Extended Data Fig. 7: Scc4 and PIP of Scc2 are both contributing to chromatin binding of the cohesin loader.
From: PCNA recruits cohesin loader Scc2 to ensure sister chromatid cohesion
a-d, Tetrad dissection analysis on the indicated diploid strains on galactose- (YP Gal) and glucose-containing (YPD) plates. N-terminally 7His8FLAG (HF)-tagged Scc2-E822K and its various PIP mutants are expressed from endogenous promoter pSCC2. Cohesin loaders HFScc2-E822K with intact PIP motifs, either endogenous (a) or replaced by the PIP of the DNA polymerase δ nonessential subunit Pol32 (c), support viability upon SCC4 shut-off on YPD plates. On the contrary, PIP mutants with conserved residues replaced by alanines (b, d) fail in providing viability. e, Subcellular fractionation of cycling cells expressing HF-tagged Scc2-E822K or its PIP mutant from strong constitutive promoter pADH1 into soluble supernatant (SUP) and chromatin-enriched (CHR) fractions by centrifugation of the whole cell extract (WCE). Chromatin binding of HFScc2-E822K-pip is decreased compared to HFScc2-E822K. Used cells had in addition SCC4 expressed from galactose-inducible pGALS promoter allowing to study HFScc2-E822K chromatin binding upon SCC4 shut-off after shift from galactose-containing media (YP GAL) to glucose (YPD). Loss of Scc4 further decreases cohesin loader binding to chromatin. To control chromatin fractionation efficiency, the levels of histone H4, replicative helicase Mcm2-7 and the cytoplasmic/plasma membrane protein Pgk1 were detected in fractions.
