PICH: a DNA translocase specially adapted for processing anaphase bridge DNA.

Generation of constructs and DNA substrates

Details of the construction of PICH expression plasmids and DNA triplexes can be found in the Supplemental Information .

Recombinant PICH protein expression and purification

In order to characterise the biochemical and biophysical properties of PICH, we purified four recombinant versions of the protein from baculovirus-infected insect cells. Two were hexa-histidine tagged, full-length PICH proteins (hereafter termed ‘native PICH’) that were not fused to GFP. Details of the purification of these proteins can be found in the Supplemental Information . ATPase assays The ATPase activity of PICH was assayed using a commercial kit (Innova Biosciences) according to the manufacturer’s instructions. Where indicated, reactions contained 500 ng of a specified DNA co-factor.

DNA binding assays Electrophoretic mobility shift assays

(EMSAs) for analyzing the binding of purified PICH or PICH-K128A protein to DNA were conducted as described in the Supplemental Information .

Translocase and branch migration assays

Details of the PICH migration assays can be found in the Supplemental Information .

Nucleosome remodeling assays

Recombinant human histone proteins were expressed and purified as described previously ( Flaus et al., 2004 ) except for human CENP-A, which was expressed as a soluble tetramer with histone H4 as described by Black et al. ( Black et al., 2004 ). Yeast RSC was expressed and purified as described by Ferreira et al. ( Ferreira et al., 2007 ). Nucleosomes were assembled using standard salt-gradient dialysis methods using recombinant human histones and PCR-generated DNA fragments. The Cy5 or Cy3 labelled PCR fragments contained the 601 or MMTV NucA nucleosome positioning sequences in the centre (47w47, 54A54) or at the end (0w47). Nucleosome sliding assays were performed in 50 mM NaCl, 50 mM Tris (pH 7.5), and 3 mM MgCl 2 plus or minus 1 mM ATP, as indicated in the Figure. The reactions were incubated at 37°C with the amount of RSC or PICH specified in the Figure. Reactions were stopped after 30 min by addition of 1 μg of plasmid DNA and sucrose to 5% (w/v) before electrophoresis on 0.2xTris-Borate-EDTA (TBE) 5% native polyacrylamide gels. The gels were scanned for the Cy-dyes signal using a PhosphorImager.

Single molecule assays

Single molecule experiments were carried out in a set-up combining fluorescence microscopy with double optical tweezers ( Candelli et al., 2011 ; Farge et al., 2012 ; van Mameren et al., 2009b ), as described in the Supplemental Information .

Supplementary Material Movie S1 Movie S1: Fluorescence movie of 50 nM PICH-eGFP DNA-binding and –unbinding at medium tension (30 pN) in high salt buffer (20 mM Tris-HCl pH 7.5, 100 mM NaCl, 2 mM MgCl 2 , 2 mM ATP). Note that the movie is speeded up by 35-fold (from 1 to 35 Hz) compared to real time, and that the interactions are much shorter than seen in low salt buffer ( Movie S2 ). The bead diameter can be used as scalebar (3.3 μm diameter). Movie S2 Movie S2: Fluorescence movie of PICH-eGFP DNA-binding and –unbinding at medium tension (30 pN) in low salt buffer (20 mM Tris-HCl pH 7.5, 25 mM NaCl, 2 mM MgCl 2 , 2 mM ATP). Note that the movie is speeded up by 35-fold (from 0.4 to 14 Hz) in order to render the slow PICH translocation more easily discernible. The bead diameter can be used as scalebar (3.3 μm diameter). Movie S3 Movie S3: Fluorescence movie of PICH-eGFP DNA-binding and –unbinding in high salt buffer (20 mM Tris-HCl [pH 7.5], 100 mM NaCl, 2 mM MgCl 2 , 2 mM ATP) at different DNA tensions; DNA tension is increased from the initial 2 pN stepwise to 16 (10 s), 30 (23), 47 (34) and finally 57 pN (47 s) by moving the right bead. Note that the movie is speeded up by 25-fold (from 1 to 25 Hz) compared to real time. The bead diameter can be used as scalebar (3.3 μm diameter). Supplementary Material