Fig. 1: Reconstitution of hDH loading.
From: MCM double hexamer loading visualized with human proteins
a, Purified human MCM loading proteins analysed by SDS-PAGE and Coomassie staining. Full-length proteins (left) and truncated proteins (right): ORC1–5 complex containing ORC1(∆N) (ORC1–5(1∆N)), CDC6(∆N) and CDT1(∆N). b, Outline of the nuclease footprinting assay. MCM loading reactions are treated with benzonase followed by quenching with EDTA, SDS and proteinase K. Then DNA is purified by phenol–chloroform–isoamyl alcohol extraction and ethanol precipitation, resolved on a TBE polyacrylamide gel, and stained with SYBR Gold. c, Timecourse of yeast (top) and human (bottom) MCM loading reactions. ∆N indicates that the assay was carried out with truncated proteins. d, Nuclease footprinting assay with truncated human proteins. Proteins were omitted as indicated. e, As d, with full-length (FL) proteins. f, Side-by-side comparison of the ORC6 dependency with full-length and truncated proteins. g, As f, testing conditions in which either ORC1, CDC6 or CDT1 was truncated and other proteins remained full-length. h, As f, with full-length ORC1 and truncated CDC6 and CDT1. i, Effect of geminin on full-length MCM loading reactions. CDT1 and geminin were pre-mixed before reactions were started. j, Salt stability of the double hexamer. MCM was loaded for 30 min and then incubated in buffers containing the indicated concentrations of sodium chloride (mM) for 15 min, followed by dilution to lower salt and benzonase treatment. The band at the bottom of the gel appears because of incomplete digestion owing to the higher salt concentration used. Gel source data for all figures can be found in Supplementary Fig. 4.
