bims-replis Biomed News
on Replisome
Issue of 2025–06–29
six papers selected by
Anna Zawada, International Centre for Translational Eye Research
  1. The DNA replication checkpoint limits Okazaki fragment accumulation to protect and restart stalled forks.
  2. The DNA replication checkpoint prevents PCNA/RFC depletion to protect forks from HLTF-induced collapse in human cells.
  3. Proteasome-dependent Orc6 removal from chromatin upon S-phase entry safeguards against MCM reloading and tetraploidy.
  4. The DNA-PKcs/JNK/p53 pathway underlies changes in cell fate decision toward death during DNA replication catastrophe.
  5. Nascent RNA at the crossroad of transcription and replication.
  6. Topoisomerase I Inhibition in ETV4-overexpressed Non-Small Cell Lung Cancer Promotes Replication and Transcription Mediated R-Loop Accumulation and DNA Damage.
  1. Mol Cell. 2025 Jun 24. pii: S1097-2765(25)00501-5. [Epub ahead of print]
    The DNA replication checkpoint limits Okazaki fragment accumulation to protect and restart stalled forks.
    Berta Canal, Agostina P Bertolin, Giselle C Lee, Lucy S Drury, Masashi Minamino, John F X Diffley.
      Understanding how DNA replication forks stall and restart and how the DNA replication checkpoint prevents irreversible fork collapse in molecular detail are crucial for understanding how cells maintain stable genomes and how they prevent the genetic instability that drives cancer. Here, we describe the reconstitution of fork stalling and restart with purified budding yeast proteins. After nucleotide depletion, leading-strand DNA synthesis quickly stops but CMG helicase continues to unwind, and Okazaki fragments continue to initiate on the lagging strand. Incomplete Okazaki fragments sequester PCNA, RFC, and DNA polymerases δ and ε, which prevents normal DNA synthesis restart and exposes nascent DNA to nuclease attack. The DNA replication checkpoint restrains fork progression, which limits this sequestration, protecting stalled forks from collapse and ensuring restart.
    Keywords:  DNA polymerase α; DNA polymerase δ; DNA polymerase ε; DNA replication checkpoint; DNA replication fork stabilization; Okazaki fragments; PCNA; RFC; RPA exhaustion; replication fork collapse
    DOI:  https://doi.org/10.1016/j.molcel.2025.06.001
  2. Mol Cell. 2025 Jun 25. pii: S1097-2765(25)00502-7. [Epub ahead of print]
    The DNA replication checkpoint prevents PCNA/RFC depletion to protect forks from HLTF-induced collapse in human cells.
    Agostina P Bertolin, Berta Canal, Mona Yekezare, Anne Early, Jingkun Zeng, Rachael Instrell, Michael Howell, John F X Diffley.
      The DNA replication checkpoint is crucial for maintaining genome stability after genotoxic stress; without it, stalled DNA replication forks cannot restart normally, excess DNA replication origins are activated, DNA damage and single-stranded DNA (ssDNA) accumulate, S phase does not finish, and cells die. Preventing excess origin firing suppresses all these effects. Here, we show in human cells that when replication is not restrained by a functional checkpoint, excess DNA synthesis sequesters the processivity factor PCNA and its loader, replication factor C (RFC), preventing normal fork restart. Nascent DNA ends unprotected by RFC/PCNA are attacked by the helicase-like transcription factor (HLTF), causing irreversible replication fork collapse and hyperaccumulation of ssDNA. This explains how the checkpoint stabilizes stalled replication forks and has implications for how origin firing is normally coordinated with fork progression. Loss of HLTF suppresses fork collapse and cell lethality in checkpoint-deficient cells, which has implications for how resistance to anti-checkpoint therapies may arise.
    Keywords:  DNA damage; DNA replication checkpoint; DNA replication fork collapse; DNA replication fork stabilization; HLTF; Okazaki fragments; PCNA; RFC; RPA exhaustion; polymerase α
    DOI:  https://doi.org/10.1016/j.molcel.2025.06.002
  3. J Cell Sci. 2025 Jun 18. pii: jcs.263596. [Epub ahead of print]
    Proteasome-dependent Orc6 removal from chromatin upon S-phase entry safeguards against MCM reloading and tetraploidy.
    Yoko Hayashi-Takanaka, Ichiro Hiratani, Tokuko Haraguchi, Yasushi Hiraoka.
