Fig. 5: S-CDK phosphorylation of Yta7 stimulates chromatin segregase function and chromatin replication.

a Reaction scheme of the analyses of Yta7 function on chromatin. b Experiment as in Fig. 3a and b but with the inclusion of Yta7 phosphorylation by S-CDK. In addition, protein samples in the supernatant were analyzed by immunoblot after the first high salt wash to detect proteins released after Yta7 treatment (ii). c Experiments as in a and b but using mutant Yta7 proteins and an S-CDK only control. d Reaction scheme of the in vitro chromatin replication assay including Yta7 and Yta7 mutants. e Effect of Yta7 on in vitro replication. Chromatin was assembled onto ARS1-containing 10.6 kb plasmid DNA in solution as described previously⁵. After purification of chromatinised templates via gel filtration, the replicative helicase Mcm2-7/Cdt1 (MCM) was loaded, phosphorylated by Dbf4-Dependent Kinase Cdc7 (DDK) and H3 was acetylated by pSAGA and acetyl-CoA followed by the addition of Yta7 and mutant versions. Chromatin replication reactions were initiated and included Ctf4, Dpb11, GINS, Cdc45, Pol ε, Mcm10, Sld2, Sld3/7, Pol α, Topo I, RPA, S-CDK, Csm3/Tof1, Mrc1, Pol δ, RFC, PCNA, and FACT/Nhp6. Reactions were stopped after 7 min and newly replicated DNA was visualized by the incorporation of [α⁻³²P] deoxycytidine triphosphate (dCTP) into nascent DNA. Products were separated through 0.8% alkaline agarose gels and visualized by phosphoimaging. Leading and lagging strands are visible because the assay does not include factors required for Okazaki fragment maturation. Asterisk indicates the end-labeling of nicked plasmid DNA. Shown are representative experiments, which have been biologically replicated three times.