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. 2023 Apr 10;222(5):e202106022. doi: 10.1083/jcb.202106022

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© 2023 Moore et al.

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Figure S5. — RFWD3 promotes the polyubiquitination of PCNA. (A–C, and E–G) U2OS cells were transfected with the indicated siRNAs, treated with 30 µM ML323 for 4 h, and then treated with 1 µg/ml 4NQO in the presence of 30 µM ML323 for another 4 h. Endogenous PCNA ubiquitination was detected in whole cell lysates using an antibody against K164-ubiquitinated PCNA. (D) U2OS cells transfected with siFF or siRFWD3-4 were treated with 2 mM HU for 2 h. Whole-cell lysates were immunoblotted for FANCD2 and RFWD3. (H) Model showing that RFWD3 promotes PCNA polyubiquitination to recruit ZRANB3 and remodel stalled replication forks. Fork reversal allows template switching and replication fork restart in BRCA2-proficient cells. In BRCA2-deficient cells, however, reversal can lead to fork degradation.