      DNA replication is tightly regulated to occur only once per cell cycle, as untimely re-initiation can lead to aneuploidy, which is associated with early senescence and cancer. The pre-replication complex (comprising Orc1-6, Cdc6, Cdt1, and MCM) is essential for the initiation of DNA replication, but the dynamics and function of Orc6 during the cell cycle remain elusive. Here, we demonstrate that Orc6 associates with chromatin during G1-phase and dissociates upon S-phase entry. The dissociation of Orc6 from chromatin is dependent on proteasome activity, and inhibition of the proteasome leads to the accumulation of chromatin-bound Orc6, which promotes abnormal MCM loading after S-phase entry without undergoing mitosis in human immortalized hTERT-RPE1 cells. Following release from proteasome inhibition, cells with elevated levels of chromatin-bound Orc6 and MCM proceed to the next replication phase as tetraploid cells. Our findings suggest that the proteasome-dependent dissociation of Orc6 after DNA replication is critical for preventing inappropriate MCM reloading and tetraploid formation.
    Keywords:  DNA replication; MCM; Orc6; Proteasome; Single-cell plot analysis; Tetraploid
    DOI:  https://doi.org/10.1242/jcs.263596
  4. Nucleic Acids Res. 2025 Jun 20. pii: gkaf573. [Epub ahead of print]53(12):
    The DNA-PKcs/JNK/p53 pathway underlies changes in cell fate decision toward death during DNA replication catastrophe.
    Jinal A Patel, Julie Rageul, Natalie Lo, Auntara Nandi, Camryn Zezelic, Cynthia T Lee, Arafat Khan, Hyungjin Kim.
      Exacerbating the DNA replication problems 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 Ataxia telangiectasia and Rad3-related (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 γH2AX, 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.
    DOI:  https://doi.org/10.1093/nar/gkaf573
  5. Trends Genet. 2025 Jun 25. pii: S0168-9525(25)00130-1. [Epub ahead of print]
    Nascent RNA at the crossroad of transcription and replication.
    Ming-Yu Zhu, Yi-Zheng Zhang, Ting Guo, Jie Ren.
      The concurrent processes of DNA replication and RNA transcription pose a significant challenge to genome integrity. Nascent RNA, the newly synthesized transcript, is emerging as a critical determinant of transcription-replication conflict (TRC) outcomes, exerting influence through its modifications, its ability to form RNA:DNA hybrids (R-loops), its regulation of chromatin structure, and its interaction with protein complexes at the transcription-replication interface. Here, we synthesize recent advances on how nascent RNA modulates transcription dynamics, replication fork progression, and genome stability. We explore its paradoxical roles - both inducing replication stress and orchestrating protective responses - highlighting how RNA processing factors mitigate TRCs. Finally, we emphasize the need for innovative technologies to dissect the dynamic and context-dependent roles of nascent RNA and therapeutic potential for genomic instability-linked diseases.
    Keywords:  R-loops; RNA modification; genome stability; nascent RNA; transcription stress; transcription–replication conflict
    DOI:  https://doi.org/10.1016/j.tig.2025.05.010
  6. Adv Sci (Weinh). 2025 Jun 23. e09307
    Topoisomerase I Inhibition in ETV4-overexpressed Non-Small Cell Lung Cancer Promotes Replication and Transcription Mediated R-Loop Accumulation and DNA Damage.
    Jiaxi Zhang, Yan Wang, Shanhu Cao, Shelly M Xie, Bei Liu, Yimeng Li, Yuqi Hou, Xue Meng, Mingzhu Ruan, Di Bu, Jia Kang, Ruxin Li, Lei Lou, Juan Wang, Lingxiao Xing.
      Coordinating transcription and replication via transcription factors (TFs) is a conserved mechanism in higher eukaryotes. The role of TFs in regulating these processes in cancers remains unclear. Here, it is shown that oncogenetic ETS transcription factor ETV4 controls DNA replication through both transcriptional and non-transcriptional mechanisms in non-small cell lung cancer (NSCLC). ETV4 localizes to specific DNA replication origins and interacts with the origin recognition complex subunits ORC1 and ORC6 during the G1/S phase, facilitating origin formation. Using quantitative in situ analysis of protein interactions at DNA replication forks (SIRF) assays, it is shown that ETV4 transiently localizes to replication forks in the S phase. It interacts with replicative helicase MCM2 N-terminal, histone H3, and histone-chaperone FACT and is involved in histone processing during replication. Additionally, ETV4 transcriptionally regulates key replisome genes MCM2, MCM4, MCM5, MCM10, and ORC1, influencing their expression and recruitment to chromatin. Due to its binding at the origin-promoter locus like the MCM4 gene, ETV4 overexpression increases R-loop formation, DNA damage, and cell death under external replication stress induced by topoisomerase I (TOP1) inhibitor. These findings highlight the dual role of ETV4 in replication and transcription and suggest that targeting TOP1 could be a synthetic-lethal approach in ETV4-overexpressed lung cancer.
    Keywords:  DNA damage; DNA replication; ETV4; NSCLC; R‐loop; TOP1 inhibitor
    DOI:  https://doi.org/10.1002/advs.202409307
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