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The DNA-PKcs/JNK/p53 pathway underlies changes in cell fate decision toward death during DNA replication catastrophe

ABSTRACT: Exacerbating the DNA replication problem of cancer cells serves as a viable therapeutic approach. Nevertheless, the cytotoxicity of cancer drugs is often hampered by therapy-induced senescence, leading to unfavorable patient outcomes. Here, we employ acute replisome dysfunction in combination with ATR inhibition as a strategy to divert senescent cells toward death by triggering DNA replication catastrophe, a form of irreversible replication fork collapse caused by excessive single-stranded DNA (ssDNA) accumulation. RNA-sequencing revealed a distinct set of p53-responsive genes responsible for death. We identify c-Jun N-terminal kinase (JNK) to be essential for augmenting p53-dependent apoptotic programs and inducing pan-nuclear distribution of H2AX, together constituting a feed-forward loop to drive cell death. Activation of DNA-PKcs initiates the signaling cascade of replication catastrophe, including CHK1-dependent JNK activation, which relies on MRE11 and PARP1 to expand and recognize ssDNA gaps, defining replication-associated gaps as an underlying basis for replication catastrophe. Our study elucidates the dynamic regulation of proximal and distal effectors along the DNA-PKcs/JNK/p53 axis that govern the cell fate decision between senescence and death. We propose that key determinants of replication catastrophe signaling are targetable vulnerabilities that can be exploited to limit senescent cell populations and increase the efficacy of anti-cancer therapies.

ORGANISM(S): Homo sapiens

PROVIDER: GSE285638 | GEO | 2025/07/02

REPOSITORIES: GEO

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The DNA-PKcs/JNK/p53 pathway underlies changes in cell fate decision toward death during DNA replication catastrophe

Project description:The DNA-PKcs/JNK/p53 pathway underlies changes in cell fate decision toward death during DNA replication catastrophe

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PARG inhibitor sensitivity correlates with accumulation of single strand DNA gaps in preclinical models of ovarian cancer

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Project description:In response to DNA replication stress, DNA replication checkpoint is activated to maintain fork stability, a process critical for maintenance of genome stability. However, how DNA replication checkpoint regulates replication forks remain elusive. Here we show that Rad53, a highly conserved replication checkpoint kinase, functions to couple leading and lagging strand DNA synthesis. In wild type cells under HU induced replication stress, synthesis of lagging strand, which contains ssDNA gaps, is comparable to leading strand DNA. In contrast, synthesis of lagging strand is much more than leading strand, and consequently, leading template ssDNA coated with ssDNA binding protein RPA was detected in rad53-1 mutant cells, suggesting that synthesis of leading strand and lagging strand DNA is uncoupled. Mechanistically, we show that replicative helicase MCM and leading strand DNA polymerase Pole move beyond actual DNA synthesis and that an increase in dNTP pools largely suppresses the uncoupled leading and lagging strand DNA synthesis. Our studies reveal an unexpected mechanism whereby Rad53 regulates replication fork stability.

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The E3 ubiquitin ligase RFWD3 is required for translesion DNA synthesis (total proteome)

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2020-12-15 | PXD021445 | Pride

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FAM111A regulates replication origin activation and cell fitness

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2023-10-03 | PXD044180 | Pride

Rtt105 promotes DNA replication by functioning as an RPA chaperone

Project description:Single-stranded DNA (ssDNA) binding protein Replication Protein A (RPA) is essential for protecting ssDNA at replication forks. However, how RPA is loaded to replication forks remains unexplored. Here, we show that Regulator of Ty1 transposition protein 105 (Rtt105) binds RPA and is required for the association of RPA with replication forks. Cells lacking Rtt105 exhibit dramatic genome instability and severe defects in DNA replication. Intriguingly, both the RPA nuclear import and its ability to bind DNA replication forks were greatly compromised in rtt105 mutant cells, however, targeting RPA to the nucleus cannot rescue the defect of RPA binding to replication forks. Importantly, Rtt105 promotes the binding of RPA to ssDNA but does not associate with the final RPA-ssDNA complex in vitro. Moreover, single-molecule studies revealed that Rtt105 affects the binding mode of RPA to ssDNA. These results support a model in which Rtt105 functions as an RPA chaperone that escorts RPA to nucleus and assemble RPA onto ssDNA at replication forks.

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