期刊
NUCLEIC ACIDS RESEARCH
卷 33, 期 22, 页码 6953-6960出版社
OXFORD UNIV PRESS
DOI: 10.1093/nar/gki995
关键词
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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 angstrom 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 (similar to 100 ps) decay component and the large increase in the amplitude of the long (similar to 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.
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