Clastogenesis by nucleotide lesions requires the completion of two cell cycles.

de Wind, N.; Jansen, J. G.; Temviriyanukul, P.; De Groot, D. C.; Szuhai, K.; Van Hees-Stuivenberg, S.; Tsaalbi-Shtylik, A.; Jacobs, H.

doi:10.64898/2025.12.16.694654

bioRxiv · 10.64898/2025.12.16.694654

Clastogenesis by nucleotide lesions requires the completion of two cell cycles.

de Wind, N.·Jansen, J. G.·Temviriyanukul, P.·De Groot, D. C.·Szuhai, K.·Van Hees-Stuivenberg, S.·Tsaalbi-Shtylik, A.·Jacobs, H.

View original source ↗Export metadata ↓

Abstract

Damaged DNA nucleotides can trigger genome rearrangements through clastogenesis, a process driven by erroneous repair of double-strand breaks (DSBs) and associated with cancer development. While DSBs are known to arise from endonuclease activity at stalled replication forks, the clastogenic potential of such DSBs has remained uncertain. Here, we identify a previously unrecognized mechanism of clastogenesis using wild-type, nucleotide excision repair (NER)-deficient and translesion synthesis (TLS)-deficient cells, combined with advanced cytogenetic analyses. We demonstrate that, single-stranded DNA (ssDNA) tracts harboring unrepaired lesions rather than DSBs at collapsed replication forks can persist through mitosis. Only during the subsequent S phase, these tracts are converted into a new class of, highly clastogenic, DSBs. Consistent with a role of this mechanism in carcinogenesis, prostate cancers exhibiting extensive genomic rearrangements frequently harbor somatic defects in NER or in error-free homologous recombination-mediated DSB repair. These findings provide critical mechanistic insight and highlight potential implications for routine clastogenicity testing. Graphical abstractNucleotide lesions (light blue triangle) can trigger double-strand breaks (DSBs) through endonucleolytic cleavage at stalled or reversed replication forks. Traditionally, these DSBs were assumed to drive genome rearrangements, a process termed clastogenesis. Here we describe a distinct, delayed, mechanism of clastogenesis. Thus, unreplicated nucleotide lesions within single-stranded (ss) DNA regions persist through mitosis into the next cell cycle. During the subsequent S phase, these ssDNA tracts collapse into DSBs, presumably via replication runoff. These delayed DSBs then promote extensive genomic reshuffling. Supporting this model, prostate cancers with high levels of genomic rearrangements are frequently associated with somatic defects in nucleotide excision repair (NER)--a pathway that normally prevents lesion-induced clastogenesis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/694654v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@7c37dborg.highwire.dtl.DTLVardef@13721aeorg.highwire.dtl.DTLVardef@842e4forg.highwire.dtl.DTLVardef@1fc7c31_HPS_FORMAT_FIGEXP M_FIG C_FIG

Explore related subjects

molecular biology Preprint

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

de Wind, N., Jansen, J. G., Temviriyanukul, P., De Groot, D. C., Szuhai, K., Van Hees-Stuivenberg, S., Tsaalbi-Shtylik, A., Jacobs, H.. 2025-12-18. Clastogenesis by nucleotide lesions requires the completion of two cell cycles.. https://doi.org/10.64898/2025.12.16.694654

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

KEEP EXPLORING

Related preprints

bioRxiv2026

Hidden Biodiversity in Wildlife Trade Networks: DNA Barcoding Reveals Fish and Crocodilian Species in Commercialized Swim Bladders

International wildlife trade represents one of the major drivers of biodiversity exploitation worldwide. However, the true taxonomic diversity embedded within commercial wildlife products often remains unknown because processing removes diagnostic morphological characteristics, preventing reliable species identification. Consequently, biodiversity assessments based solely on product labels may substantially underestimate the diversity of species involved in trade networks. To investigate hidden biodiversity within wildlife trade products, we applied DNA barcoding based on the mitochondrial cytochrome c oxidase subunit I (COI) gene to 77 products commercialized as fish swim bladders and seized at Guarulhos International Airport, Brazil. Molecular analyses successfully identified all samples and revealed the presence of four species: Plagioscion auratus (n = 38), Cynoscion acoupa (n = 7), Melanosuchus niger (n = 17), and Caiman crocodilus (n = 15). Fish species accounted for 71.4% of all samples, whereas crocodilians represented 28.6%, demonstrating that products marketed under a single commercial category may conceal substantial taxonomic diversity. Notably, the occurrence of two Amazonian crocodilian species within a trade chain traditionally associated with fish products reveals a previously undocumented component of the international wildlife trade. Our findings demonstrate that DNA barcoding is an effective tool for uncovering hidden biodiversity within processed wildlife products and provide evidence that wildlife trade networks may involve a broader spectrum of species than suggested by commercial labels. These results highlight the importance of molecular surveillance for biodiversity monitoring, wildlife trade regulation, and conservation planning.

