13 research outputs found

    The Spectroscopic Basis of Fluorescence Triple Correlation Spectroscopy

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    We have developed fluorescence triple correlation spectroscopy (F3CS) as an extension of the widely used fluorescence microscopy technique fluorescence correlation spectroscopy. F3CS correlates three signals at once and provides additional capabilities for the study of systems with complex stoichiometry, kinetic processes, and irreversible reactions. A general theory of F3CS was developed to describe the interplay of molecular dynamics and microscope optics, leading to an analytical function to predict experimental triple correlations of molecules that freely diffuse through the tight focus of the microscope. Experimental correlations were calculated from raw fluorescence data using triple correlation integrals that extend multiple-tau correlation theory to delay times in two dimensions. The quality of experimental data was improved by tuning specific spectroscopic parameters and employing multiple independent detectors to minimize optoelectronic artifacts. Experiments with the reversible system of freely diffusing 16S rRNA revealed that triple correlation functions contain symmetries predicted from time-reversal arguments. Irreversible systems are shown to break these symmetries, and correlation strategies were developed to detect time-reversal asymmetries in a comprehensive way with respect to two delay times, each spanning many orders of magnitude in time. The correlation strategies, experimental approaches, and theory developed here enable studies of the composition and dynamics of complex systems using F3CS

    Single-Molecule Förster Resonance Energy Transfer Reveals an Innate Fidelity Checkpoint in DNA Polymerase I

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    Enzymatic reactions typically involve complex dynamics during substrate binding, conformational rearrangement, chemistry, and product release. The noncovalent steps provide kinetic checkpoints that contribute to the overall specificity of enzymatic reactions. DNA polymerases perform DNA replication with outstanding fidelity by actively rejecting noncognate nucleotide substrates early in the reaction pathway. Substrates are delivered to the active site by a flexible fingers subdomain of the enzyme, as it converts from an open to a closed conformation. The conformational dynamics of the fingers subdomain might also play a role in nucleotide selection, although the precise role is currently unknown. Using single-molecule Förster resonance energy transfer, we observed individual Escherichia coli DNA polymerase I (Klenow fragment) molecules performing substrate selection. We discovered that the fingers subdomain actually samples through three distinct conformationsopen, closed, and a previously unrecognized intermediate conformation. We measured the overall dissociation rate of the polymerase–DNA complex and the distribution among the various conformational states in the absence and presence of nucleotide substrates, which were either correct or incorrect. Correct substrates promote rapid progression of the polymerase to the catalytically competent closed conformation, whereas incorrect nucleotides block the enzyme in the intermediate conformation and induce rapid dissociation from DNA. Remarkably, incorrect nucleotide substrates also promote partitioning of DNA to the spatially separated 3′–5′ exonuclease domain, providing an additional mechanism to prevent misincorporation at the polymerase active site. These results reveal the existence of an early innate fidelity checkpoint, rejecting incorrect nucleotide substrates before the enzyme encloses the nascent base pair

    Dynamics of Site Switching in DNA Polymerase

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    DNA polymerases replicate DNA by catalyzing the template-directed polymerization of deoxynucleoside triphosphate (dNTP) substrates onto the 3′ end of a growing DNA primer strand. Many DNA polymerases also possess a separate 3′-5′ exonuclease activity that is used to remove misincorporated nucleotides from the nascent DNA (proofreading). The polymerase (pol) and exonuclease (exo) activities are spatially separated in different enzyme domains, indicating that a mechanism must exist to transfer the growing primer terminus from one site to the other. Here we report a single-molecule Förster resonance energy transfer (smFRET) system that directly monitors the movement of a DNA substrate between the pol and exo sites of DNA polymerase I Klenow fragment (KF). FRET trajectories recorded during the encounter between single polymerase and DNA molecules reveal that DNA can channel between the pol and exo sites in both directions while remaining closely associated with the enzyme (intramolecular transfer). In addition, it is evident from the trajectories that DNA can also dissociate from one site and subsequently rebind at the other (intermolecular transfer). Rate constants for each pathway have been determined by dwell-time analysis, revealing that intramolecular transfer is the faster of the two pathways. Unexpectedly, a mispaired primer terminus accesses the exo site more frequently when dNTP substrates are also present in solution, which is expected to enhance proofreading. Together, these results explain how the separate pol and exo activities of KF are physically coordinated to achieve efficient proofreading

    <i>In vivo</i> RNA binding by Rev mutants.

