Fig. 5. MCMs are recruited by ORC to nucleosomes independently of ARS DNA.

a Cartoon (top), an example kymograph (middle) and the corresponding fluorescence intensities (bottom) of the pre-RC assembly experiment using Cy3-labeled X.l. nucleosomes (green), LD650-labeled MCM (red), unlabeled ORC, Cdc6 and Cdt1. Yellow arrowhead in the kymograph indicates the time when the MCM fluorescence signal appeared at the nucleosomal site. b Cartoon (top), an example kymograph (middle) and the corresponding fluorescence intensities (bottom) of the pre-RC assembly experiment using A488-labeled S.c. nucleosomes (blue), LD650-labeled MCM (red), unlabeled ORC, Cdc6 and Cdt1. In both examples in a, b the nucleosomes were at non-ARS1 positions on the DNA. c Fraction of nucleosomes (X.l. or S.c.) that were observed to have colocalized MCM signals in the presence or absence of ORC. n indicates the number of nucleosomes analyzed for each condition. d Fraction of MCM complexes on a nucleosome-loaded (X.l. or S.c.) tether that colocalized with a nucleosome vs. with nucleosome-free DNA. n indicates the number of MCM complexes analyzed. e Fraction of MCM-nucleosome (X.l. or S.c.) colocalization events observed at ARS1 vs. non-ARS1 positions. n indicates the number of events analyzed. f Cartoon (top) and an example kymograph (bottom) of the three-color experiment using A488-labeled S.c. nucleosomes (blue), both LD650-labeled MCM (red) and Cy3-labeled MCM (green), unlabeled ORC, Cdc6 and Cdt1. The colocalization of a dual-color MCM with a nucleosome indicates MCM DH recruitment to the nucleosomal site. Individual lasers were occasionally turned off to confirm the fluorescence signals from the other channels. Source data are provided as a Source Data file.