molecular biology↗

bioRxiv2026

Plasmid architecture determines the stability of inverted terminal repeats in adeno-associated virus vectors

Recombinant vectors derived from adeno-associated viruses (rAAVs) are a mainstay of human gene therapy. rAAVs are produced from plasmids containing transgene cassettes flanked by inverted terminal repeats (ITRs), which form structured DNA elements that stabilize the ends of the single-stranded viral genome and are the only viral sequences required in cis for genome packaging. For decades, it has been recognized that propagation of ITR-containing plasmids can result in deletions and other mutations, prompting the use of specialized bacterial strains, modified growth conditions, and truncated or altered ITRs. Despite these practices, ITR instability remains a persistent source of plasmid heterogeneity. To identify determinants of ITR stability, we evaluated ITR integrity in one of the original cloned AAV2 genome isolates, a reconstructed AAV2 plasmid, and a synthetic rAAV vector containing full-length native AAV2 ITRs. We established a quantitative bioinformatic workflow for analyzing ITR-containing plasmids and virus preparations from raw Oxford Nanopore sequencing data. These experiments showed that ITRs were highly stable during short-term culture, whereas prolonged culture revealed strong positional effects, with preferential loss or mutation of the ITR nearest the plasmid origin of replication. Consistent with this model, a survey of 7,041 sequence-verifiable AAV plasmids from the Addgene repository identified a widely disseminated 11-bp ITR deletion in 4,773 plasmids; among analyzable two-ITR plasmids, this deletion was located in the origin-proximal ITR in 95.3% of cases. Guided by these findings, we constructed a novel rAAV entry vector with stable full-length native AAV2 ITRs that enabled efficient packaging of a 4,750-bp all-in-one CRISPR-Cas9 cassette. Finally, we developed a cell-based strategy to compare the effects of ITR mutations on rAAV genome integration, providing preliminary evidence that ITR sequence variation can influence integration outcomes. Together, these findings show that ITR instability is a preventable, position-dependent property of plasmid architecture and identify ITR integrity as an important variable in rAAV vector design and quality control.

molecular biology↗

bioRxiv2026

Single-point mutation alters odorant receptor sensitivity associated with host plant specialization in Spodoptera moths

Host specialization in herbivorous insects is often associated with divergence in chemosensory abilities. Here, we investigated the possible contribution of odorant receptors (ORs) in host plant restriction in the lily moth Spodoptera picta, a species specialized on Amaryllidaceae. Manual annotation of S. picta ORs in its genome revealed a repertoire similar in size and composition to those of its polyphagous sister species, S. littoralis and S. litura, suggesting that specialization did not involve major gene loss or expansion in the lily moth. To assess functional divergence beyond gene number, we applied a large scaled structure-based virtual screening approach to the entire OR repertoires of these three Spodoptera species, generating ligand-binding profiles for 120,591 volatile compounds. Among 69 1:1:1 OR orthologs, 24 exhibited divergent predicted binding spectra. We pinpointed OR29 that we also found to be highly expressed in both male and female antennae of S. picta through a RNAseq approach. Functional assays demonstrated that S. picta OR29 acquired heightened sensitivity to limonene enantiomers, volatiles emitted by host Amaryllidaceae inflorescences. Site-directed mutagenesis revealed that a single amino acid substitution within the predicted binding region underlies this shift in sensitivity. These results show that host specialization in S. picta has not been accompanied by significant OR repertoire remodeling, but rather by subtle molecular changes that fine-tune receptor sensitivity to host-derived volatiles.

molecular biology↗