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Active and passive mechanisms of helicases
M. Manosas, X. G. Xi, D. Bensimon and V. Croquette
Nucleic Acids Res. - 38(16) :5518–26 - DOI:10.1093/nar/gkq273 - 2010
In this work, we discuss the active or passive character of helicases. In the past years, several studies have used the theoretical framework proposed by Betterton and Julicher [Betterton, M.D. and Julicher, F. (2005) Opening of nucleic-acid double strands by helicases: active versus passive opening. Phys. Rev. E, 71, 11904-11911.] to analyse the unwinding data and assess the mechanism of the helicase under study (active versus passive). However, this procedure has given rise to apparently contradictory interpretations: helicases exhibiting similar behaviour have been classified as both active and passive enzymes [Johnson, D.S., Bai, L. Smith, B.Y., Patel, S.S. and Wang, M.D. (2007) Single-molecule studies reveal dynamics of DNA unwinding by the ring-shaped T7 helicase. Cell, 129, 1299-1309; Lionnet, T., Spiering, M.M., Benkovic, S.J., Bensimon, D. and Croquette, V. (2007) Real-time observation of bacteriophage T4 gp41 helicase reveals an unwinding mechanism Proc. Natl Acid. Sci., 104, 19790-19795]. In this work, we show that when the helicase under study has not been previously well characterized (namely, if its step size and rate of slippage are unknown) a multi-parameter fit to the afore-mentioned model can indeed lead to contradictory interpretations. We thus propose to differentiate between active and passive helicases on the basis of the comparison between their observed translocation velocity on single-stranded nucleic acid and their unwinding rate of double-stranded nucleic acid (with various GC content and under different tensions). A threshold separating active from passive behaviour is proposed following an analysis of the reported activities of different helicases. We study and contrast the mechanism of two helicases that exemplify these two behaviours: active for the RecQ helicase and passive for the gp41 helicase.
Mechanism of strand displacement synthesis by DNA replicative polymerases
M. Manosas, M. M. Spiering, F. Ding, D. Bensimon, J.-F. Allemand, S. J. Benkovic and V. Croquette
Nucleic Acids Res. - 40(13) :6174-86 - PMID:22434889 - 2010
Replicative holoenzymes exhibit rapid and processive primer extension DNA synthesis, but inefficient strand displacement DNA synthesis. We investigated the bacteriophage T4 and T7 holoenzymes primer extension activity and strand displacement activity on a DNA hairpin substrate manipulated by a magnetic trap. Holoenzyme primer extension activity is moderately hindered by the applied force. In contrast, the strand displacement activity is strongly stimulated by the applied force; DNA polymerization is favoured at high force, while a processive exonuclease activity is triggered at low force. We propose that the DNA fork upstream of the holoenzyme generates a regression pressure which inhibits the polymerization-driven forward motion of the holoenzyme. The inhibition is generated by the distortion of the template strand within the polymerization active site thereby shifting the equilibrium to a DNA-protein exonuclease conformation. We conclude that stalling of the holoenzyme induced by the fork regression pressure is the basis for the inefficient strand displacement synthesis characteristic of replicative polymerases. The resulting processive exonuclease activity may be relevant in replisome disassembly to reset a stalled replication fork to a symmetrical situation. Our findings offer interesting applications for single-molecule DNA sequencing.
Pre-Dispositions and Epigenetic Inheritance in the Escherichia Coli Lactose Operon Bistable Switch
L. Robert, G. Paul, Y. Chen, F. Taddei, D. Baigl and A. B. Lindner
Molecular Systems Biology - 6 :357 - DOI:10.1038/msb.2010.12 - 2010
The lactose operon regulation in Escherichia coli is a primary model of phenotypic switching, reminiscent of cell fate determination in higher organisms. Under conditions of bistability, an isogenic cell population partitions into two subpopulations, with the operon's genes turned on or remaining off. It is generally hypothesized that the final state of a cell depends solely on stochastic fluctuations of the network's protein concentrations, particularly on bursts of lactose permease expression. Nevertheless, the mechanisms underlying the cell switching decision are not fully understood. We designed a microfluidic system to follow the formation of a transiently bimodal population within growing microcolonies. The analysis of genealogy and cell history revealed the existence of pre-disposing factors for switching that are epigenetically inherited. Both the pre-induction expression stochasticity of the lactose operon repressor LacI and the cellular growth rate are predictive factors of the cell's response upon induction, with low LacI concentration and slow growth correlating with higher switching probability. Thus, stochasticity at the local level of the network and global physiology are synergistically involved in cell response determination.
