Fig. 4: Defective Okazaki fragment maturation results in nascent strand degradation.

a, b DNA fiber combing assays showing that depletion of LIG1 a or FEN1 b results in faster replication fork progression under normal conditions, and induce nascent strand degradation upon fork arrest. The quantification of the IdU tract length is presented, with the median values marked on the graph and listed at the top. Asterisks indicate statistical significance (Mann–Whitney test, two-sided). Schematic representations of the assay conditions are also presented. Western blots confirming the knockdowns are shown in Supplementary Fig. 6a. c Chromatin fractionation experiment showing increased PAR chain formation in KR cells under normal growth conditions, indicative of defective Okazaki fragment maturation. Cells were treated as indicated with a PARG inhibitor (PARGi) for 45 min prior to harvesting to block PAR chain removal. LIG1 depletion was used as positive control for defective Okazaki fragment maturation. Chromatin-associated LaminB1 was used as loading control. d Loss of LIG1 is epistatic with the PCNA-K164R mutation for fork progression and HU-induced nascent strand degradation in 293T cells. The quantification of the IdU tract length 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 assay conditions is also presented. Similar results for LIG1 and FEN1 depletion in RPE1 cells are presented in Supplementary Fig. 6c, d. e BrdU-alkaline comet assay showing accumulation of ssDNA gaps under normal replication conditions in 293T-K164R and RAD18-knockout 293T and U2OS cells. 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, and representative micrographs, are also presented. Western blots confirming RAD18 knockout, and presenting the levels of PCNA ubiquitination in these cells, are shown in Supplementary Fig. 6e, f. Source data are provided as a Source Data file.