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    <p>(A) Schematic representation of the pSLIIB/CAT reporter used to analyze Rev:RRE RNA binding <i>in vivo</i>. (B) <i>In vivo</i> RRE RNA-binding phenotypes of various <i>trans</i>-activators are shown. HeLa cells were transiently cotransfected with the Rev reporter construct pSLIIB/CAT, pBC12/CMV/β-Gal (internal control) and the indicated Tat-Rev fusion constructs. At 48 h post-transfection, CAT and β-Gal expression was analysed by ELISA. CAT values were adjusted for transfection efficiency to the β-Gal level in each culture. Data are expressed as a percentage of wildtype Rev activity (set to 100%).</p

    Nucleocytoplasmic shuttling of Rev.

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    <p>Standard interspecies heterokaryon fusion assay for detection of protein nuclear export. HeLa cells were transiently transfected with the indicated Rev expression plasmids and fused at 24 h post-transfection with untransfected NIH3T3 cells (indicated by an asterisk). CRM1-mediated protein shuttling was blocked by treatment of the cultures with leptomycin B (LMB). Localization and nuclear export of Rev proteins was visualized by indirect immunofluorescence microscopy using specific anti-Rev antibody (red label; panel b, e, h, k, n, q, t). Nuclei were stained with Hoechst 33258 (blue label; panel a, d, g, j, m, p, s) and merged with the fluorescence and bright light pictures (panel c, f, i, l, o, r, u).</p

    FRET measurement of Rev oligomer formation.

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    <p>(A) Expression level and <i>trans</i>-activation capacity of Rev-CFP and Rev-YFP fusion proteins. COS cells were transiently cotransfected with the Rev reporter plasmid pGPV-RRE and the indicated <i>trans</i>-activator constructs. At 24 h post-transfection Rev-dependent expression of HIV-1 structural proteins p55<sup>Gag</sup> and p24<sup>Gag</sup>, the respective Rev <i>trans</i>-activators and actin (gel loading control) was detected by Western blot analysis using specific antibodies. (B) Representative FACS plots illustrating the percentage of FRET positive cells. COS cells were transiently transfected with expression vectors for control CFP and RevWT-YFP, or the indicated combinations of Rev donor and acceptor constructs. To provide RRE RNA the plasmid pGPV-RRE was included in each transfection. At 24 h post-transfection cells were harvested and analyzed by flow cytometry for Rev:Rev interaction, represented as FRET positive cells. (C) Mean percentage of FRET-positive cells (adjusted to the background) determined in FRET-FACS experiments.</p

    Analysis of Rev-mediated nuclear RNA export.

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    <p>(A) Schematic representation of the nuclease S1 protection assay. The Rev reporter, the end-labelled input probe and the protected sequences, recognizing unspliced and spliced RNA are depicted. (B) Nucleocytoplasmic distribution of Rev-regulated unspliced and spliced RNA determined by nuclease S1 assay. COS cells were transiently cotransfected with Rev reporter DNA pDM128/CMV and the indicated Rev expression vectors. Nuclear and cytoplasmic RNA was isolated at 48 h post-transfection. Unspliced and spliced reporter-derived RNA was detected by autoradiography using the <sup>32</sup>P-labeled input probe. (N, nuclear RNA; C, cytoplasmic RNA). (C) Quantification of Rev-regulated cytoplasmic and nuclear viral RNA. HeLa cells were transiently cotransfected with pHXB2Δ<i>rev</i> and the indicated Rev constructs. At 48 h post-transfection nuclear and cytoplasmic RNAs were isolated and the accumulation of Rev-regulated unspliced <i>gag</i> RNA was quantified by real-time PCR. Total viral copy numbers were adjusted to the endogenous <i>gapdh</i> mRNA level in each sample.</p

    Regulated homodimerization of HIV-1 Rev.

