A 'one-pot' assay for the accessibility of DNA in a nucleosome core particle

Abstract

The accessibility of nucleosomal DNA to transcription factors and other sequence-specific DNA binding proteins is of importance in the consideration of mechanisms of transcriptional control. Here, we report a simple novel assay which determines this accessibility at eight different rotationally equivalent sites on nucleosomal DNA and shows that linker histones and the chromosomal HMGB proteins, HMG-D and HMG-Z, have opposite effects on the accessibility of nucleosomal DNA. We compare this assay to previously described methods.

Figure 1

(a) A digestion time course of cleavage using 500 U/ml HaeIII. (b) Product bands from experiment shown in (a) were quantified and expressed as uncut fractions. (c) Parallel experiment with a 10-fold higher enzyme concentration.

Figure 2

Parallel experiments (conditions identical to Figure 1) were conducted using four enzyme concentrations (from top to bottom: 5000 U/ml, 500 U/ml, 50 U/ml and 5 U/ml). For clarity, experimental data points as well as best fit curves are shown for three sites.

Figure 3

k₁ and k₂ obtained from experiments described in Figure 2 were plotted in pairs for all sites.

Figure 4

Effects of linker histone H1 (a), HMG-D (b) and HMG-Z (c) compared in terms of normalized apparent rate constants. The final concentrations for H1, HMG-D and HMG-Z in the reaction mixtures were 19 nM, 2.1 μM and 2.3 μM, respectively.