FIGURE 2.

Catalytic activity of Mot1 on complexes formed on gapped DNA probes. A, EMSA using a fully duplex, radiolabeled TATA-containing DNA (<1 nm), 10 nm TBP, and Mot1 as indicated with or without 50 μm ATP. The positions of the free DNA, TBP-DNA, and Mot1-TBP-DNA complexes are indicated by the arrows. B, quantitation of relative TBP-DNA complex levels measured by EMSAs as in A and containing 2.9 nm Mot1 and using fully duplex DNA (WT) or each of the 12 indicated gapped DNA probes (diagrammed in Fig. 1A). The level of TBP-DNA complex formed on each probe in the absence of Mot1 was set to 1, and the relative levels of TBP-DNA present in reactions containing Mot1 ± ATP were normalized to those levels. Note that addition of Mot1 without ATP reduced the TBP-WT DNA complex level to about 30% of the level present in the absence of Mot1 coincident with the formation of the Mot1-TBP-DNA complex. In contrast, the extent of TBP-DNA complex formation was reduced to a comparable level when ATP was added because Mot1 catalyzed TBP-DNA complex disassembly. Gapped DNA probes impaired for ATP-dependent TBP-DNA dissociation are indicated by the pink bars (p < 0.005; those probes not significantly affected had p values >0.05). Complexes formed on probe 7 (green bar) were cleared to significantly greater extent than on the WT probe. For one probe (probe 6), the level of TBP-DNA complex formation was less affected by addition of Mot1 than for the other probes (asterisk; see text). C, relative Mot1-TBP-DNA complex formation measured in reactions containing the indicated gapped DNA probes, TBP, Mot1, and ATP compared with results obtained using the WT probe. Relative Mot1-TBP-DNA complex levels remaining in reactions containing ATP were higher using probes 1, 2, and 5 than when using WT DNA. The difference obtained using probe 1 was significant (p = 0.04) using p < 0.05 as the cutoff. However, the difference obtained using probe 5 was just outside the 0.05 cutoff (p = 0.065) and quite possibly also significant. Comparisons of results from all other probes with WT had p values >0.1. p values were determined using a two-tailed Student's t test. D, relative TBP-DNA complex levels present in reactions formed using fully duplex WT probe or probes with a 1-base gap or a single strand nick as indicated in the inset diagram. The strand discontinuity is located 5 bases upstream from the TATA sequence. E, summary of the main findings from EMSA analysis using the gapped DNA probes. Sequences highlighted in pink indicate gap positions that interfere with Mot1-mediated, ATP-dependent dissociation of TBP-DNA complexes. The sequence in red indicates the gap at position 1 that supports ternary complex assembly but is less affected by ATP addition than the other probes. Error bars represent S.D.