Influence of ionic strength on the dichroism properties of polynucleosomal fibers

Abstract

SynopsisWe report electric-dichroism and electron-microscopic studies of chromatin fibers fixed by protein-protein crosslinking at salt concentrations ranging from 10 to 100 mM. The results confirm a progressive disorganization of the fiber as the salt concentration is lowered. The positive dichroism and large polarizability anisotropy characteristic of the 300-A diameter fiber found in 100 mM salt are replaced by negative dichroism and smaller effective polarizability anisotropy or dipole moment for samples fixed at lower salt concentration. We interpret the results in terms of segmental, field-induced orientation of the disorganized structure which is present in low salt concentrations. We also observed a field-induced absorbance decrease in chromatin fibers fixed at salt concentration at and below 100 mM. All three optical effects, namely overall orientation of the high-salt fixed fiber, segmental orientation of the low-salt fixed fiber, and field-induced absorbance decrease, occur on roughly the same time scale, 20-100 ps for 50 nucleosome polynucleosomes. The polarizability anisotropy of fibers fixed in 100 mM salt was found to be proportional to the length of the fragment and to the reciprocal square root of the conductivity of the solution used for electric-dichroism measurements. Addition of Mg2+ to the measurement buffer affected the dichroism amplitude of samples fixed below 100 mM salt but not those fixed at 100 mM salt. The results reinforce the need for caution in interpreting electric-dichroism measurements on chromatin fibers because of possible field-induced distortion effects.

Reinterpretation of Linear Dichroism of Chromatin Supports a Perpendicular Linker Orientation in the Folded State

“…At intermediate ionic strength (Region II) the FLO signal goes through a positive maximum, after which it decreases and eventually vanishes. The ELO signal in this region becomes slowly less negative and is usually observed to reach the zero line (30,34,41 ). At high ionic strength (Region III) the FLO signal is zero in NaCl but is found largely positive in the presence ofMg 2 + (30).…”

“…LD reflects the average orientation of the light absorbing transition dipoles in a macroscopically oriented sample, and is expected to depend sensitively on the DNA arrangement in chromatin. Despite intense study, however, the interpretation of LD results on chromatin has been subject to a remaining ambiguity, since electrically oriented and flow oriented chromatin, in an intermediate range of ionic strengths, persistently show different features: flow LD (FLD) being positive (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30) and electric LD (ELD) generally negative (11,(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41). This incongruity has been suggested to reside in different preparation procedures, buffer conditions or in experimental artifacts (28).…”

Linear dichroism spectroscopy of nucleic acids

“…Because of the requirement of low conductivity in electric field orientation, in most of the early ELD studies the chromatin was condensed by di-and multivalent cations, and under these conditions, positive dichroism of condensed chromatin was not observed. However, chromatin condensed with monovalent cations has positive dichroism under all conditions, independently of orientation technique (Lee et al 1981;Lee & Crothers, 1982;Yabuki et al 1982;Roche et al 1984;Mithieux et al 1984;Marion, 1984;Chauvin et al 1985;Marion et al 1985;Marquet et al 1990). It is not clear why positive LD is not observed in electric fields for chromatin condensed with di-and multivalent cations, but one possible explanation is as follows (Kubista et al 1990 a): if chromatin orientation in electric fields is caused by polarization of DNA counter ions, as is the case for the orientation of DNA (Fredericq & Houssier, 1973;Porschke, 1985), the orientation of chromatin in the electric field will depend on its local structure.…”

Salt‐Induced Conformational Transitions in Chromatin

“…The latter two methods have been extensively used to study the structure of both isolated nucleosomes and high-molecular-weight chromatin fragments. These methods, however, suffer from two serious disadvantages : (a) limited application to high-ionic-strength solutions, due to the thermal and electrochemical processes in the Kerr's cell, and (b) the electric field used for orientation of the molecules probably induces structural distortions in the chromatin fibrils [15,27]. These limitations can be overcome by flow orientation of the chromatin fibrils [15,16].…”