bims-replis 2025-06-01 papers

Elife. 2025 May 29. pii: e107379. [Epub ahead of print]14

Godefroid Charbon, Anders Løbner-Olesen.

A protein called PriC allows DNA replication to proceed in Escherichia coli when the complex that usually initiates this process is compromised.

Keywords: DNA replication; E. coli; helicase loading; infectious disease; microbiology; primosome; replication initiation; replication origin; replication stress

DOI: https://doi.org/10.7554/eLife.107379

Cell Rep. 2025 May 22. pii: S2211-1247(25)00510-8. [Epub ahead of print]44(6): 115739

USP37 protects mammalian cells during DNA replication stress by counteracting CUL2LRR1 and TRAIP.

Fabrizio Villa, Johanna Ainsworth, Karim P M Labib.

The USP37 deubiquitylase is essential for mammalian cells to survive DNA replication stress, but the underlying mechanisms are unknown. Here, we demonstrate that USP37 binds the CDC45-MCM-GINS (CMG) helicase, which forms the stable core of the replisome until DNA replication termination when CMG is ubiquitylated and disassembled. USP37 contacts CDC45, and structure-guided mutations that displace USP37 from CMG cause sensitivity to DNA synthesis defects or topological stress. Binding to CDC45 at replication forks enables USP37 to counteract CMG ubiquitylation by the CUL2LRR1 ligase, which subsequently induces replisome disassembly during termination. Correspondingly, depletion of CUL2LRR1 suppresses the sensitivity of Usp37 mutants to DNA synthesis defects and ATR checkpoint kinase inhibitors. In contrast, mutation of the TRAIP ubiquitin ligase specifically suppresses the sensitivity of Usp37 mutants to topological stress. We propose that USP37 protects mammalian cells from replication stress by reversing the untimely action of the CUL2LRR1 and TRAIP ubiquitin ligases.

Keywords: ATR checkpoint kinase; CMG helicase; CP: Molecular biology; CUL2(LRR1); DNA replication; TRAIP; USP37; deubiquitylase; ubiquitylation

DOI: https://doi.org/10.1016/j.celrep.2025.115739

Biology (Basel). 2025 Apr 26. pii: 478. [Epub ahead of print]14(5):

New Insights into the Geometry and Topology of DNA Replication Intermediates.

Victor Martínez, Edith Ruiz-Díaz, Delia Cardozo, Cristian Cappo, Christian E Schaerer, Jorge Cebrián, Dora B Krimer, María José Fernández-Nestosa.

The regulation of superhelical stress, mediated by the combined action of topoisomerases and fork rotation, is crucial for DNA replication. The conformational changes during DNA replication are still experimentally challenging, mainly due to the rapid kinetics of the replication process. Here, we present the first molecular dynamics simulations of partially replicated circular DNA molecules, with stalled replication forks at both early and late stages of DNA replication. These simulations allowed us to map the distribution of superhelical stress after deproteinization. We propose a five-component model that determines the linking number difference of replication intermediates. At a thermodynamic equilibrium, the contribution of these five components was correlated to the progress of the replication forks. Additionally, we identified four types of segment collision events in replication intermediates, characterized by their geometric properties, including chirality and topological sign. The distribution of these collision events between the early and late stages of DNA replication provides new insights into the coordinated function of topoisomerases, warranting further discussion.

Keywords: DNA replication; DNA topology; chirality; collision events; computational biology; superhelical stress

DOI: https://doi.org/10.3390/biology14050478

Proc Natl Acad Sci U S A. 2025 Jun 03. 122(22): e2507232122

The proofreading mechanism of the human leading-strand DNA polymerase ε holoenzyme.

Feng Wang, Qing He, Michael E O'Donnell, Huilin Li.

The eukaryotic leading-strand DNA polymerase ε (Polε) is a dual-function enzyme with a proofreading 3'-5' exonuclease (exo) site located 40 Å from the DNA synthesizing pol site. Errors in Polε proofreading can cause various mutations, including C-to-G transversions, the most prevalent mutation in cancers and genetic diseases. Polε interacts with all three subunits of the PCNA ring to assemble a functional holoenzyme. Despite previous studies on proofreading of several Pol's, how Polε-or any Pol complexed with its sliding clamp-proofreads a mismatch generated in situ has been unknown. We show here by cryo-EM that a template/primer DNA substrate with a preexisting mismatch cannot enter the exo site of Polε-PCNA holoenzyme, but a mismatch generated in situ in the pol site yields three bona fide proofreading intermediates of Polε-PCNA holoenzyme. These intermediates reveal how the mismatch is dislodged from the pol site, how the DNA unwinds six base pairs, and how the unpaired primer 3'-end is inserted into the exo site for cleavage. These results unexpectedly demonstrate that PCNA imposes strong steric constraints that extend unwinding and direct the trajectory of mismatched DNA and that this trajectory is dramatically different than for Polε in the absence of PCNA. These findings suggest a physiologically relevant proofreading mechanism for the human Polε holoenzyme.

