FIG 6.

Interactions of recombinant Spt5 domains with various forms of NA. (A) Sequences of the single-stranded and double-stranded NAs. The 5′ end of a single strand and the top strand of a double strand were synthesized (IDT) with a TAMRA fluorescent group for detection. (B) Gel shift images from the binding assays. The reaction mixtures each contained 5 μM NA probe, 80 μM protein domain, and the buffer. A name of an NA form (bottom left side) indicates a free probe, while the label “bound” indicates a species of protein-NA complex. The protein domain used in a binding reaction is identified above the gels. (C) Direct comparison between two forms of NA for K1L1 binding in the same binding mixture. NA forms (5 μM) are indicated (bottom left side), and protein concentrations varied from 0 to 80 μM, as indicated. Binding curves for the NA forms are shown below the gels, with each data point averaged from multiple experiments (see Materials and Methods). The error bars indicate mean errors. (D) Binding competition of unlabeled dsDNAs of the same length but different sequences. Gel bands of the unbound 20-mer probe and bound species are indicated, and the probe and protein concentrations are given at the top.