Analysis of hcDNA with single-strand-specific nucleases. hcDNA radioactively labeled at one end was digested with S1 and P1 nucleases and the digestion products were analyzed under denaturing conditions on a polyacrylamide-urea gel. Note the two sensitive regions inside the poly(CA)·poly(TG) tract. A G+A sequencing reaction on the same fragment was used as a size marker. In the experiment with S1 nuclease, hcDNA was 3'-labelled at the Eco RI site. The samples were: G+A: size marker; hcDNA1: upper major band of hcDNA (see Fig. 2A); hcDNA2: lower major band (id.); hcDNA: total hcDNA; ds: double strand. In the experiment with P1 nuclease, hcDNA was 5'-labelled at the Eco RI site. Samples were: G+A: size marker; ds: P1 digestion of the linear double strand; hcDNA: digestion of hcDNA with P1 nuclease in the absence of HMGB1; hcDNA+HMG: digestion of hcDNA with P1 nuclease in the presence of HMGB1; ss: P1 digestion of the single strand. Scans of the samples digested with P1 nuclease in the presence or absence of HMGB1 are also shown.