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Responses to replication stress in human cells
Author(s) -
Bielinsky AnjaKatrin,
Leung Wendy,
Shetty Mihir,
Starr Tim,
Moldovan GeorgeLucian
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.345.1
Subject(s) - proliferating cell nuclear antigen , mutant , dna damage , telomere , dna replication , biology , microbiology and biotechnology , immortalised cell line , dna repair , telomerase , mutation , postreplication repair , homologous recombination , cell culture , cell growth , dna , genetics , dna mismatch repair , gene
Genome integrity relies on faithful genome inheritance. DNA damage induced replication stress leads to the generation of single‐stranded (ss) DNA, which triggers the ubiquitination (Ub) of the replication clamp, proliferating cell nuclear antigen (PCNA) at lysine 164 (K164). This activates two DNA damage tolerance (DDT) pathways: 1) error‐prone translesion synthesis, or 2) error‐free template switching. We generated PCNAK164R knock‐in cell lines via CRISPR/Cas9‐mediated gene targeting in HCT116, a colorectal cancer cell line and hTERT‐RPE1, an immortalized nontransformed epithelial cell line. RPE1 PCNAK164R hemizygous and homozygous mutants showed an increase in fork stalling and a decrease in new origin firing under unperturbed conditions. Moreover, these cells also exhibited accelerated fork speed. Although homologous recombination was enhanced in the mutants, it was not further upregulated in the presence of genotoxic drugs. We are currently testing the replication dynamics of these RPE1 mutants in response to DNA damaging agents. We hypothesized that cancer cells are more dependent on PCNA K164 for survival than nontransformed cells. Indeed, we were unable to isolate homozygous cell lines in HCT116. Heterozygous PCNA K164R/+ mutants displayed a slow‐growth phenotype, increased DNA damage sensitivity and chromosome breaks following UV‐light exposure, indicating a reduced capacity to tolerate extrinsic replication stress. These mutants also exhibited massive telomere erosion that was not caused by a decrease in telomerase activity, suggesting that PCNA‐K164 is essential in HCT116 because it regulates telomere length. In contrast, in a HCT116 PCNA K164R/+ mutant xenograft mouse model tumor burden was increased compared to wildtype. Thus, the slow‐growth phenotype in vitro is not predictive of the growth behavior in vivo . RNA sequencing revealed that tumor tissues arising from PCNA K164R/+ mutant cells drastically downregulated several DNA repair pathways, providing a mechanistic explanation for the enhanced tumor burden. Together, our data argue that DDT pathways are important for preserving genome stability and protecting against tumor growth. Support or Funding Information Supported by NIH R01 GM074917 (AKB). This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .