Fig. 6: Genetic interaction between PCNA ubiquitination and the BRCA pathway.

a DNA fiber combing assay showing that concomitant loss of BRCA2 and PCNA ubiquitination enhances nascent strand degradation upon HU-induced fork arrest. The ratio of CldU to IdU tract lengths is presented, with the median values marked on the graph and listed at the top. Asterisks indicate statistical significance (Mann–Whitney test, two-sided). A schematic representation of the fiber combing assay conditions is also presented. b Immunofluorescence experiment showing synergistic increase in 53BP1 foci formation in unsynchronized K164R cells upon BRCA2 depletion. At least 100 cells were quantified for each condition (pooled from two experiments). The mean values are marked on the graph, and asterisks indicate statistical significance (t-test, two-tailed, unequal variance). Representative micrographs are also shown. c BrdU-alkaline comet assay measuring ssDNA gaps under normal replication conditions in BRCA2-depleted 293T cells at 1 and 5 h after BrdU pulse. At least 100 cells were quantified for each condition. Center line indicates the median, bounds of box indicate the first and third quartile, and whiskers indicate the 10th and 90th percentile. Asterisks indicate statistical significance (t-test, two-tailed, unequal variance). A schematic representation of the assay conditions is also presented. d, e Clonogenic survival experiments showing that loss of PCNA ubiquitination does not result in olaparib sensitivity, but it drastically increases the olaparib sensitivity of BRCA1-depleted d and BRCA2-depleted e cells. The average of three experiments, with standard deviations indicated as error bars, is shown. Asterisks indicate statistical significance (t-test, two-tailed, unequal variance) comparing WT siBRCA1/2 to KR siBRCA1/2. Representative images of the clonogenic assays are presented in Supplementary Fig. 8c, d. f, g Clonogenic survival experiments showing that depletion of RAD18 f or ATAD5 g increases the olaparib sensitivity of BRCA2-knockout HeLa cells. The average of three experiments, with standard deviations indicated as error bars, is shown. Asterisks indicate statistical significance (t-test, two-tailed, unequal variance) comparing BRCA2^KO siControl to BRCA2^KO siRAD18/siATAD5. Source data are provided as a Source Data file.