Figure 1: Two distinct DNA–protein interactions at replicator sequences.

(a) Map of the HBB locus (top row), Rep-P (second row) and sequences of the intact (AG WT) and mutant (AG1, AG2 and AG mut) oligonucleotides used in this study. Only one strand is shown. The unshaded nucleotides indicate changes from the AG WT oligo. (b) EMSA analyses were used to measure interactions between proteins from K562 cells and biotin-labelled oligos of AG WT, mutated AG1 and mutated AG2 with sequences shown in a. Two DNA–protein complexes were detected with AG WT oligos, but only one complex was detected for AG1 mutant oligos (lower motility—interaction at the AG2 site) and AG2 mutant oligos (higher motility—interaction with the AG1 site). Arrowheads point to specific activities termed AG1 and AG2 and to free oligonucleotides. (c) Specificity of AG1 complex formation. Biotin-labelled double-stranded AG2-mutated oligonucleotides, which contain an intact AG1 site and a mutated AG2 site, interacted with K562 nuclear protein extracts in the presence and absence of specific competing unlabelled oligonucleotides (AG2) and nonspecific competing unlabelled oligonucleotides (AG mut, which could not participate in either AG1 or AG2 complexes). Increasing concentrations of unlabelled AG2, but not AG mut, competed for the AG1 complex. The molecular ratios of specific competitor and probe were 1:1, 1:10 and 1:100, and unspecific competitor and probe were 1:1 and 1:100. The ‘+’ symbol indicates that the reagent was added to the binding reaction, whereas the ‘−’ symbol indicates that the reagent was not included.