Bending Step Research Articles

Non-cognate Enzyme–DNA Complex: Structural and Kinetic Analysis of EcoRV Endonuclease Bound to the EcoRI Recognition Site GAATTC

The crystal structure of EcoRV endonuclease bound to non-cognate DNA at 2.0 angstroms resolution shows that very small structural adaptations are sufficient to ensure the extreme sequence specificity characteristic of restriction enzymes. EcoRV bends its specific GATATC site sharply by 50 degrees into the major groove at the center TA step, generating unusual base-base interactions along each individual DNA strand. In the symmetric non-cognate complex bound to GAATTC, the center step bend is relaxed to avoid steric hindrance caused by the different placement of the exocyclic thymine methyl groups. The decreased base-pair unstacking in turn leads to small conformational rearrangements in the sugar-phosphate backbone, sufficient to destabilize binding of crucial divalent metal ions in the active site. A second crystal structure of EcoRV bound to the base-analog GAAUTC site shows that the 50 degrees center-step bend of the DNA is restored. However, while divalent metals bind at high occupancy in this structure, one metal ion shifts away from binding at the scissile DNA phosphate to a position near the 3'-adjacent phosphate group. This may explain why the 10(4)-fold attenuated cleavage efficiency toward GAATTC is reconstituted by less than tenfold toward GAAUTC. Examination of DNA binding and bending by equilibrium and stopped-flow florescence quenching and fluorescence resonance energy transfer (FRET) methods demonstrates that the capacity of EcoRV to bend the GAATTC non-cognate site is severely limited, but that full bending of GAAUTC is achieved at only a threefold reduced rate compared with the cognate complex. Together, the structural and biochemical data demonstrate the existence of distinct mechanisms for ensuring specificity at the bending and catalytic steps, respectively. The limited conformational rearrangements observed in the EcoRV non-cognate complex provide a sharp contrast to the extensive structural changes found in a non-cognate BamHI-DNA crystal structure, thus demonstrating a diversity of mechanisms by which restriction enzymes are able to achieve specificity.

Simultaneous DNA binding and bending by EcoRV endonuclease observed by real-time fluorescence.

The complete catalytic cycle of EcoRV endonuclease has been observed by combining fluorescence anisotropy with fluorescence resonance energy transfer (FRET) measurements. Binding, bending, and cleavage of substrate oligonucleotides were monitored in real time by rhodamine-x anisotropy and by FRET between rhodamine and fluorescein dyes attached to opposite ends of a 14-mer DNA duplex. For the cognate GATATC site binding and bending are found to be nearly simultaneous, with association and bending rate constants of (1.45-1.6) x 10(8) M(-1) s(-1). On the basis of the measurement of k(off) by a substrate-trapping approach, the equilibrium dissociation constant of the enzyme-DNA complex in the presence of inhibitory calcium ions was calculated as 3.7 x 10(-12) M from the kinetic constants. Further, the entire DNA cleavage reaction can be observed in the presence of catalytic Mg(2+) ions. These measurements reveal that the binding and bending steps occur at equivalent rates in the presence of either Mg(2+) or Ca(2+), while a slow decrease in fluorescence intensity following bending corresponds to k(cat), which is limited by the cleavage and product dissociation steps. Measurement of k(on) and k(off) in the absence of divalent metals shows that the DNA binding affinity is decreased by 5000-fold to 1.4 x 10(-8) M, and no bending could be detected in this case. Together with crystallographic studies, these data suggest a model for the induced-fit conformational change in which the role of divalent metal ions is to stabilize the sharply bent DNA in an orientation suitable for accessing the catalytic transition state.

Bending and axial strain dependence of the critical current in superconducting BSCCO tapes

A comparison is made between the critical current (Ic) versus bending strain and axial strain of superconducting multi-filamentary Bi2Sr2Ca2Cu3Ox (Bi-2223) AgMg sheathed tapes. For the bending strain measurement the tape is sandwiched between a curved base and cover plate. Six sets of bending plates introduce bending strains ranging from 0% to 1.0%. The measurements show a slight decrease in Ic after the first bending step after which the degradation becomes more pronounced. The Ic in a bent conductor is calculated assuming a linear axial strain profile inside the conductor. For this calculation the Ic degradation determined in an axial compression and elongation experiment is used. The model predicts an immediate decrease of Ic, caused by the compressive strain dependence. There is a good agreement (within 5%) between the measured data and the calculated values. Based on this good agreement it can be concluded that a possible shift in the neutral line or the formation of additional cracks due to bending has no significant influence on the Ic degradation. It is concluded that the influence of thermal contraction is crucial for a good calculation.

The Bauschinger Effect of Sheet Metal Under Cyclic Reverse Pure Bending

AbstractIn some sheet metal forming operations, metal flow involves a series of bending and unbending steps. In any such bending sequence the occurrence of the Bauschinger effect is likely. Correct process modelling of such forming operations therefore requires inclusion of the Bauschinger effect. This paper presents results of an experimental procedure developed for the determination of the Bauschinger effect. Sheet strips were subjected to a series of bending and reverse bending steps by means of a pure moment bending device. Results show the Bauschinger effect in steel and aluminum sheet as a function of strain amplitude, prestrain, and sheet thickness.