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

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-amino...

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Autores principales: Neely, Robert K., Daujotyte, Dalia, Grazulis, Saulius, Magennis, Steven W., Dryden, David T. F., Klimašauskas, Saulius, Jones, Anita C.
Formato: Texto
Lenguaje:English
Publicado: Oxford University Press 2005
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1310896/
https://www.ncbi.nlm.nih.gov/pubmed/16340006
http://dx.doi.org/10.1093/nar/gki995
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author Neely, Robert K.
Daujotyte, Dalia
Grazulis, Saulius
Magennis, Steven W.
Dryden, David T. F.
Klimašauskas, Saulius
Jones, Anita C.
author_facet Neely, Robert K.
Daujotyte, Dalia
Grazulis, Saulius
Magennis, Steven W.
Dryden, David T. F.
Klimašauskas, Saulius
Jones, Anita C.
author_sort Neely, Robert K.
collection PubMed
description 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 Å 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 (∼100 ps) decay component and the large increase in the amplitude of the long (∼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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spelling pubmed-13108962005-12-12 Time-resolved fluorescence of 2-aminopurine as a probe of base flipping in M.HhaI–DNA complexes Neely, Robert K. Daujotyte, Dalia Grazulis, Saulius Magennis, Steven W. Dryden, David T. F. Klimašauskas, Saulius Jones, Anita C. Nucleic Acids Res Article 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 Å 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 (∼100 ps) decay component and the large increase in the amplitude of the long (∼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. Oxford University Press 2005 2005-12-09 /pmc/articles/PMC1310896/ /pubmed/16340006 http://dx.doi.org/10.1093/nar/gki995 Text en © The Author 2005. Published by Oxford University Press. All rights reserved
spellingShingle Article
Neely, Robert K.
Daujotyte, Dalia
Grazulis, Saulius
Magennis, Steven W.
Dryden, David T. F.
Klimašauskas, Saulius
Jones, Anita C.
Time-resolved fluorescence of 2-aminopurine as a probe of base flipping in M.HhaI–DNA complexes
title Time-resolved fluorescence of 2-aminopurine as a probe of base flipping in M.HhaI–DNA complexes
title_full Time-resolved fluorescence of 2-aminopurine as a probe of base flipping in M.HhaI–DNA complexes
title_fullStr Time-resolved fluorescence of 2-aminopurine as a probe of base flipping in M.HhaI–DNA complexes
title_full_unstemmed Time-resolved fluorescence of 2-aminopurine as a probe of base flipping in M.HhaI–DNA complexes
title_short Time-resolved fluorescence of 2-aminopurine as a probe of base flipping in M.HhaI–DNA complexes
title_sort time-resolved fluorescence of 2-aminopurine as a probe of base flipping in m.hhai–dna complexes
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1310896/
https://www.ncbi.nlm.nih.gov/pubmed/16340006
http://dx.doi.org/10.1093/nar/gki995
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