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. 2017 Feb 13;45(8):4564–4576. doi: 10.1093/nar/gkx107

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© The Author(s) 2017. 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-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 1. — The SprT domain is sufficient to suppress the cell-cycle defects, but not the lesion bypass deficiency, in Sprtn knockout cells. (A) Schematic representation of full-length and truncated Spartan proteins used in this study. The truncated Spartan mutant, denoted as SprT, contains the SprT domain but lacks the C-terminal half. SprT, a zinc metalloprotease-like domain; SHP, a p97/VCP-interacting motif; PIP, PCNA-interacting peptide; UBZ4, ubiquitin-binding zinc-finger 4. (B) (Upper panel) Schematic representation of DNA fiber assays. To visualize ongoing replication, mouse embryonic fibroblasts (MEFs) treated with MeOH or 4-OHT for 48 h were sequentially labeled with IdU and CldU with or without UV irradiation (40 J m⁻²) between the labeling. (Lower panel) DNA fiber assays were performed in Sprtn^F/F; Cre-ER^T2 MEFs expressing full-length human Spartan (FL) or the SprT domain only (amino acids 1–219). Distribution of replication forks at different CldU/IdU ratios is shown. At least 100 fibers were scored for each sample. Horizontal red lines indicate median values. (C) Cell-cycle profiling of Sprtn^F/F; Cre-ER^T2 MEFs expressing full-length human Spartan (FL) or the SprT domain only (amino acids 1–219). Cells were treated with MeOH or 4-OHT for 48 h, stained with PI and analyzed by flow cytometry. (D) Isolation of proteins on nascent DNA (iPOND) assays showing the localization of Spartan at replication forks. 293T cells were pulsed with EdU for 15 min and then chased with thymidine for 10 and 30 min. Eluted proteins were separated by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted for the indicated proteins. PCNA, POLD1 and POLD3 are shown as controls for replisome proteins that are enriched only at replication forks. Histone H2A is shown as a chromatin protein that is not enriched at replication forks. (E) iPOND assays showing that the isolated SprT domain of Spartan can localized at the fork. iPOND was performed in 293T cells expressing full-length human Spartan (FL) or the SprT domain only after pulse labeling with EdU for 20 min. Proteins at EdU-labeled nascent DNA were isolated and assessed using Western blotting. PCNA is shown as a control for replisome protein.