Castelli, S. | Search bioRxiv

bioRxiv2026

Structural basis for continuous DNA-end protection during ligation of double-strand breaks in yeast Non-Homologous End-Joining

Non-homologous end joining (NHEJ) repairs DNA double-strand breaks by synapsing and ligating DNA ends. In vertebrates, DNA-PKcs promotes end alignment and processing, yet several eukaryotes - including budding yeast - perform NHEJ without DNA-PKcs, and the underlying mechanism remains unclear. Here we report cryo-electron microscopy structures of reconstituted Saccharomyces cerevisiae NHEJ synaptic complexes assembled on DNA ends bearing either 4-bp terminal microhomologies or blunt termini. On the microhomology substrate with a single 5-P, we capture a ligation-competent short-range complex in which a single Dnl4 catalytic core is fully ordered and engaged at the 5'-adenylated nick. When two 5-P are present, we resolve two predominant, coexisting DNA-aligned protective states in which both Dnl4 DBD-NTD modules occupy the break region with reciprocal geometries, supporting an alternating engagement model for sequential sealing of the two strands while maintaining continuous end association. In contrast, blunt-ended substrates yield a non-aligned protective configuration in which two Dnl4 catalytic modules constrain the DNA ends [~]30 [A] apart in a ligation-incompatible arrangement, providing a structural explanation for slow blunt-end joining in yeast. Together, these structures define architectural and mechanistic principles of DNA-PKcs-independent NHEJ.

molecular biology↗

bioRxiv2025

IL-9 signaling redirects CAR T cell fate toward CD8+ memory and CD4+ cycling states, enhancing anti-tumor efficacy

The success of chimeric antigen receptor T cell therapies targeting solid tumors is limited by the immunosuppressive tumor microenvironment. We demonstrate that endowing CAR T cells with ectopic interleukin-9 (IL-9) signaling by co-expressing an IL-9 receptor, rewires CAR T cell fate under antigen stress to enhance anti-tumor efficacy. In preclinical solid tumor models, IL-9-signaling CAR T cells exhibit increased expansion, persistence, and tumor infiltration, resulting in superior tumor control at significantly lower doses than conventional products. Trajectory and RNA velocity analyses of single-cell RNA sequencing data reveal that IL-9 signaling alters CAR T cell differentiation under antigen stress away from dysfunction, favoring a multipotent transition toward CD8+ cell memory and effector states, and promoting a CD4+ cell proliferative state. Interrogation of transcription factor pathways indicates that IL-9-mediated activation of STAT1 and STAT4 drives the superior phenotype of IL-9-signaling CAR T cells, providing a promising therapeutic strategy for targeting solid cancers.

immunology↗