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    <p>(A) Expression of FKBP-Rev fusion constructs. HeLa cells were transiently transfected with plasmids expressing the indicated Rev fusion proteins. Levels of protein expression were determined by Western blot analysis at 48 h post-transfection using specific anti-Rev and anti-actin (gel loading control) antibodies. (B) <i>Trans</i>-activation capacity of Rev fusion proteins. A provirus rescue assay was performed by transient cotransfection of HeLa cells with pHXB2Δ<i>rev</i> DNA, the internal control plasmid pBC12/CMV/SEAP and the indicated Rev expression vectors in the absence or presence of the chemical homodimerizer AP20187. Culture supernatants were collected at 48 h post-transfection and the accumulation of HIV-1 particles was quantified by p24<sup>Gag</sup> antigen ELISA. Values of p24<sup>Gag</sup> antigen were adjusted for transfection efficiency to SEAP activity in each culture supernatant.</p

    Regulated heterodimerization of HIV-1 Rev.

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    <p>(A) Transient expression of various Rev proteins containing the FKBP (N1) or modified FRAP (RH) heterodimerization motif. COS cells were transiently transfected with the indicated Rev vector constructs, cultured in medium supplemented with the heterodimerizer AP21967, and analyzed by Western blot using specific anti-Rev and anti-GAPDH (gel loading control) antibodies. (B) <i>Trans</i>-activation capacity of heterodimeric Rev proteins. COS cells were transiently transfected with the Rev-dependent reporter vector pGPV-RRE, the indicated Rev fusion constructs and pBC12/CMV/SEAP for internal transfection control. Release of HIV-1 particles into the culture supernatant was determined in the absence or presence of the heterodimerizer AP21967 by p24<sup>Gag</sup> antigen ELISA. For control of transfection efficiency, all p24<sup>Gag</sup> antigen values were adjusted to the SEAP level present in the supernatant of each culture. (C) <i>Trans</i>-activation model of heterodimeric Rev proteins. A Rev dimer needs to exhibit two functional activation domains containing two NES for the biological activity. The remaining ability of RevM10 to multimerize leads to higher order complexes of RevM10:RevSLT40 which contain more than one functional NES and therefore exhibit biological activity.</p

    Half-life determination of Rev-regulated transcripts.

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    <p>(A) Expression of Rev-dependent <i>gag</i> (unspliced) and <i>env</i> (single spliced) mRNAs in HeLa-tTA cells. HeLa-tTA cells were transiently cotransfected with the Rev-deficient doxycyline-regulated proviral construct pUHC-HXB2Δ<i>rev</i> and Rev expression plasmid or the parental control vector pBC12/CMV. Transcription of viral RNA was blocked where indicated by 100 ng/ml doxycycline treatment at 5 h post-transfection. Subsequently, the viral RNA levels were quantified at 24 h posttransfection by real-time PCR. Results for viral RNA copies were adjusted to the level of endogenous <i>gapdh</i> mRNA. (B) Inhibition of replication by doxycycline treatment. HeLa-tTA cells were transiently cotransfected with pUHC-HXB2Δ<i>rev,</i> pBC12/CMV/SEAP (internal control) and the indicated Rev constructs. The accumulation of p24<sup>Gag</sup> antigen in the culture supernatants was quantified by ELISA at 48 h post-transfection. All values were adjusted for transfection efficiency to the SEAP activity present in each supernatant. Viral expression was blocked when the culture medium was supplemented with 100 ng/ml doxycycline. (C) Effect of the indicated Rev mutants on the half-life of unspliced full-length (<i>gag</i>) HIV-1 RNA. Transfection experiments were performed in HeLa-tTA cells using pUHC-HXB2Δ<i>rev</i> DNA and the indicated <i>trans</i>-activator constructs. Total RNA was isolated and viral <i>gag</i> RNA copy numbers were quantified after transcriptional pulse at the indicated time points by real-time PCR and adjusted to the endogenous <i>gapdh</i> mRNA level in each sample. The relative RNA levels are show in comparison to the respective initial level (after transcriptional pulse) at time point 0 h (set to 1.0).</p
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