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. 2019 May 22;47(13):6826–6841. doi: 10.1093/nar/gkz441

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© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 5. — Over-retained PCNA over-recruits Msh2–Msh6 to chromatin, affecting mismatch repair. (A) Msh6 accumulates on chromatin in elg1Δ, compared to ELG1⁺. WCE and chromatin-enriched fractions (Chromatin) were prepared from cells expressing Msh6–6HA in log phase. Msh6–6HA, PCNA and histone H3 (loading control) were detected by western blotting. (B) Quantification of chromatin-bound Msh6, normalized to histone H3 (average of two independent clones shown in panel A) and chromatin-bound Msh2 and Msh6 in cells in hydroxyurea by SILAC quantitative proteomics performed previously (46). Error bars, SDs. (C) Msh6PIP does not accumulate on chromatin in elg1Δ, compared to ELG1⁺. WCE and chromatin-enriched fractions (Chromatin) were prepared from msh6PIP msh3PIP mutants expressing Msh6PIP-6HA in log phase. Msh6PIP-6HA, PCNA and histone H3 (loading control) were detected by Western blotting. (D) Quantification of chromatin-bound Msh6PIP (normalized to histone H3) shown in panel C. Average of two independent clones is shown. Error bars, SDs. (E) ChIP-seq analysis of PCNA performed previously (11). PCNA distribution around ARS607 on chromosome VI is shown. PCNA is unloaded behind replication forks in ELG1⁺ but retained in elg1Δ in S phase (15 min after release from a cdc7–1 block at 16°C). Black square, region quantified by ChIP-qPCR in panels F and G. (F) ChIP-qPCR analysis of Msh6 and PCNA for early origin ARS607 in the presence and absence of Elg1. ChIP was performed using cells arrested in alpha-factor (G1) or collected 15 min after release from a cdc7–1 block into S phase at 16°C (S). Error bars, SDs of three technical replicates. (G) ChIP-qPCR analysis of Msh6 for ARS607 in the msh6PIP mutant or the disassembly-prone PCNA mutant pol30-D150E backgrounds. ChIP was performed using cells collected 15 min after release from a cdc7–1 block into S phase at 16°C. Three independent isolates of msh6PIP elg1Δ were shown. Error bars, SDs of three technical replicates. (H) Effect of over-expression of Msh2, Msh3 and Msh6 or their PIP mutants on mutation rate at the lys2–10A locus. Msh2, Msh3 and Msh6 or their PIP mutants are over-expressed under their own promoters from three multicopy plasmids. Error bars, 95% confidence intervals. (I) Mutation rates of ELG1⁺ and elg1Δ in wild-type and exo1Δ backgrounds at the lys2–10A locus. Error bars, 95% confidence intervals.