Micropillar array chips toward new immunodiagnosis
H.Y. Li, V. Dauriac, V. Thibert, H. Senechal, G. Peltre, X.X. Zhang, S. Descroix
Lab. Chip - 10(19) :2597-604 - DOI:10.1039/c005034b - 2010
In this paper, we demonstrate the possibility to use a micropillar array to perform molecular immunodiagnosis. A polydimethylsiloxane (PDMS) microdevice consisting of a rectangular array of micropillars (45 µm in height, 100 × 100 µm square cross section) was used to replace microchannels or gels (polyacrylamide or agarose) to perform electrokinetic separation. This microarray was used to mimic highly diluted gel and to maintain electrolyte within the pillar zone by capillary effect. The electrolyte composition (glycerol and agarose content) was investigated in order to improve protein separation by isoelectric focusing (IEF). The influence of glycerol on focusing time and on the different evaporative contributions was further evaluated. In order to perform an immunodiagnostic of milk allergy, different surface treatments were optimized to prevent milk allergen adsorption on PDMS surface. Poly(dimethylacrylamide)-co-allyl glycidyl ether (PDMA-AGE) as well as gelatin led to a satisfactory signal to noise ratio. Finally the possibility to perform protein mixture separation using this micropillar array chip followed by immunoblotting was demonstrated by using the serum from an allergic individual, confirming the great potential of this analytical platform in the field of immunodiagnosis.
Total on-line analysis of a target protein from plasma by immunoextraction, digestion and liquid chromatography-mass spectrometry
A. Cingöz, F. Hugon-Chapuis, V. Pichon
J. Chrom. B - 878(2) :213-21 - DOI:10.1016/j.jchromb.2009.07.032 - 2010
A total on-line analysis of a target protein from a plasma sample was made using a selective immunoextraction step coupled on-line to an immobilized enzymatic reactor (IMER) for the protein digestion followed by LC-MS/MS analysis. For the development of this device, cytochrome c was chosen as model protein due to its well-known sequence. An immunosorbent (IS) based on the covalent immobilization of anti-cytochrome c antibodies on a solid support was made and an immunoextraction procedure was carefully developed to assess a selective extraction of the target protein from plasma. For the first time, IS was easily coupled on-line with a laboratory-made IMER based on pepsin. The whole on-line device (IS-IMER-LC-MS/MS) allowed the quantification of cytochrome c from 8.5pmol to 1.7nmol in buffer medium. Finally, this device was applied to the analysis of only 85pmol of cytochrome c from plasma with a RSD value lower than 10% (n=3).
Supercritical fluid chromatography hyphenated with twin comprehensive two-dimensional gas chromatography for ultimate analysis of middle distillates
F. Adam, D. Thiébaut, F. Bertoncini, M. Courtiade, M.C. Hennion
J. Chrom. A - 1217(8) :1386-94 - DOI:10.1016/j.chroma.2009.11.092 - 2010
This paper reports the conditions of online hyphenation of supercritical fluid chromatography (SFC) with twin comprehensive two-dimensional gas chromatography (twin-GCxGC) for detailed characterization of middle distillates; this is essential for a better understanding of reactions involved in refining processes. In this configuration, saturated and unsaturated compounds that have been fractionated by SFC are transferred on two different GC x GC columns sets (twin-GCxGC) placed in the same GC oven. Cryogenic focusing is used for transfer of fractions into the first dimension columns before simultaneous GCxGC analysis of both saturated and unsaturated fractions. The benefits of SFC-twin-GC x GC are demonstrated for the extended alkane, iso-alkane, alkene, naphthenes and aromatics analysis (so-called PIONA analysis) of diesel samples which can be achieved in one single injection. For that purpose, saturated and unsaturated compounds have been separated by SFC using a silver loaded silica column prior to GC x GC analysis. Alkenes and naphthenes are quantitatively recovered in the unsaturated and saturated fractions, respectively, allowing their identification in various diesel samples. Thus, resolution between each class of compounds is significantly improved compared to a single GCxGC run, and for the first time, an extended PIONA analysis of diesel samples is presented.