Keywords: DNA polymerase; DNA polymerase epsilon; DNA proofreading; leading strand; replication fidelity

DOI: https://doi.org/10.1073/pnas.2507232122

Elife. 2025 May 29. pii: RP103340. [Epub ahead of print]13

Primosomal protein PriC rescues replication initiation stress by bypassing the DnaA-DnaB interaction step for DnaB helicase loading at oriC.

Ryusei Yoshida, Kazuma Korogi, Qinfei Wu, Shogo Ozaki, Tsutomu Katayama.

In Escherichia coli, replisome and replication fork assembly is initiated by DnaB helicase loading at the chromosomal origin oriC via its interactions with the DnaA initiator and the DnaC helicase loader. Upon replication fork arrest, the replisome including DnaB dissociates from the stalled fork. Replication fork progression is rescued by primosomal protein PriA- or PriC-dependent pathway in which PriA and PriC promote reloading of DnaB in different mechanisms. However, the mechanism responsible for rescue of blocked replication initiation at oriC remains unclear. Here, we found that PriC rescued blocked replication initiation in cells expressing an initiation-specific DnaC mutant, in mutant cells defective in DnaA-DnaB interactions, and in cells containing truncated oriC sequence variants. PriC rescued DnaB loading at oriC even in the absence of Rep helicase, a stimulator of the PriC-dependent replication fork restart pathway. These results of in vitro reconstituted assays concordantly suggest that this initiation-specific rescue mechanism provides a bypass of the DnaA-DnaB interaction for DnaB loading by PriC-promoted loading of DnaB to the unwound oriC region. These findings expand understanding of mechanisms sustaining the robustness of replication initiation and specific roles for PriC in the genome maintenance.

Keywords: DNA replication; E. coli; helicase loading; infectious disease; microbiology; primosome; replication initiation; replication origin; replication stress

DOI: https://doi.org/10.7554/eLife.103340

Exp Hematol Oncol. 2025 May 26. 14(1): 79

Targeting DNA helicase CMG complex and NFκB2-driven drug-resistant transcriptional axis to effectively treat KRASG12D-mutated pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating malignancy with a 5-year overall survival (OS) rate of approximately 12%. More than 90% of PDAC patients harbor oncogenic mutations in the Kirsten rat sarcoma viral homolog (KRAS) gene. MRTX1133 (MRTX), a novel inhibitor of KRASG12D (the most common KRAS mutation found in pancreatic and colon cancers) has shown promise as a therapeutic agent. To address reported resistance to MRTX, we adapted our anti-leukemia co-targeting strategy and evaluated a combination of MRTX and Bedaquiline (BED), an FDA-approved inhibitor of mitochondrial ATP production, in in vitro human PDAC models. The combination of MRTX and BED demonstrated enhanced cytotoxic effects by disrupting all 11 genes within the DNA helicase family (CMG complex: CDC45-MCM-GINS), which are essential for initiating DNA replication and regulating cell cycle progression. Notably, real-world data analysis from Caris Life Sciences and NCI-TCGA database revealed that low transcriptomic expression of the DNA helicase CMG complex was significantly associated with prolonged survival (e.g., low CDC45 expression and low GINS2 expression with greater than 8 months longer overall survival) in PDAC patients with KRASG12 mutations (N = 9,717; P < 0.00001). However, this combination therapy also triggered strong pro-survival nuclear reprogramming. This effect was mediated by significant genetic activation of an NFκB2-DDIT (DNA damage-induced transcript) axis, which supported tumor chromosomal integrity and DNA repair mechanisms. To overcome NFκB2-driven resistance mechanisms, we explored a triple-targeting strategy that addresses metabolic and genomic plasticity in addition to actively intercepting cell division. This approach combines MRTX1133, Bedaquiline, and the NFκB2 inhibitor SN52, offering a novel therapeutic avenue to treat aggressive pancreatic cancer and potentially improve patient outcomes.

Keywords: ATP; Bedaquiline; DDIT; Helicase; KRAS; MRTX1133; Mitochondrion; NFκB2; PDAC; S100P

DOI: https://doi.org/10.1186/s40164-025-00669-w