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

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

Nature communications (2020-05-03)
Tanay Thakar, Wendy Leung, Claudia M Nicolae, Kristen E Clements, Binghui Shen, Anja-Katrin Bielinsky, George-Lucian Moldovan
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Poly-L-lysine solution, 0.1 % (w/v) in H2O
Sigma-Aldrich
Anti-DNA Antibody, single stranded, clone 16-19, clone 16-19, Chemicon®, from mouse
Sigma-Aldrich
PDD00017273, ≥98% (HPLC)