Biology subjects

Spagnolo, L.

Publications and source records attributed to Spagnolo, L..

5 recordsLinked to original sources

bioRxiv2026

NANOG assembles into self-limiting aging micelles that drive a sol-gel transition and modulate DNA dynamics

Proteins and nucleic acids form non-Newtonian liquids with complex rheological properties that contribute to their function in vivo. Here we investigate the rheology of the transcription factor NANOG, a key protein to maintain embryonic stem cell pluripotency. We find that at high concentrations, NANOG forms macroscopic aging gels that are dependent on its intrinsically disordered domain. By combining molecular dynamics simulations, mass photometry and Cryo-EM, we also discover that - in contrast with unbounded condensates formed by other intrinsically disordered proteins - NANOG forms self-limiting micelles with exposed DNA-binding domains. We show that these micelles can stabilize DNA entanglements and in turn modulate DNA dynamics. Based on our findings, we conjecture that NANOG may contribute to regulate gene expression by creating local gel-like environments that restrict genome dynamics and that its aging may ingrain mechanical memory in gene regulatory networks.

biophysics↗

bioRxiv2025

Structural basis of DNA recombination catalysis and regulation by phiC31 integrase

Large serine integrases catalyse the integration and excision of bacteriophage (phage) DNA genomes into and from the genomes of their bacterial hosts by site-specific recombination1. These recombination reactions are notable for their unidirectionality: integrase recognises and efficiently recombines short (40-50-bp) sequences in the phage (attP) and bacterial (attB) genomes, but is inactive on the product sites attL and attR flanking the inserted prophage, ensuring stable integration. The reverse reaction (excision) only occurs in the presence of a second phage-encoded protein, the Recombination Directionality Factor (RDF). Their strict recombination directionality has made serine integrases versatile tools in emerging genome-editing technologies2-5; however, the structural basis of directionality remains. Here we report structures of {phi}C31 integrase, the most-studied and most widely used member of the serine integrase family, in complexes with its DNA recombination sites, with and without its RDF. These structures correspond to four key mechanism steps: integration of the phage DNA genome (attP x attB); excision of the integrated prophage DNA, mediated by integrase and RDF (attL x attR); inhibition of integrase-catalysed excision in the absence of RDF (attL dimer complex); and inhibition of integrase-catalysed integration in the presence of RDF (attB dimer complex). Our data provide a mechanistic understanding of how the serine integrase recombination system establishes and regulates phage lysogeny, and lay the foundation for future development of integrase-based genome-editing technologies.

biochemistry↗

bioRxiv2022

Structure of the Saccharolobus solfataricus type III-D CRISPR effector

CRISPR-Cas is a prokaryotic adaptive immune system, classified into six different types, each characterised by a signature protein. Type III systems, classified based on the presence of a Cas10 subunit, are rather diverse multi-subunit assemblies with a range of enzymatic activities and downstream ancillary effectors. The broad array of current biotechnological CRISPR applications is mainly based on proteins classified as Type II, however recent developments established the feasibility and efficacy of multi-protein Type III CRISPR-Cas effector complexes as RNA-targeting tools in eukaryotes. The crenarchaeon Saccharolobus solfataricus has two type III system subtypes (III-B and III-D). Here, we report the cryo-EM structure of the Csm Type III-D complex from S. solfataricus (SsoCsm), which uses CRISPR RNA to bind target RNA molecules, activating the Cas10 subunit for antiviral defence. The structure reveals the complex organisation, subunit/subunit connectivity and protein/guide RNA interactions of the SsoCsm complex, one of the largest CRISPR effectors known.

microbiology↗

bioRxiv2022

Cyclic nucleotide-induced superhelical structure activates a bacterial TIR immune effector

Cyclic nucleotide signalling is a key component of anti-viral defence in all domains of life, from bacteria to humans. Viral detection activates a nucleotide cyclase to generate a second messenger, resulting in activation of effector proteins. This is exemplified by the metazoan cGAS-STING innate immunity pathway 1, which originated in bacteria 2. These defence systems require a sensor domain such as STING or SAVED to bind the cyclic nucleotide, coupled with an effector domain that causes cell death when activated by destroying essential biomolecules 3. One example is the TIR (Toll/interleukin-1 receptor) domain, which degrades the essential cofactor NAD+ when activated in response to pathogen invasion in plants and bacteria 2,4,5 or during nerve cell programmed death 6. Here, we show that a bacterial anti-viral defence system generates a cyclic tri-adenylate (cA3) signal which binds to a TIR-SAVED effector, acting as the "glue" to allow assembly of an extended superhelical solenoid structure. Adjacent TIR subunits interact to organise and complete a composite active site, allowing NAD+ degradation. Our study illuminates a striking example of large-scale molecular assembly controlled by cyclic nucleotides and reveals key details of the mechanism of TIR enzyme activation.

biochemistry↗

bioRxiv2020

Differential functions of FANCI and FANCD2 ubiquitination stabilize ID2 complex on DNA

The Fanconi Anemia (FA) pathway is a dedicated pathway for the repair of DNA interstrand crosslinks, and which is additionally activated in response to other forms of replication stress. A key step in the activation of the FA pathway is the monoubiquitination of each of the two subunits (FANCI and FANCD2) of the ID2 complex on specific lysine residues. However, the molecular function of these modifications has been unknown for nearly two decades. Here we find that ubiquitination of FANCD2 acts to increase ID2s affinity for double stranded DNA via promoting/stabilizing a large-scale conformational change in the complex, resulting in a secondary "Arm" ID2 interphase encircling DNA. Ubiquitination of FANCI, on the other hand, largely protects the ubiquitin on FANCD2 from USP1/UAF deubiquitination, with key hydrophobic residues of FANCIs ubiquitin being important for this protection. In effect, both of these post-translational modifications function to stabilise a conformation in which the ID2 complex encircles DNA.

biochemistry↗