Unusual 2-aminopurine fluorescence from a complex of DNA and the EcoKI methyltransferase

Su, Ting; Connolly, Bernard A.; Darlington, C. D.; Mallin, Rosie L.; Dryden, David T. F.

doi:10.1093/nar/gkh531

Cited by 30 publications

(35 citation statements)

References 47 publications

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“…Accordingly, dipole-dipole interactions between 2AP and polar solvents will divert energy from 2AP, resulting in red-shifted emission. Blue-shifted emission has also been reported for a 2AP-labeled DNA substrate upon addition of the DNA methyltransferase EcoKI [33].…”

Section: Effect Of Microenvironment On 2ap Lifetime Distributionmentioning

confidence: 75%

“…Furthermore, the large FWHM value for 2AP measured at the shorter wavelength ( Figure 8) is consistent with increased heterogeneity of 2AP emission from the F state versus the R state. In previous studies, differences in fluorescence lifetime properties from F versus R states of the single tryptophan residue in staphylococcal nuclease revealed that local intramolecular dynamics create a polar environment around tryptophan rather than exposure to aqueous solvent [33]. Similarly, 2AP intensity decays from F and R states may help to predict the microenvironment around 2AP in different states.…”

Section: Effect Of Spectral Relaxation On 2-ap Lifetime Distributionmentioning

confidence: 97%

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Fluorescence intensity decays of 2-aminopurine solutions: Lifetime distribution approach

Bharill

Sarkar

Ballin

et al. 2008

Analytical Biochemistry

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The fluorescent adenine analogue 2-aminopurine (2AP) has been used extensively to monitor conformational changes and macromolecular binding events involving nucleic acids because its fluorescence properties are highly sensitive to changes in chemical environment. Furthermore, site-specific incorporation of 2AP permits local DNA and RNA conformational events to be discriminated from the global structural changes monitored by UV/Vis spectroscopy and circular dichroism. However, while the steady-state fluorescence properties of 2AP have been well defined in diverse settings, interpretation of 2AP fluorescence lifetime parameters has been hampered by the heterogeneous nature of multi-exponential 2AP intensity decays observed across populations of microenvironments. To resolve this problem, we have tested the utility of multi-exponential versus continuous Lorentzian lifetime distribution models to describe fluorescence intensity decays from 2AP in diverse chemical backgrounds and within the context of RNA. Heterogeneity was introduced into 2AP intensity decays by mixing solvents of differing polarities, or by adding quenchers under high viscosity to evaluate the transient effect. Heterogeneity of 2AP fluorescence within the context of a synthetic RNA hairpin was introduced by structural remodeling using a magnesium salt. In each case, except folded RNA which required a bimodal distribution, 2AP lifetime properties were well described by single Lorentzian distribution functions, abrogating the need to introduce additional discrete lifetime subpopulations. Rather, heterogeneity in fluorescence decay processes was accommodated by the breadth of each distribution. This approach also permitted solvent relaxation effects on 2AP emission to be assessed by comparing lifetime distributions at multiple wavelengths. Together, these studies provide a new perspective for the interpretation of 2AP fluorescence lifetime properties, which will further the utility of this Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript fluorophore in analyses of the complex and heterogeneous structural microenvironments associated with nucleic acids.

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Section: Effect Of Microenvironment On 2ap Lifetime Distributionmentioning

confidence: 75%

Section: Effect Of Spectral Relaxation On 2-ap Lifetime Distributionmentioning

confidence: 97%

Fluorescence intensity decays of 2-aminopurine solutions: Lifetime distribution approach

Bharill

Sarkar

Ballin

et al. 2008

Analytical Biochemistry

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“…The annealing efficiency of all duplexes or gapped duplexes (i.e. those with two short oligonucleotides annealed to a single long oligonucleotide) was checked using analytical high-performance liquid chromatography as described elsewhere (6). A molar ratio of 1:1 was obtained for the annealed duplexes and of 1:1:1 for the gapped duplexes, as required.…”

