Extended Data Fig. 5: Yeast cohesin loader Scc2 interacts with PCNA in vivo.
From: PCNA recruits cohesin loader Scc2 to ensure sister chromatid cohesion
a, GST pull-down using recombinant GST-Pol30 (yeast PCNA) in lysates from yeast cells expressing N-terminally 7His8FLAG (HF)-tagged Scc2-E822K from a strong constitutive promoter pADH1. b, Ni-NTA pull-down (Ni PD) of HF-tagged Scc2-E822K or its PIP mutant under denaturing conditions from yeast cells after formaldehyde crosslinking. HFScc2-E822K but not its PIP mutant variant binds to cross-linked PCNA species (Pol30 X-linked). PCNA was detected using polyclonal anti-Pol30 antibody (the asterisk indicates a cross-reactive protein), untagged scc2-E822K strain was used as control. Ni PD efficiency was assayed using Ponceau S staining (nonspecifically pulled-down protein of ≈15 kDa visualized), Pgk1 served as loading control. c, Cross-linked PCNA species (Pol303MYC X-linked) detected using anti-MYC antibody after formaldehyde crosslinking of yeast cells expressing C-terminally 3MYC-tagged Pol30. d, Residues Q1475 and T1493 in the vicinity of Scc2 PIP selected to be replaced with BPA for in vivo cross-linking experiments. e, In vivo BPA cross-linking of Scc2-Q1475BPA and Scc2-T1493BPA to PCNA. Cross-links are detected in cells expressing C-terminally 3MYC-tagged Pol30 (yeast PCNA) and N-terminally 3HA-tagged Scc2-Q1475BPA or Scc2-T1493BPA specifically following UV irradiation. Proteins were isolated by immunoprecipitation.
