Linear Dichroism Measurements for the Study of Protein-DNA Interactions

Abstract

Linear dichroism (LD) is a differential polarized light absorption spectroscopy used for studying filamentous molecules such as DNA and protein filaments. In this study, we review the applications of LD for the analysis of DNA-protein interactions. LD signals can be measured in a solution by aligning the sample using flow-induced shear force or a strong electric field. The signal generated is related to the local orientation of chromophores, such as DNA bases, relative to the filament axis. LD can thus assess the tilt and roll of DNA bases and distinguish intercalating from groove-binding ligands. The intensity of the LD signal depends upon the degree of macroscopic orientation. Therefore, DNA shortening and bending can be detected by a decrease in LD signal intensity. As examples of LD applications, we present a kinetic study of DNA digestion by restriction enzymes and structural analyses of homologous recombination intermediates, i.e., RecA and Rad51 recombinase complexes with single-stranded DNA. LD shows that the DNA bases in these complexes are preferentially oriented perpendicular to the filament axis only in the presence of activators, suggesting the importance of organized base orientation for the reaction. LD measurements detect DNA bending by the CRP transcription activator protein, as well as by the UvrB DNA repair protein. LD can thus provide information about the structures of protein-DNA complexes under various conditions and in real time.

Keywords: DNA/protein complex; Rad51; RecA; UvrB nucleotide excision repair protein; catabolism activator protein (CAP); cyclic AMP receptor protein (CRP); homologous recombination; linear dichroism (LD); restriction enzyme; transcription regulation.

Figure 1

Transition dipole moments in a chromophore (a) and principle of linear dichroism (b).

Figure 2

Linear dichroism observation of DNA digestion by endonuclease I in the presence of various concentrations of ethidium bromide. From [5].

Figure 3

Schematic presentation of the geometry of groove binding and intercalation ligands. From [15].

Figure 4

Couette cell and flow-through cell for LD measurements.

Figure 5

ATP cofactor dependence of RecA/ssDNA complex LD spectra. The LD spectrum of the RecA-ssDNA complex (dots) shows a positive signal around 280 nm, while that of the ATPγS-RecA-ssDNA complex shows a strong negative signal around 260 nm (continuous line). From [9].

Figure 6

Use of poly(dεA) for assigning changes in the LD signal to the DNA bases in the RecA/ssDNA complex. Poly(dεA) absorbs light above 310 nm, while RecA does not. Furthermore, poly(dεA) absorbs strongly around 240 nm. The negative LD signals above 310 nm and around 240 nm indicate the perpendicular orientation of DNA bases in the complex. From [4] (Copyright Elsevier).

Figure 7

Determination of the orientation of one tryptophan residue of RecA in the RecA-ssDNA complex. The LD spectra of the complex formed with poly(dεA) and wild-type RecA, and that of the complex formed with poly(dεA) and engineered RecA, in which tryptophan 290 is replaced with histidine, were measured. The tryptophan 290 signal was estimated by subtracting the spectra with RecAW290H from those with wild-type RecA. From [56] (Copyright Elsevier).