Fluorescence characteristics of 5-carboxytetramethylrhodamine linked covalently to the 5' end of oligonucleotides: multiple conformers of single-stranded and double-stranded dye-DNA complexes

Vámosi, György; Gohlke, C.; Clegg, Robert M.

doi:10.1016/s0006-3495(96)79300-1

Cited by 134 publications

(109 citation statements)

References 63 publications

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“…To address the possibility of decreased energy transfer as a result of an increase in interfluorophore distance and potentially obtain some evidence for bend directionality, we performed steady state fluorescence experiments by moving the dye attachment from the 5Ј-to 3Ј-ends of the duplex. With evidence for TAMRA stacking on the end of duplex DNA (46,47), we assumed the TAMRA position would not change when moved from a 5Ј-to 3Ј-attachment point. Oligonucleotides with either the 5Ј-or 3Ј-end coupled to fluorophore were used to prepare duplexes modified at both 5Ј-or 3Ј-ends.…”

Section: Resultsmentioning

confidence: 99%

Simultaneous DNA Binding, Bending, and Base Flipping

Hopkins

Reich

2004

Journal of Biological Chemistry

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We measured the kinetics of DNA bending by M.EcoRI using DNA labeled at both 5-ends and observed changes in fluorescence resonance energy transfer. Although known to bend its cognate DNA site, energy transfer is decreased upon enzyme binding. This unanticipated effect is shown to be robust because we observe the identical decrease with different dye pairs, when the dye pairs are placed on the respective 3-ends, the effect is cofactor-and protein-dependent, and the effect is observed with duplexes ranging from 14 through 17 base pairs. The same labeled DNA shows the anticipated increased energy transfer with EcoRV endonuclease, which also bends this sequence, and no change in energy transfer with EcoRI endonuclease, which leaves this sequence unbent. We interpret these results as evidence for an increased end-to-end distance resulting from M.EcoRI binding, mediated by a mechanism novel for DNA methyltransferases, combining DNA bending and an overall expansion of the DNA duplex. The M.EcoRI protein sequence is poorly accommodated into well defined classes of DNA methyltransferases, both at the level of individual motifs and overall alignment. Interestingly, M.EcoRI has an intercalation motif observed in the FPG DNA glycosylase family of repair enzymes. Enzyme-dependent changes in anisotropy and fluorescence resonance energy transfer have similar rate constants, which are similar to the previously determined rate constant for base flipping; thus, the three processes are nearly coincidental. Similar fluorescence resonance energy transfer experiments following AdoMet-dependent catalysis show that the unbending transition determines the steady state product release kinetics.Structural transitions involving substrate-induced changes in enzyme conformation and protein-induced changes in substrate conformation occur in diverse classes of enzymes. The quantitative importance of such mechanisms toward catalysis and specificity has been demonstrated for DNA polymerases (1), phytase (2), integration host factor (3), pyridoxal kinase (4), restriction enzymes, DNA repair enzymes (5), and DNA modifying enzymes (6). Yet, conformational mechanisms are difficult to study because the reaction intermediates are largely inaccessible to classical kinetic analysis. Spectroscopic probes and, in particular, the highly distant-dependent method of fluorescence resonance energy transfer (FRET) 1 provide a viable solution-based approach to characterize conformational intermediates (7). Only through the quantitative analysis of stable conformational intermediates and the kinetic transitions involving their interconversion can a mechanistic understanding of catalysis and specificity be approached.DNA methyltransferases provide a rich system to investigate the mechanisms and importance of conformational transitions. The enzymes are structurally characterized, carry out both DNA bending and base flipping (8), and the bacterial and human enzymes are the targets of novel antibiotic (9) and cancer drug development efforts respectively (10). These l...

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Section: Resultsmentioning

confidence: 99%

Simultaneous DNA Binding, Bending, and Base Flipping

Hopkins

Reich

2004

Journal of Biological Chemistry

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“…Sometimes the sequence of the oligonucleotide can affect the properties of the attached fluorophore. Time-resolved fluorescence studies of various tetramethylrhodamine (TMR)-or TAMRA-labeled oligonucleotides demonstrate that the fluorophore can have multiple fluorescent lifetimes, suggesting that the probe exists in multiple environments (Edman et al, 1996;Eggeling et al, 1998;Harley et al, 2002;Vamosi et al, 1996). One of these lifetimes may involve an interaction between fluorophore and DNA bases, which can potentially quench fluorescence emission or otherwise affect the measured values (Sauer et al, 1995;Seidel et al, 1996;Sevenich et al, 1998).…”

Section: Designing the Oligonucleotidementioning

confidence: 99%

“…Properties of fluorophores, however, are often sensitive to pH and salt. For example, the fluorescence intensity of a TAMRA-labeled oligonucleotide can decrease with increasing NaCl concentrations, while its fluorescence anisotropy increases (Vamosi et al, 1996). Both fluorescence intensity and anisotropy of a TAMRA-labeled oligonucleotide decrease with increasing temperature (Vamosi et al, 1996).…”

