Fluorescence
  • Time Factors


Dive into the research topics of 'Time-resolved fluorescence of 2-aminopurine as a probe of base flipping in M.HhaI-DNA complexes'. Together they form a unique fingerprint.

Cite this