Extended Data Fig. 5: Additional control experiments of chromatin replication.
From: Coordination of cohesin and DNA replication observed with purified proteins
a, Purified core histones, Nap1, ISW1a complex, FACT complex and Nhp6A were analysed by SDS-PAGE followed by Coomassie Brilliant Blue (CBB) staining. b, Micrococcal nuclease (MNase) digestion of the chromatinized circular DNA template used for chromatin replication. nuc. denotes nucleosome. The digested products were separated by agarose gel electrophoresis and stained with Sybr Gold. This confirmed the evenly spaced nucleosome assembly. A representative result from three independent experiments is shown. c, Schematic of in vitro replication with the chromatinized template. d, Denaturing and native agarose analyses of the experiments depicted in c with the indicated proteins. e, Quantification of full-length products in d (n = 3 independent experiments; means ± s.d.). Omission of Csm3-Tof1 or Mrc1 reduced the full-length products even in the presence of Pif1, suggesting that these cohesion establishment factors promote DNA replication per se in a chromatin context. f, Nucleosomes limit cohesin loading. In vitro cohesin loading reactions (IP assay) were carried out with chromatinized circular DNA. Representative gel images of the experiment performed with naked (top) or chromatinized DNA (bottom) in the presence of the indicated proteins are shown. The graph represents quantification of cohesin-bound DNA (n = 3 independent experiments; means ± s.d.). g, Confirmation of chromatin assembly using cohesin-loaded circular DNA templates. Cohesin loading was first carried out in the presence of Scc2-Scc4 and ATP, and then was sequentially subjected to nucleosome assembly using Nap1 and ISW1a. MNase digestion confirmed that the nucleosomes were efficiently and evenly assembled on cohesin-loaded DNA, as on naked DNA. A representative result from two independent experiments is shown. h, Schematic of the “cohesin-first” replication reaction with the chromatinized template. i, Denaturing and native gel analyses of the experiments depicted in h. Like replication with naked DNA, replicated DNA was recovered by cohesin-IP after chromatin replication, whereas no detectable replicated DNA was seen in the absence of cohesin. A representative result from two independent experiments is shown. Note that Pol δ was included in chromatin replication because nucleosomes limit the strand displacement synthesis by Pol δ37,38.