Section: Designing the Oligonucleotidementioning

confidence: 99%

Chapter 12 Using Fluorophore-Labeled Oligonucleotides to Measure Affinities of Protein–DNA Interactions

Anderson

Larkin

Guja

et al. 2008

Methods in Enzymology

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Changes in fluorescence emission intensity and anisotropy can reflect changes in the environment and molecular motion of a fluorophore. Researchers can capitalize on these characteristics to assess the affinity and specificity of DNA-binding proteins using fluorophore-labeled oligonucleotides. While there are many advantages to measuring binding using fluorescent oligonucleotides, there are also some distinct disadvantages. Here we describe some of the relevant issues for the novice, illustrating key points using data collected with the F plasmid relaxase domain and a variety of labeled oligonucleotides. Topics include selection of a fluorophore, experimental design using a fluorometer equipped with an automatic titrating unit, and analysis of direct binding and competition assays.

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“…Even with a collection efficiency of 10%, this is 100 μs∕photon. Because the excited state lifetime of the donor is <10 ns (41) and the 10 nearest neighbors contribute ∼90% of the FRET signal in our geometry (SI Methods), the probability that two donors will be simultaneously excited is <10 −3 . Based on the assumption that only one donor is excited at a time, the FRET efficiency per acceptor molecule is calculated by summing the contributions of each donor:…”

Section: Methodsmentioning

confidence: 99%

FRET measurements of kinesin neck orientation reveal a structural basis for processivity and asymmetry

Martin

Fathi²,

Mitchison

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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As the smallest and simplest motor enzymes, kinesins have served as the prototype for understanding the relationship between protein structure and mechanochemical function of enzymes in this class. Conventional kinesin (kinesin-1) is a motor enzyme that transports cargo toward the plus end of microtubules by a processive, asymmetric hand-over-hand mechanism. The coiled-coil neck domain, which connects the two kinesin motor domains, contributes to kinesin processivity (the ability to take many steps in a row) and is proposed to be a key determinant of the asymmetry in the kinesin mechanism. While previous studies have defined the orientation and position of microtubule-bound kinesin motor domains, the disposition of the neck coiled-coil remains uncertain. We determined the neck coiled-coil orientation using a multidonor fluorescence resonance energy transfer (FRET) technique to measure distances between microtubules and bound kinesin molecules. Microtubules were labeled with a new fluorescent taxol donor, TAMRA-X-taxol, and kinesin derivatives with an acceptor fluorophore attached at positions on the motor and neck coiled-coil domains were used to reconstruct the positions and orientations of the domains. FRET measurements to positions on the motor domain were largely consistent with the domain orientation determined in previous studies, validating the technique. Measurements to positions on the neck coiled-coil were inconsistent with a radial orientation and instead demonstrated that the neck coiled-coil is parallel to the microtubule surface. The measured orientation provides a structural explanation for how neck surface residues enhance processivity and suggests a simple hypothesis for the origin of kinesin step asymmetry and "limping."is a dimeric motor enzyme involved in microtubule-based intracellular transport (1). Kinesin utilizes ATP hydrolysis to move toward the plus ends of microtubules via a hand-over-hand mechanism in which each motor domain (or "head") alternately steps forward (2, 3). The mechanism is asymmetrical, in the sense that alternate steps are different structurally and kinetically (2, 4-7), and is processive, in that kinesin can take hundreds of steps along a microtubule before dissociating (8). Both of these mechanistic features may be important to biological function: Asymmetry allows kinesin to use energy for cargo transport rather than dissipating it in cargo rotation; processivity allows individual kinesin molecules to transport cargoes through the viscoelastic cytoplasm.Processive movement requires that kinesin maintain a tight grip on the microtubule as it moves. To this end, the catalytic cycle is gated to ensure that at least one head remains tightly bound to the microtubule at all times (9-11). However, the kinesin-1 neck, a five heptad repeat coiled-coil connected to the two heads by the short nonhelical neck linkers (Fig. 1), also appears to play a role in keeping the enzyme bound to the microtubule: Mutations on the neck surface can significantly increase or decrease processi...

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Fluorescence characteristics of 5-carboxytetramethylrhodamine linked covalently to the 5' end of oligonucleotides: multiple conformers of single-stranded and double-stranded dye-DNA complexes

Cited by 134 publications

References 63 publications

Simultaneous DNA Binding, Bending, and Base Flipping

Simultaneous DNA Binding, Bending, and Base Flipping

Chapter 12 Using Fluorophore-Labeled Oligonucleotides to Measure Affinities of Protein–DNA Interactions

FRET measurements of kinesin neck orientation reveal a structural basis for processivity and asymmetry

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