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PDBsum entry 2c7q
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Transferase/DNA
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PDB id
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2c7q
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References listed in PDB file
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Key reference
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Title
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Time-Resolved fluorescence of 2-Aminopurine as a probe of base flipping in m.Hhai-Dna complexes.
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Authors
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R.K.Neely,
D.Daujotyte,
S.Grazulis,
S.W.Magennis,
D.T.Dryden,
S.Klimasauskas,
A.C.Jones.
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Ref.
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Nucleic Acids Res, 2005,
33,
6953-6960.
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PubMed id
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Abstract
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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 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.
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Figure 2.
Detailed view of the H-bond interactions between the M.HhaI enzyme and the
APtarget duplex. DNA and protein residues are shown as sticks, a bound solvent molecule
(presumed water) is shown as a red ball.
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Figure 3.
Interaction of M.HhaI with duplexes where AP is opposite or adjacent to the
target base. (a) The sequences of the 10 bp at the centre of the APadj duplex (left) and
APopp duplex (right). Bases in the M.HhaI recognition sequence are shown in
boldface/italic; the target base is circled; AP is denoted P and is in red; M is 5-methyl
cytosine (used to direct enzyme binding to the opposite strand of the duplex). (b) Crystal
structures of the complexes of the M.HhaI enzyme with the APadj duplex (left) and the
APopp duplex (right), showing the molecular structure in the vicinity of the recognition
sequence. (c) Detailed view of the H-bond interactions between the M.HhaI enzyme and the
APadj duplex (left) and the APopp duplex (right).
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