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, George Lucian Moldovan

Research output: Contribution to journalArticle

3 Scopus citations

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 languageEnglish (US)
Article number2147
JournalNature communications
Volume11
Issue number1
DOIs
StatePublished - Dec 1 2020

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural

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