Restriction Enzyme Kinetics Monitored by UV Linear Dichroism

Abstract

The use of linear dichroism (LD) spectroscopy for biological applications has been brought to the forefront recently by our development of thermostated microvolume Couette cells. We present a method for following the digestion of DNA by restriction endonucleases in real time without the use of any extrinsic dyes or labels. This is accomplished using linear dichroism spectroscopy (the differential absorbance of light polarized parallel and perpendicular to the sample orientation axis). The differential absorbance signal depends on the degree of alignment of the molecules. In this case the DNA is aligned by Couette flow (flowing the solution in the annular gap between two concentric cylinders), and we monitor the increase in alignment upon linearization of a circular DNA molecule. In addition, we observe a decrease in alignment upon further digestion and subsequent shortening of the DNA. Ten enzymes were investigated: seven enzymes with a single cut site (EcoRI, KpnI, NdeI, NotI, NruI, SmaI, XbaI), two enzymes with two cut sites (BstZ17I, EagI), and one enzyme with no cut site (ClaI). LD, as implemented in this new assay, is broadly applicable across a wide range of DNA-modifying enzymes and compounds and, as such, is a useful addition to the toolbox of biological characterization.

Fine tuning of the catalytic activity of colicin E7 nuclease domain by systematic N‐terminal mutations

“…Flow linear dichroism (FLD) as a noninvasive technique was simultaneously applied for monitoring the digestion of long linear calf thymus DNA chains. The signal intensity of the FLD spectra depends on the ability of the long filiform DNA molecules to align, that is, on the length, conformation, and rigidity of the double helix . Due to the cleavage by nucleases, the DNA chains shortened and aquired a lower degree of orientation in the Couette flow cell.…”

Real-time detection of Fe{middle dot}EDTA/H2O2-induced DNA cleavage by linear dichroism

“…The LD measurement method has been described elsewhere (20,23–26). The measured LD is divided by the isotropic absorption spectrum to give the reduced LD (LD r ), which is related to the ability of orientation, via the following equation: where A iso denotes the isotropic absorption spectrum and S the orientation factor, such that S = 1 or 0 for perfectly oriented or randomly orientated samples, respectively.…”

“…The absorption spectra of DNA in the presence or absence of various cofactors were not altered to any great extent; therefore, the measured LD was considered to be a direct reflection of the flexibility and length of the DNA in this work. The method used for the measurement of the LD was identical to the previously reported method (20), with the exception of the microvolume of the thermostatically controlled Couette cell. A conventional Couette cell containing 2.5 ml of the sample was used.…”

“…The magnitude of the LD was reported to increase upon the linearization of a circular DNA, brought about by the use of restriction enzymes, due to its elongation. The further digestion and subsequent shortening of the DNA resulted in a decrease in the magnitude of the LD (20). A decrease in the magnitude of the LD reflecting the shortening of the DNA due to double strand cleavage by deoxyribonuclease I has also been reported (22).…”

Oxidative DNA cleavage by Cu(II) complexes: Effect of periphery substituent groups

“…The cleavage of both strands at a similar place shortens the DNA molecule, which also results in a lower LD magnitude in the DNA absorption region. Using this concept, LD is an excellent tool for probing the cleavage of sc and dsDNA in real-time[19,[33][34][35]39,[43][44][45][46].…”