Figure 6. Sites of DNA synthesis are... | Rockefeller University Press

Figure 6.

$Figure 6. Sites of DNA synthesis are sites of low chromatin and Mcm4 density. (A) Lack of colocalization of PCNA and Mcm proteins in living cells. Mcm4-mEm/RFP-PCNA–expressing cells in very early S phase were subjected to FLIP to reduce the fluorescence of the soluble pool of molecules as in Fig. 4. After FLIP, colocalization analysis of the immobile Mcm4-mEm and RFP-PCNA fractions was performed, and the correlation coefficient is indicated in the Post-FLIP overlay. (B and C) Similar FLIP colocalization analysis for RFP-PCNA/H2B-mCherry (B)- and Mcm4-mEm/H2B-mCherry (C)-expressing cells. The yellow circles represent where the FLIP laser (488 nm) was directed. (D) Strip FRAP was performed in cells stably expressing Mcm4-mEm/RFP-PCNA (top), Mcm4-mEm/H2B-mCherry (middle), and H2B-eGFP/RFP-PCNA (bottom) in early S and G2 phase. In each experiment, the IF was measured along the length of the bleach strip (third column), and this was correlated (via Pearson’s correlation coefficient, fourth column) with the prebleach fluorescence along the bleach strip (first two columns). Mean results are shown together with the standard error of the mean (error bars with number of cells for each displayed) in the bar plot below. Bars, 10 µm.$

Sites of DNA synthesis are sites of low chromatin and Mcm4 density. (A) Lack of colocalization of PCNA and Mcm proteins in living cells. Mcm4-mEm/RFP-PCNA–expressing cells in very early S phase were subjected to FLIP to reduce the fluorescence of the soluble pool of molecules as in Fig. 4. After FLIP, colocalization analysis of the immobile Mcm4-mEm and RFP-PCNA fractions was performed, and the correlation coefficient is indicated in the Post-FLIP overlay. (B and C) Similar FLIP colocalization analysis for RFP-PCNA/H2B-mCherry (B)- and Mcm4-mEm/H2B-mCherry (C)-expressing cells. The yellow circles represent where the FLIP laser (488 nm) was directed. (D) Strip FRAP was performed in cells stably expressing Mcm4-mEm/RFP-PCNA (top), Mcm4-mEm/H2B-mCherry (middle), and H2B-eGFP/RFP-PCNA (bottom) in early S and G2 phase. In each experiment, the IF was measured along the length of the bleach strip (third column), and this was correlated (via Pearson’s correlation coefficient, fourth column) with the prebleach fluorescence along the bleach strip (first two columns). Mean results are shown together with the standard error of the mean (error bars with number of cells for each displayed) in the bar plot below. Bars, 10 µm.