Indirect Grafting of Acetonitrile-Derived Films on Metallic Substrates
A. Berisha, C. Combellas, F. Kanoufi, J. Pinson, S. Ustaze, F.I. Podvorica
Chem. Mater - 22(9) :2962-9 - DOI:10.1021/cm100295n - 2010
Strongly bonded organic films with amino groups are obtained on gold, copper, and silicon surfaces by reduction of 2,6-dimethyl benzenediazonium in acetonitrile (ACN). The sterically hindered 2,6-dimethylphenyl radical is unable to attach to the surface, but it abstracts an hydrogen atom from ACN to give the cyanomethyl radical (·CH2CN) that reacts with the surface. A spontaneous reaction is also possible on copper. The film is characterized by IR spectroscopy, scanning electron microscopy, ellipsometry, water contact angles, and cyclic voltammetry. A mechanism is elaborated that accounts for the formation, grafting of the cyanomethyl radical, and finally formation of amino multilayers.
Dansyl-peptides matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) and tandem mass spectrometric (MS/MS) features improve the liquid chromatography/MALDI-MS/MS analysis of the proteome
Chiappetta G, Ndiaye S, Demey E, Haddad I, Marino G, Amoresano A, Vinh J.
Rapid Commun Mass Spectrom - 24(20) :3021-32 - DOI:10.1002/rcm.4734 - 2010
Peptide tagging is a useful tool to improve matrix-assisted laser desorption/ionization tandem mass spectrometric (MALDI-MS/MS) analysis. We present a new application of the use of the dansyl chloride (DNS-Cl). DNS-Cl is a specific primary amine reagent widely used in protein biochemistry. It adds a fluorescent dimethylaminonaphthalene moiety to the molecule. The evaluation of MALDI-MS and MS/MS analyses of dansylated peptides shows that dansylation raises the ionization efficiency of the most hydrophilic species compared with the most hydrophobic ones. Consequently, higher Mascot scores and protein sequence coverage are obtained by combining MS and MS/MS data of native and tagged samples. The N-terminal DNS-Cl sulfonation improves the peptide fragmentation and promotes the generation of b-fragments allowing better peptide sequencing. In addition, we set up a labeling protocol based on the microwave chemistry. Peptide dansylation proved to be a rapid and cheap method to improve the performance of liquid chromatography (LC)/MALDI-MS/MS analysis at the proteomic scale in terms of peptide detection and sequence coverage.
Cell shape and cell division in fission yeast
Piel M, Tran PT
Curr Biol. - 17(19) :R823-7 - DOI:10.1016/j.cub.2009.08.012 - 2009
The fission yeast Schizosaccharomyces pombe has served as an important model organism for investigating cellular morphogenesis. This unicellular rod-shaped fission yeast grows by tip extension and divides by medial fission. In particular, microtubules appear to define sites of polarized cell growth by delivering cell polarity factors to the cell tips. Microtubules also position the cell nucleus at the cell middle, marking sites of cell division. Here, we review the microtubule-dependent mechanisms that regulate cell shape and cell division in fission yeast.
Simple and rapid process for single cell micro-patterning
Ammar Azioune, Marko Storch, Michel Bornens, Manuel Théry and Matthieu Piel
Lab. Chip - 9(11) :1640-2 - DOI:10.1039/b821581m - 2009
We present a simple and environmentally friendly process for cell patterning on glass covered with an ultrathin layer of poly-l-lysine-grafted-polyethylene glycol (PLL-g-PEG) by exposure to deep UV light. The patterned substrates are stable for months in the lab atmosphere before incubation with proteins. Incubation with proteins resulted in well defined patterns, with high feature resolution. RPE-1 cells seeded on fibronectin/fibrinogen-Alexa 488 patterns were constrained for days on the deep UV exposed regions. Finally, large glass plates were patterned with high homogeneity enabling the assembly of micro-patterned microplates in 96-well format.

347 publications.