Rapid Freeze-Quench EPR Spectroscopy: Improved Collection of Frozen Particles

Nami, Faezeh; Gast, P.; Groenen, E. J. J.

doi:10.1007/s00723-016-0783-7

Cited by 11 publications

(17 citation statements)

References 31 publications

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“…Accordingly, it is possible that these findings may represent an artifact of the structural technique. Therefore, we expect that advanced spectroscopic methods such as electroparamagnetic resonance (EPR) spectroscopy 236 will be necessary to fully validate and further elucidate the function of the third divalent metal ion during polymerase catalysis under a more physiological context.…”

Section: Future Characterization Of the Third Divalent Metal Ionmentioning

confidence: 99%

Kinetic Mechanism of DNA Polymerases: Contributions of Conformational Dynamics and a Third Divalent Metal Ion

2018

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Faithful transmission and maintenance of genetic material is primarily fulfilled by DNA polymerases. During DNA replication, these enzymes catalyze incorporation of deoxynucleotides into a DNA primer strand based on Watson-Crick complementarity to the DNA template strand. Through the years, research on DNA polymerases from every family and reverse transcriptases has revealed structural and functional similarities, including a conserved domain architecture and purported two-metal-ion mechanism for nucleotidyltransfer. However, it is equally clear that DNA polymerases possess distinct differences that often prescribe a particular cellular role. Indeed, a unified kinetic mechanism to explain all aspects of DNA polymerase catalysis, including DNA binding, nucleotide binding and incorporation, and metal-ion-assisted nucleotidyltransfer (i.e., chemistry), has been difficult to define. In particular, the contributions of enzyme conformational dynamics to several mechanistic steps and their implications for replication fidelity are complex. Moreover, recent time-resolved X-ray crystallographic studies of DNA polymerases have uncovered a third divalent metal ion present during DNA synthesis, the function of which is currently unclear and debated within the field. In this review, we survey past and current literature describing the structures and kinetic mechanisms of DNA polymerases from each family to explore every major mechanistic step while emphasizing the impact of enzyme conformational dynamics on DNA synthesis and replication fidelity. This also includes brief insight into the structural and kinetic techniques utilized to study DNA polymerases and RTs. Furthermore, we present the evidence for the two-metal-ion mechanism for DNA polymerase catalysis prior to interpreting the recent structural findings describing a third divalent metal ion. We conclude by discussing the diversity of DNA polymerase mechanisms and suggest future characterization of the third divalent metal ion to dissect its role in DNA polymerase catalysis.

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Section: Future Characterization Of the Third Divalent Metal Ionmentioning

confidence: 99%

Kinetic Mechanism of DNA Polymerases: Contributions of Conformational Dynamics and a Third Divalent Metal Ion

2018

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“…Ten RFQ samples (named Mb1 to Mb10) were prepared with the RFQ apparatus (Update Instruments) and method described in [8] (2-mL syringes, ram velocity 3.2 cm s −1 , displacement 3 mm), at a mixing temperature of 21.5˚C, and at reaction times ranging between 2 and 50 ms. These ten samples were initially measured at 9.5 GHz, and later the same were used for measurements at 275 GHz.…”

Section: Sample Preparationmentioning

confidence: 99%

“…The preparation of the RFQ samples for 9.5 GHz EPR in essence follows the procedure described by Nami [8]. Briefly, a 3 mm open quartz tube, equipped with a polypropylene filter at one end and connected to a syringe was submerged into the cold isopentane containing the minute particles from the RFQ.…”

Section: Sample Packing For 95 Ghz Eprmentioning

confidence: 99%

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Effective coupling of rapid freeze-quench to high-frequency electron paramagnetic resonance

Panarelli¹,

Meer²,

Gast³

et al. 2020

PLoS ONE

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We report an easy, efficient and reproducible way to prepare Rapid-Freeze-Quench samples in sub-millimeter capillaries and load these into the probe head of a 275 GHz Electron Paramagnetic Resonance spectrometer. Kinetic data obtained for the binding reaction of azide to myoglobin demonstrate the feasibility of the method for high-frequency EPR. Experiments on the same samples at 9.5 GHz show that only a single series of Rapid-Freeze-Quench samples is required for studies at multiple microwave frequencies.

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“…The quenched sample concerns frozen particles, which are dispersed in the cryo medium isopentane and are difficult to handle. [8][9][10] This is all the more true when the RFQ technique has to follow the developments in EPR spectroscopy towards microwave frequencies higher than 9 GHz. [11][12][13][14][15] At high microwave frequencies the resonant cavities become smaller in size and so do the corresponding sample volumes and sample holders.…”

Section: Introductionmentioning

confidence: 99%

Temperature-cycle electron paramagnetic resonance

Panarelli

Gast

Groenen

2020

Phys. Chem. Chem. Phys.

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We report on a novel approach to the study of rates and short-lived intermediates of (bio)chemical reactions that involve paramagnetic species. Temperature-cycle Electron Paramagnetic Resonance (EPR) concerns the repeated heating of a reaction mixture in the cavity of an EPR spectrometer by pulsed irradiation with a near-infrared diode laser combined with intermittent characterization of the sample by 275 GHz EPR at a lower temperature at which the reaction does not proceed. The new technique is demonstrated for the reduction of TEMPOL with sodium dithionite in aqueous solution down to the sub-second time scale. We show that a single sample suffices to obtain a complete kinetic trace.Variation of the length and power of the laser pulse offers great flexibility as regards the time scale of the experiment and the temperature at which the reaction can be studied. For water/glycerol mixtures we introduce a simple way to obtain and load an unreacted sample into the spectrometer at low temperature. Experimental SamplesOxygen-free mixtures of TEMPOL (4-hydroxy-TEMPO, Sigma-Aldrich) and sodium dithionite (Na 2 S 2 O 4 , sodium hydrosulphite 85%, Sigma-Aldrich) were prepared from batch solutions at concentrations of 2 and 100 mM, respectively, in a mixture of PCCP Paper

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Rapid Freeze-Quench EPR Spectroscopy: Improved Collection of Frozen Particles

Cited by 11 publications

References 31 publications

Kinetic Mechanism of DNA Polymerases: Contributions of Conformational Dynamics and a Third Divalent Metal Ion

Kinetic Mechanism of DNA Polymerases: Contributions of Conformational Dynamics and a Third Divalent Metal Ion

Effective coupling of rapid freeze-quench to high-frequency electron paramagnetic resonance

Temperature-cycle electron paramagnetic resonance

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