Fig. 5: Abnormal retention of PCNA on chromatin drives replication fork degradation by altering nucleosome deposition. | Nature Communications

Fig. 5: Abnormal retention of PCNA on chromatin drives replication fork degradation by altering nucleosome deposition.

From: Ubiquitinated-PCNA protects replication forks from DNA2-mediated degradation by regulating Okazaki fragment maturation and chromatin assembly

Fig. 5

a PCNA immunofluorescence showing increased PCNA retention on chromatin in 293T-K164R cells, or upon LIG1 depletion. At least 65 cells were quantified for each condition. The mean values are marked on the graph, and asterisks indicate statistical significance (t-test, two-tailed, unequal variance). Representative micrographs are also shown. b ATAD5 knockdown results in DNA2-mediated nascent strand degradation upon HU-induced replication fork arrest, which is epistatic to the K164R mutation. 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). Confirmation of ATAD5 knockdown is shown in Supplementary Fig. 7b. c Microccocal nuclease sensitivity assay showing altered nucleosome deposition in 293T-K164R cells, as well as upon depletion of LIG1, ATAD5, or CHAF1A. A quantification of the signal intensity is also shown. Confirmation of CHAF1A knockdown is shown in Supplementary Fig. 7c. d CHAF1A depletion results in HU-induced degradation of nascent DNA by DNA2 nuclease. 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). e Schematic representation of the proposed model depicting the UbiPCNA–LIG1–ATAD5–CAF-1 genetic pathway. Under unperturbed growth conditions, PCNA ubiquitination is required for efficient lagging strand synthesis by mediating gap-filling behind progressing replication forks. In the absence of PCNA ubiquitination, persistent gaps are formed between the lesion and the previous OF. These gaps interfere with OF maturation and subsequent PCNA unloading by ATAD5. By sequestering the CAF-1 chromatin assembly complex, PCNA retention on the lagging strand alters the efficiency of chromatin establishment which results in replication forks encountering a sparse chromatin organization. Upon fork arrest and reversal, the abnormal structure generated is a substrate for uncontrolled resection by DNA2. Source data are provided as a Source Data file.

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