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. 2016 Aug 22;6:30819. doi: 10.1038/srep30819

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Copyright © 2016, The Author(s)

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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(a) Mutlicolor SR image of a U2OS cell nucleus in early S-phase with nascent DNA, PCNA, and RPA stained in magenta, cyan, and yellow, respectively. (b) Two magnified images showing examples of the internal spatial organization of a replication focus. The experimental localization accuracies for the different colors used in these measurements are: 15 nm, 37 nm, and 44 nm for magenta (Alexa Fluor 647), yellow (Alexa Fluor 568), and cyan (Alexa Fluor 488), respectively (see Data Analysis in Methods section). (c) Triple-Pair-Correlation maps obtained by averaging the Triple-Correlation of 13 nuclei. Distances from DNA to RPA (r_D−R) and from DNA to PCNA (r_D−P) are ~149.7 ± 0.6 and 132.0 ± 0.7 nm, respectively; angles in between r_D−R and r_D−P follows a Gaussian distribution centering at 0 with standard deviation of ~29 deg. (Supplementary Figure S6). (d) Mutlicolor SR image of a U2OS cell nucleus treated with 1 mM HU in early S-phase. (e) Two magnified images showing examples of the internal spatial organization of a replication focus under replication stress. (f) Triple-Pair-Correlation maps obtained by averaging the Triple-Pair-Correlation of 18 nuclei. Distances from DNA to RPA (r_D−R) and from DNA to PCNA (r_D−P) are ~100 ± 1 and 109 ± 1 nm, respectively. Angles in between r_D−R and r_D−P follows a Gaussian distribution centering at 0 with standard deviation of ~20 deg. (Supplementary Figure S8, all errors are propagated fitting errors). (g) 1D correlation of r_D−R (top) and r_D−P (bottom) obtained via integration of r_D−R − r_D−P correlation map (c(iii),f(iii)) along r_D−P and r_D−R, respectively. Both graphs display a decrease of correlation distances when cells treated with 1 mM HU. Dots and lines represent the raw data and modified Gaussian fit, respectively (Supplementary Note 3). We note that the overlapped portion between nascent DNA and replisome proteins resulted in a high Triple-Correlation response at r_D−R (or r_D−P) of ~0 nm covering all , which further resulted in a high correlation at (or ) of ~0 nm after integration through in (c(iii),f(iii),g) (Supplementary Note 3 and Supplementary Figure S7). (h) Schematic illustration of the internal organization of a single replication fork obtained from the TPC analysis. Straight and Dashed arrows represent regular and stalled replication processing, respectively.

Inline graphic — (a) Mutlicolor SR image of a U2OS cell nucleus in early S-phase with nascent DNA, PCNA, and RPA stained in magenta, cyan, and yellow, respectively. (b) Two magnified images showing examples of the internal spatial organization of a replication focus. The experimental localization accuracies for the different colors used in these measurements are: 15 nm, 37 nm, and 44 nm for magenta (Alexa Fluor 647), yellow (Alexa Fluor 568), and cyan (Alexa Fluor 488), respectively (see Data Analysis in Methods section). (c) Triple-Pair-Correlation maps obtained by averaging the Triple-Correlation of 13 nuclei. Distances from DNA to RPA (r_D−R) and from DNA to PCNA (r_D−P) are ~149.7 ± 0.6 and 132.0 ± 0.7 nm, respectively; angles in between r_D−R and r_D−P follows a Gaussian distribution centering at 0 with standard deviation of ~29 deg. (Supplementary Figure S6). (d) Mutlicolor SR image of a U2OS cell nucleus treated with 1 mM HU in early S-phase. (e) Two magnified images showing examples of the internal spatial organization of a replication focus under replication stress. (f) Triple-Pair-Correlation maps obtained by averaging the Triple-Pair-Correlation of 18 nuclei. Distances from DNA to RPA (r_D−R) and from DNA to PCNA (r_D−P) are ~100 ± 1 and 109 ± 1 nm, respectively. Angles in between r_D−R and r_D−P follows a Gaussian distribution centering at 0 with standard deviation of ~20 deg. (Supplementary Figure S8, all errors are propagated fitting errors). (g) 1D correlation of r_D−R (top) and r_D−P (bottom) obtained via integration of r_D−R − r_D−P correlation map (c(iii),f(iii)) along r_D−P and r_D−R, respectively. Both graphs display a decrease of correlation distances when cells treated with 1 mM HU. Dots and lines represent the raw data and modified Gaussian fit, respectively (Supplementary Note 3). We note that the overlapped portion between nascent DNA and replisome proteins resulted in a high Triple-Correlation response at r_D−R (or r_D−P) of ~0 nm covering all , which further resulted in a high correlation at (or ) of ~0 nm after integration through in (c(iii),f(iii),g) (Supplementary Note 3 and Supplementary Figure S7). (h) Schematic illustration of the internal organization of a single replication fork obtained from the TPC analysis. Straight and Dashed arrows represent regular and stalled replication processing, respectively.