FIG. 6.

Representative gel mobility shift assay used to establish which components of the 47-bp penta 2, 4 + AT assembly unit are required for hexamer and double-hexamer formation. (A) Reactions were conducted in the presence of AMP-PNP with 6 pmol of T-ag and 25 fmol of the indicated oligonucleotide. As a positive control, one reaction was conducted with the 47-bp penta 2, 4 + AT oligonucleotide (lane 2). To test the role of the AT in these assembly events, a reaction was conducted with the 47-bp penta 2, 4 + ATm oligonucleotide (lane 4). Reactions conducted with the single pentanucleotide containing 47-bp penta 4 + AT and 47-bp penta 4 + ATm oligonucleotides are presented in lanes 6 and 8, respectively. Similar reactions, conducted with the 47-bp 2 + AT and 47-bp penta 2 + ATm oligonucleotides, are presented in lanes 10 and 12, respectively. To assay for non-sequence-specific binding events, an additional reaction was conducted with the 47-bp control oligonucleotide (lane 14). Reactions in the odd-numbered lanes were conducted in the absence of protein. The positions of T-ag hexamers (H) and double hexamers (DH) are indicated by arrows, while the position of free DNA is indicated by a bracket. (B) The reactions shown in Fig. 6A and similar reactions conducted in the presence of ADP, ATP, and no exogenous nucleotides (data not shown) were quantitated with a Molecular Dynamics PhosphorImager. Histogram 2 displays the percentage of input DNA, containing single pentanucleotides, that is shifted into hexamers. The nucleotide cofactor used in a given set of reactions is shown to the right of the figure, the names of the individual oligonucleotides are shown on the x axis, and the percentage of input DNA shifted into hexamers is shown on the y axis. Histogram 1 reveals the quantitative impact of mutating the AT on T-ag oligomerization on the penta 2, 4 + AT assembly unit.