Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2025.05.13.652823

An ATM-PPM1D Circuit Controls the Processing and Restart of DNA Replication Forks

Cao, Y.·Wang, Y.·Badar, J.·Palek, M.·Jaber, K.·Faca, V. M.·Huang, T.·Celik, E.·Macurek, L.·Smolka, M. B.
View original source ↗Export metadata ↓

Abstract

In response to DNA replication stress, DNA damage signaling kinases inhibit origin firing and promote the remodeling and stabilization of replication forks, leading to a systemic reduction in DNA synthesis, which collectively protects genomic integrity. Little is understood about the regulatory mechanisms of replication stress recovery, including the mechanisms involved in the restart of stalled replication forks. Here, we identify the oncogenic phosphatase PPM1D/WIP1 as a regulator of replication fork restart. During recovery from replication stress, PPM1D prevents excessive MRE11- and DNA2-dependent nucleolytic degradation of stalled forks. Loss of PPM1D function leads to defects in RAD51 recruitment to chromatin and impairs RAD51-dependent fork restart. Phosphoproteomic analysis reveals that PPM1D regulates a network of ATM substrates, several of which are phosphorylated at an S/T-Q-(E/D)n motif. Strikingly, inhibition of ATM suppresses the deleterious consequences of impaired PPM1D function at replication forks, enabling timely fork restart. The dominant effect of ATM hyper-signaling in suppressing fork restart occurs, in part, through the excessive engagement of 53BP1 and consequent RAD51 antagonization. These findings uncover a new mode of ATM signaling responding to fork stalling and highlight the need for PPM1D to restrain ATM signaling and facilitate proper fork restart.

Explore related subjects

biochemistryPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Cao, Y., Wang, Y., Badar, J., Palek, M., Jaber, K., Faca, V. M., Huang, T., Celik, E., Macurek, L., Smolka, M. B.. 2025-05-15. An ATM-PPM1D Circuit Controls the Processing and Restart of DNA Replication Forks. https://doi.org/10.1101/2025.05.13.652823

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Biochemical reassessment of the activation of PANK1 by SGLT2 inhibitors

Although SGLT2 inhibitors (SGLT2i) show cardiovascular benefits in addition to glycemic control, their molecular target for cardiovascular effect remains unclear. A recent study reported that two SGLT2i, empagliflozin and dapagliflozin, directly activate pantothenate kinase 1 (PANK1), which mediate their cardioprotective effect. PANK1 phosphorylates pantothenate with ATP to yield 4'-phosphopantothenate and ADP, representing the rate-limiting enzyme of coenzyme A (CoA) biosynthesis. We therefore reassessed this claim using purified recombinant human PANK1. The malachite green assay detected free inorganic phosphate (Pi) but neither ADP nor 4'-phosphopantothenate and its signals do not reflect PANK1 activity. In the ADP-Glo assay, empagliflozin and dapagliflozin did not stimulate PANK1 under either basal conditions or acetyl-CoA feedback inhibition, while the activator PZ-2891 enhanced the activity of PANK1. These results show that SGLT2i do not directly activate PANK1, challenging the proposed PANK1-dependent cardioprotection mechanism.

biochemistry↗
bioRxiv2026

Intraspecific glucosinolate chemodiversity in Cardamine hirsuta

Within some plant species, a high intraspecific chemical variation, termed chemodiversity, can be found in the composition of specialized metabolites. For Brassicales, glucosinolates are highly characteristic. Their composition is genetically determined but variation has been found within species and between organs in different species. Cardamine hirsuta is increasingly being used as genetic model species, but until now, little was known about its genetically-based variation in glucosinolates. Thus, we aimed to characterize glucosinolate profiles, their putative genetic variation and their chemodiversity in established accessions and wild-collected material. We analyzed the glucosinolate profiles of leaves and roots from offspring, grown under standardized conditions, of 18 accessions collected in nine countries. Glucosinolates were quantified by HPLC-DAD and identities were confirmed by UHPLC-QToF-MS/MS. Glucosinolate composition was analyzed in biosynthetic terms, compared across samples with principal component analysis and chemodiversity was quantified as glucosinolate richness and Shannon diversity. In total, 14 glucosinolates were confirmed across samples. Two main sources of variation among accessions were the relative levels of but-3-enyl- versus pent-4-enylglucosinolate and of benzylglucosinolate versus phenethylglucosinolate. An almost universal presence of 1-methoxyindol-3-ylmethylglucosinolate in roots but not leaves was observed, along with quantitative contrasts between leaves and roots. The glucosinolate composition varied profoundly among accessions and between leaves and roots, while offspring within accessions were mostly consistent in their profiles. Even accessions collected in close proximity produced distinct profiles, with no clear separation along latitude or longitude. Our study highlights two independent and chemically simple traits of apparently genetic nature, namely the extent of chain elongation of methionine-derived and of phenylalanine-derived glucosinolates. Their independent occurrence is a main factor shaping the glucosinolate profiles among accessions. High variation was also observed at a local scale, highlighting complex genetic variation in glucosinolate biosynthesis in the species throughout Europe. Moreover, within individuals different organs showed clearly distinct profiles, indicating organ-specific biosynthesis. The genetic basis and ecological consequences of this variation need to be explored in future studies.

biochemistry↗
bioRxiv2026

Expanding Plasma Proteome Coverage While Preserving Biological Signatures: A Systematic Characterization of Enrichment and Preanalytical Variables

Plasma is a rich and accessible source for biomarkers. The analytical challenge of the steep dynamic range was recently addressed using enrichment technologies. Enrichment technologies have been shown to be sensitive to preanalytical variables, making this an active area of research. We established the P2 enrichment system as high-performing and robust for large scale studies, with reproducible identifications and precision across batches, days, operators and laboratories. We characterized P2-based enrichment for preanalytical variables (eight blood collection types, centrifugation, freeze-thaw and pathogen inactivation). K2EDTA-plasma resulted in the deepest proteome coverage with about 6000 protein groups on the timsTOF HT. We spiked 16 heavy-labelled proteins into platelet-poor and platelet-contaminated plasma. Importantly, linearity, accuracy, and precision were high in both platelet-poor and platelet-contaminated plasma for both P2-enriched and neat preparations. In a cancer cohort, we demonstrated that 97% of differentially abundant proteins retained the same direction of regulation after P2-based enrichment compared with the neat preparation. Differentially abundant proteins identified in a colorectal cancer pilot cohort remained largely conserved after platelet contamination, including previously reported biomarkers. We demonstrated that P2-based enrichment enables deep, large scale proteome profiling and preserves biological information despite preanalytical variation. Candidates emerging from P2-based discovery can be validated in independent cohorts with targeted assays.

biochemistry↗