Section: Methodsmentioning

confidence: 99%

“…This communication of methylation status from one adenine target to the other presumably occurs via conformational changes induced by the recognition of the presence or absence of methyl groups on the adenines by the enzyme via steric clashes between the enzyme–S-adenosyl-methionine (SAM) cofactor complex and the DNA (4). It is likely that the methyl group on the cofactor is the sensor for the methyl group on the DNA, and it is highly probable that, in common with all other DNA methyltransferases (5), recognition of adenine methylation requires flipping of the target base out of the DNA helix into the enzyme methylation pocket, where it can interact with the cofactor (6). …”

Section: Introductionmentioning

confidence: 99%

DNA bending by M.EcoKI methyltransferase is coupled to nucleotide flipping

Su¹

2005

Nucleic Acids Research

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The maintenance methyltransferase M.EcoKI recognizes the bipartite DNA sequence 5 0 -AACNNN-NNNGTGC-3 0 , where N is any nucleotide. M.EcoKI preferentially methylates a sequence already containing a methylated adenine at or complementary to the underlined bases in the sequence. We find that the introduction of a single-stranded gap in the middle of the non-specific spacer, of up to 4 nt in length, does not reduce the binding affinity of M.EcoKI despite the removal of non-sequencespecific contacts between the protein and the DNA phosphate backbone. Surprisingly, binding affinity is enhanced in a manner predicted by simple polymer models of DNA flexibility. However, the activity of the enzyme declines to zero once the single-stranded region reaches 4 nt in length. This indicates that the recognition of methylation of the DNA is communicated between the two methylation targets not only through the protein structure but also through the DNA structure. Furthermore, methylation recognition requires base flipping in which the bases targeted for methylation are swung out of the DNA helix into the enzyme. By using 2-aminopurine fluorescence as the base flipping probe we find that, although flipping occurs for the intact duplex, no flipping is observed upon introduction of a gap. Our data and polymer model indicate that M.EcoKI bends the non-specific spacer and that the energy stored in a doublestranded bend is utilized to force or flip out the bases. This energy is not stored in gapped duplexes. In this way, M.EcoKI can determine the methylation status of two adenine bases separated by a considerable distance in double-stranded DNA and select the required enzymatic response.

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“…Thus, 2AP functions as a probe for interactions between neighboring bases. 2AP has been used to study mispair recognition, [13][14][15] base flipping, [16,17] local melting, [1,6,7] protein binding, [18][19][20] and electron transfer. [21][22][23][24] A four-exponential decay of 2AP fluorescence is often observed in DNA fragments.…”

Section: Introductionmentioning

confidence: 99%

Structural Heterogeneity in DNA: Temperature Dependence of 2‐Aminopurine Fluorescence in Dinucleotides

et al. 2005

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The fluorescent base analogue 2-aminopurine is a sensitive probe for local dynamics of DNA. Its fluorescence is quenched by interaction with the neighboring bases, but the underlying mechanisms are still under investigation. We studied 2-aminopurine fluorescence in dinucleotides with each of the natural bases. Consistently, two of the four fluorescence-decay components depend strongly on temperature. Our results indicate that these components are due to the excited-state dynamics of a single conformational state. We propose a variation of the gating model in which transient unstacking occurs in the excited state.

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Unusual 2-aminopurine fluorescence from a complex of DNA and the EcoKI methyltransferase

Cited by 30 publications

References 47 publications

Fluorescence intensity decays of 2-aminopurine solutions: Lifetime distribution approach

Fluorescence intensity decays of 2-aminopurine solutions: Lifetime distribution approach

DNA bending by M.EcoKI methyltransferase is coupled to nucleotide flipping

Structural Heterogeneity in DNA: Temperature Dependence of 2‐Aminopurine Fluorescence in Dinucleotides

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