Extended Data Fig. 11: Chl1 facilitates topological cohesin loading on bubble DNA.
From: Coordination of cohesin and DNA replication observed with purified proteins
a, Helicase activity of Chl1 was measured at 100 mM of the indicated salt using Y-fork structured DNA. In all conditions tested, Chl1 displayed higher helicase activity in the presence of 5 mM magnesium ion. As shown in Fig. 4g, the helicase activity of Chl1 appears to be critical for stimulation of cohesin loading during DNA replication. Thus, we carried out IP assays at 5 mM magnesium ion (and 5 mM ATP) concentration to assess the effect of Chl1 on cohesin loading. A representative result from two independent experiments is shown. b, IP assays were performed with the bubble DNA in the presence of the indicated protein at 100 mM NaCl (n = 4 independent experiments; means ± s.d.). c, Gel image of the XL-assays performed with bubble DNA in the presence of Chl1. A representative result from two independent experiments is shown. d, Schematic of the IP assay containing RPA and Chl1 using bubble DNA. e, Gel image and f, quantification of the IP assay depicted in d. The experiment was performed once. g, Schematic of the Order 1 reaction performed with the indicated concentration of RPA. h, Native agarose gel analysis and i, quantitative comparison of the experiments performed as depicted in g, expressed relative to the −Pif1 / −Chl1 reaction using 50 nM RPA (denoted by stars). Means ± s.d. from three independent experiments are shown. Note that Polδ was omitted from the experiments shown in Extended Data Fig. 11h, i.
