Fig 3. PCNA ubiquitylation contributes to the interaction of Polδ with the clamp.

(A) Assessing co-immunoprecipitation of Polδ and PCNA. Top: experimental scheme for co-immunoprecipitation experiments. Cells were either treated, or not, with hydroxyurea (HU) and TCA extracts prepared to monitor PCNA modification (whole cell) or soluble extracts prepared for immunoprecipitation with either anti-PCNA or anti-GFP (the catalytic subunit of Polδ is GFP tagged). Bottom: comparison of the Polδ - PCNA interaction in rad18Δ (Δ), wild-type (+) or urg1-rad18 cells (++) cells that exhibiting distinct levels of PCNA ubiquitylation (see Fig 2A). (B) Cell-cycle profiles. Open histograms -HU, grey histograms +HU. (C) Quantification of modified forms of PCNA that was co-immunoprecipitated with Polδ (“IP with αGFP” in a) and in whole cell extracts. (D) Co-immunoprecipitation of Polδ in P_urg1-rad18 cells is partially dependent on ubc13. (E) Quantification of DNA-associated Polδ by single molecule PALM imaging (see Fig 2F for details). (F). Increased chromatin association of PCNA in the elg1Δ genetic background. Chromatin was fractionated from the indicated strains and probed for Pcn1 and a histone H3. (G) Co-immunoprecipitation of Polδ with PCNA in elg1⁺ (+) and elg1Δ (Δ) cells in the rad18⁺ and rad18Δ genetic backgrounds.