We employ real-time PCR to allow us to quantify the sensitivity of chromatin to digestion by DNaseI. This approach has three clear advantages over the more conventional use of the Southern hybridization assay: the accuracy of quantification is improved; the resolution of the assay is enhanced, by designing primers to amplify small amplicons it is possible to analyze sequences both co-incident and proximal to sites of DNaseI-hypersensitivity; less material is needed, as little as 5 ng of treated genomic DNA. We applied this method in an analysis of the chromatin structure of the previously described mouse β-globin locus control region (LCR) using fetal liver cells. The four hypersensitive sites of the canonical mouse LCR, HS1 to HS4, are shown to have kinetics of digestion consistent with these sequences being nucleosome-free in vivo. A different pattern was seen for HS6, a recently described "weak" hypersensitive site. The site was also rapidly lost but more of the sites proved resistant, we interpreted this to show that this hypersensitive was only forming in a portion of the erythroid cells. This finding implies that in vivo the LCR is structurally heterogeneous. Sequences proximal to the hypersensitive sites show a third pattern of intermediate sensitivity, consistent with the chromatin being unfolded but the sites still bound by a continual nucleosomal array. Our results demonstrate that this method has the potential to achieve accurate and detailed mapping of chromatin structure from small amounts of tissue samples.

Alpha and beta protein subunits of the telomere end binding protein from Oxytricha nova (OnTEBP) combine with telomere single strand DNA to form a protective cap at the ends of chromosomes. We tested how protein-protein interactions seen in the co-crystal structure relate to DNA binding through use of fusion proteins engineered as different combinations of domains and subunits derived from OnTEBP. Joining alpha and beta resulted in a protein that bound single strand telomere DNA with high affinity (K D-DNA =1.4 nM). Another fusion protein, constructed without the C-terminal proteinprotein interaction domain of alpha, bound DNA with 200-fold diminished affinity (K D-DNA =290 nM) even though the DNA-binding domains of alpha and beta were joined through a peptide linker. Adding back the alpha C-terminal domain as a separate protein restored high-affinity DNA binding. The binding behaviors of these fusion proteins and the native protein subunits are consistent with cooperative linkage between protein-association and DNA-binding equilibria. Linking DNA-protein stability to protein-protein contacts at a remote site may provide a trigger point for DNA-protein disassembly during telomere replication when the single strand telomere DNA must exchange between a very stable OnTEBP complex and telomerase.