Figure 4.

Analysis of the processivity of Sen1 translocation on ssDNA. (A) Sequence and structure of the substrate used in these experiments. (B) Unwinding experiments performed in single-round conditions with Sen1 HD. An experiment performed with full-length Sen1 gave identical results (data not shown). Top: Schematic description of the assay. The presence of heparin in the reaction prevents re-association of Sen1 molecules that have dissociated from the substrate, thus precluding new rounds of unwinding. Bottom-left: Representative gels of unwinding experiments. An asterisk indicates the radioactive label at the 5΄ end of the long DNA molecule. The first three lanes show different nucleic acid molecules that could be detected in these experiments. –ATP, control reaction incubated with Sen1 HD in the absence of ATP. H, control reaction in which heparin was added before challenging the substrates with Sen1 HD. Bottom-right: Graphs showing the fraction of oligonucleotide dissociated by Sen1 HD as a function of time. Values represent the average and SD from three independent experiments. (C) Control multiple-round unwinding assay. The reaction proceeded for 5 min. In the absence of heparin, Sen1 can undertake multiple cycles of association-translocation-dissociation that allow carrying the unwinding reaction to completion (89% of the substrate fully unwound).