Abstract
Upon genotoxic stress, PCNA ubiquitination allows for replication of damaged DNA by recruiting lesion-bypass DNA polymerases. However, PCNA is also ubiquitinated during normal S-phase progression. By employing 293T and RPE1 cells deficient in PCNA ubiquitination, generated through CRISPR/Cas9 gene editing, here, we show that this modification promotes cellular proliferation and suppression of genomic instability under normal growth conditions. Loss of PCNA-ubiquitination results in DNA2-dependent but MRE11-independent nucleolytic degradation of nascent DNA at stalled replication forks. This degradation is linked to defective gap-filling in the wake of the replication fork and incomplete Okazaki fragment maturation, which interferes with efficient PCNA unloading by ATAD5 and subsequent nucleosome deposition by CAF-1. Moreover, concomitant loss of PCNA-ubiquitination and the BRCA pathway results in increased nascent DNA degradation and PARP inhibitor sensitivity. In conclusion, we show that by ensuring efficient Okazaki fragment maturation, PCNA-ubiquitination protects fork integrity and promotes the resistance of BRCA-deficient cells to PARP-inhibitors.
Original language | English (US) |
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Article number | 2147 |
Journal | Nature communications |
Volume | 11 |
Issue number | 1 |
DOIs | |
State | Published - Dec 1 2020 |
Bibliographical note
Funding Information:We would like to thank Drs. Alessandro Vindigni, David Cortez, Alberto Ciccia, James Broach, and Mark Hedglin for materials and advice; and the Penn State College of Medicine Flow Cytometry and Imaging cores. This work was supported by: NIH R01GM134681 (to A.-K.B. and G.-L.M.); NIH R01ES026184 (to G.-L.M.); NIH R01GM074917 and NIH T32-CA009138 (to A.-K.B.); NIH R01CA073764 (to B.S.).
Publisher Copyright:
© 2020, The Author(s).