Fluorescence Anisotropy Controlled by Light Quenching

Gryczyński, Ignacy; Gryczyński, Zygmunt; Lakowicz, Joseph R.

doi:10.1562/0031-8655(1998)067<0641:facblq>2.3.co;2

Cited by 3 publications

(3 citation statements)

References 10 publications

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“…Steady-State Anisotropy Measurement. Equilibrium DNA binding in solution was assessed by fluorescence anisotropy (35)(36)(37). Fluorescence measurements were performed on a steady-state photon counting spectrofluorometer, PC1, from ISS Instruments (Champaign, IL) equipped with a Hamamatsu R928P photomultiplier tube.…”

Section: Methodsmentioning

confidence: 99%

Mechanisms of DNA Binding and Regulation of Bacillus anthracis DNA Primase

Biswas¹,

Wydra

Biswas

2009

Biochemistry

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DNA primases are pivotal enzymes in chromosomal DNA replication in all organisms. In this article, we report unique mechanistic characteristics of recombinant DNA primase from Bacillus anthracis (B. anthracis). The mechanism of action of B. anthracis DNA primase (DnaGBA) may be described in several distinct steps as follows. Its mechanism of action is initiated when it binds to single-stranded DNA (ssDNA) in the form of a trimer. Although DnaGBA binds to different DNA sequences with moderate affinity (as expected of a mobile DNA binding protein), we found that DnaGBA bound to the origin of bacteriophage G4 (G4ori) with approximately eight-fold higher affinity. DnaGBA was strongly stimulated (≥75-fold) by its cognate helicase, DnaBBA during RNA primer synthesis. With the G4ori ssDNA template, DnaGBA formed short (≥ 20 nucleotides) primers in the absence of DnaBBA. The presence of DnaBBA increased the rate of primer synthesis. The observed stimulation of primer synthesis by cognate DnaBBA is thus indicative of a positive effector role for DnaBBA. By contrast, E. coli DnaB helicase (DnaBEC) did not stimulate DnaGBA and inhibited primer synthesis to near completion. This observed effect of E. coli DnaBEC is indicative of a strong negative effector role for heterologous DnaBEC. We conclude that DnaGBA is capable of interacting with DnaB proteins from both B. anthracis and E. coli; however, between DnaB proteins derived from these two organisms, only the homologous DNA helicase (DnaBBA) acted as a positive effector of primer synthesis.

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Section: Methodsmentioning

confidence: 99%

Mechanisms of DNA Binding and Regulation of Bacillus anthracis DNA Primase

Biswas¹,

Wydra

Biswas

2009

Biochemistry

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“…Fluorescence anisotropy was measured to investigate DNA binding by SSB BA in solution [40,52]. Fluorescence measurements were carried out using a steady-state-photon counting spectrofluorometer, PC1 with Vinci software, from ISS Instruments (Champaign, IL) and Fluoromax4-TCSPC with time-resolved fluorescence from Horiba Instruments Inc. (Edison, NJ).…”

Section: Methodsmentioning

confidence: 99%

Thermodynamic analysis of DNA binding by a Bacillus single stranded DNA binding protein

Biswas-Fiss

Kukiratirat²,

Biswas³

2012

BMC Biochemistry

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BackgroundSingle-stranded DNA binding proteins (SSB) are essential for DNA replication, repair, and recombination in all organisms. SSB works in concert with a variety of DNA metabolizing enzymes such as DNA polymerase.ResultsWe have cloned and purified SSB from Bacillus anthracis (SSBBA). In the absence of DNA, at concentrations ≤100 μg/ml, SSBBA did not form a stable tetramer and appeared to resemble bacteriophage T4 gene 32 protein. Fluorescence anisotropy studies demonstrated that SSBBA bound ssDNA with high affinity comparable to other prokaryotic SSBs. Thermodynamic analysis indicated both hydrophobic and ionic contributions to ssDNA binding. FRET analysis of oligo(dT)70 binding suggested that SSBBA forms a tetrameric assembly upon ssDNA binding. This report provides evidence of a bacterial SSB that utilizes a novel mechanism for DNA binding through the formation of a transient tetrameric structure.ConclusionsUnlike other prokaryotic SSB proteins, SSBBA from Bacillus anthracis appeared to be monomeric at concentrations ≤100 μg/ml as determined by SE-HPLC. SSBBA retained its ability to bind ssDNA with very high affinity, comparable to SSB proteins which are tetrameric. In the presence of a long ssDNA template, SSBBA appears to form a transient tetrameric structure. Its unique structure appears to be due to the cumulative effect of multiple key amino acid changes in its sequence during evolution, leading to perturbation of stable dimer and tetramer formation. The structural features of SSBBA could promote facile assembly and disassembly of the protein-DNA complex required in processes such as DNA replication.

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“…It was demonstrated that both types of IFE can greatly enhance the sensitivity of optical sensing for analytes, and the detection limit was lowered by 1–3 orders of magnitude with respect to the absorption‐based scheme . However, the use of IFEs for determination of analytes was also limited by interferences of quenching (dynamic or static quenching) and interactions between analytes and fluorescers .…”

Section: Introductionmentioning

confidence: 99%

The use of outer filter effects for Cu²⁺ quantitation: a unique example for monitoring nonfluorescent molecule with fluorescence

Zong

Cao

et al. 2011

Luminescence

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This article concerns a new and precise strategy for the determination of Cu(2+) based on a color reaction and outer filter effects (OFEs). Cu(2+) can react with sodium diethyldithiocarbamate trihydrate (DDTC) to form a DDTC-Cu(2+) complex with a significant absorption at 447 nm. Being positively correlated with Cu(2+), the absorption could be treated as the basis for the determination of Cu(2+). When cuvettes containing the complex were fixed in the light path of a fluorescence spectrophotometer, the excitation/emitted light were absorbed by the OFEs, similar to absorption mechanisms of inner filter effects. Under suitable conditions, OFEs from the complex could quantitatively reduce the fluorescence intensities of quinine sulfate and acridine yellow by absorbing the excitation or emission light. Compared with traditional absorption spectroscopy (with a detection limit at 0.9 µmol/L), indirect OEF techniques showed increased sensitivities by about 1 order of magnitude. The strategy could be extended to many different systems where components absorb the excitation wavelength and/or emission wavelength of fluorescers.

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Fluorescence Anisotropy Controlled by Light Quenching

Cited by 3 publications

References 10 publications

Mechanisms of DNA Binding and Regulation of Bacillus anthracis DNA Primase

Mechanisms of DNA Binding and Regulation of Bacillus anthracis DNA Primase

Thermodynamic analysis of DNA binding by a Bacillus single stranded DNA binding protein

The use of outer filter effects for Cu²⁺ quantitation: a unique example for monitoring nonfluorescent molecule with fluorescence

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Fluorescence Anisotropy Controlled by Light Quenching

Cited by 3 publications

References 10 publications

Mechanisms of DNA Binding and Regulation of Bacillus anthracis DNA Primase

Mechanisms of DNA Binding and Regulation of Bacillus anthracis DNA Primase

Thermodynamic analysis of DNA binding by a Bacillus single stranded DNA binding protein

The use of outer filter effects for Cu2+ quantitation: a unique example for monitoring nonfluorescent molecule with fluorescence

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The use of outer filter effects for Cu²⁺ quantitation: a unique example for monitoring nonfluorescent molecule with fluorescence