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

Tanay Thakar, Wendy Leung, Claudia M. Nicolae, Kristen E. Clements, Binghui Shen, Anja-Katrin Bielinsky and George-Lucian Moldovan ()
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Tanay Thakar: The Pennsylvania State University College of Medicine
Wendy Leung: University of Minnesota
Claudia M. Nicolae: The Pennsylvania State University College of Medicine
Kristen E. Clements: The Pennsylvania State University College of Medicine
Binghui Shen: Beckman Research Institute of City of Hope
Anja-Katrin Bielinsky: University of Minnesota
George-Lucian Moldovan: The Pennsylvania State University College of Medicine

Nature Communications, 2020, vol. 11, issue 1, 1-14

Abstract: 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.

Date: 2020
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DOI: 10.1038/s41467-020-16096-w

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