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. 2015 Sep 9;43(20):9835–9855. doi: 10.1093/nar/gkv880

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© The Author(s) 2015. 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 4. — RAD51 paralogs suppress nascent DNA degradation at stalled forks. (A) Experimental design of fork protection assay. Length of nascent replication tracts (IdU labeled) were measured by DNA spreading after 5 h of replication stalling with 4 mM HU. Representative DNA fiber image is shown. Measurement of IdU labeled DNA fiber in RAD51 paralog-defective and parental cells. (B) Experimental protocol for the parental ssDNA assay. Parental DNA of indicated cells were labeled by the addition of 10 μM BrdU for 20 h followed by a 2 h chase in the presence of 4 mM HU and later recovered in fresh media for 2 h. Cells were fixed and stained with BrdU antibodies without DNA denaturation to selectively detect parental ssDNA. Representative image for BrdU intensity is shown (BrdU-green and DNA-blue). Graph represents the mean BrdU intensity from indicated cells. P-values are obtained for paralog deficient cells in comparison with wt V79 cells. (C) Representative images of HU-induced RPA foci in the indicated cells after 4 h of recovery. Scale bar 10 μm. Quantitative analysis of the RPA-foci/nucleus in the indicated cells at indicated recovery time points following 4 mM HU treatment for 2 h (RPA-green and DNA-red). Bar graph represents average of three independent experiments (±SD). (CL-V4B- RAD51C^−/−, irs1- XRCC2^−/−, irs1SF- XRCC3^−/−, and V79B, V79 and CHO-AA8 are parental cell lines for CL-V4B, irs1 and irs1SF respectively).