RAD51 separation of function mutation disables replication fork maintenance but preserves DSB repair.

Homologous recombination (HR) protects replication forks (RFs) and repairs DNA double-strand breaks (DSBs). Within HR, BRCA2 regulates RAD51 via two interaction regions: the BRC repeats to form filaments on single-stranded DNA and exon 27 (Ex27) to stabilize the filament. Here, we identified a RAD51 S181P mutant that selectively disrupted the RAD51-Ex27 association while maintaining interaction with BRC repeat and proficiently forming filaments capable of DNA binding and strand invasion. Interestingly, RAD51 S181P was defective for RF protection/restart but proficient for DSB repair. Our data suggest that Ex27-mediated stabilization of RAD51 filaments is required for the protection of RFs, while it seems dispensable for the repair of DSBs.

Recognition and coacervation of G-quadruplexes by a multifunctional disordered region in RECQ4 helicase.

Biomolecular polyelectrolyte complexes can be formed between oppositely charged intrinsically disordered regions (IDRs) of proteins or between IDRs and nucleic acids. Highly charged IDRs are abundant in the nucleus, yet few have been functionally characterized. Here, we show that a positively charged IDR within the human ATP-dependent DNA helicase Q4 (RECQ4) forms coacervates with G-quadruplexes (G4s). We describe a three-step model of charge-driven coacervation by integrating equilibrium and kinetic binding data in a global numerical model. The oppositely charged IDR and G4 molecules form a complex in the solution that follows a rapid nucleation-growth mechanism leading to a dynamic equilibrium between dilute and condensed phases. We also discover a physical interaction with Replication Protein A (RPA) and demonstrate that the IDR can switch between the two extremes of the structural continuum of complexes. The structural, kinetic, and thermodynamic profile of its interactions revealed a dynamic disordered complex with nucleic acids and a static ordered complex with RPA protein. The two mutually exclusive binding modes suggest a regulatory role for the IDR in RECQ4 function by enabling molecular handoffs. Our study extends the functional repertoire of IDRs and demonstrates a role of polyelectrolyte complexes involved in G4 binding.

Assembly principle of a membrane-anchored nuclear pore basket scaffold.

Nuclear pore complexes (NPCs) are membrane-embedded gatekeepers of traffic between the nucleus and cytoplasm. Key features of the NPC symmetric core have been elucidated, but little is known about the NPC basket, a prominent structure with numerous roles in gene expression. Studying the basket was hampered by its instability and connection to the inner nuclear membrane (INM). Here, we reveal the assembly principle of the yeast NPC basket by reconstituting a recombinant Nup60-Mlp1-Nup2 scaffold on a synthetic membrane. Nup60 serves as the basket's flexible suspension cable, harboring an array of short linear motifs (SLiMs). These bind multivalently to the INM, the coiled-coil protein Mlp1, the FG-nucleoporin Nup2, and the NPC core. We suggest that SLiMs, embedded in disordered regions, allow the basket to adapt its structure in response to bulky cargo and changes in gene expression. Our study opens avenues for the higher-order reconstitution of basket-anchored NPC assemblies on membranes.

Nuclear pore basket proteins are tethered to the nuclear envelope and can regulate membrane curvature.

Nuclear pore complexes (NPCs) are selective transport channels embedded in the nuclear envelope. The cylindrical NPC core forms a protein coat lining a highly curved membrane opening and has a basket-like structure appended to the nucleoplasmic side. How NPCs interact with lipids, promoting membrane bending and NPC integrity, is poorly understood. Here we show that the NPC basket proteins Nup1 and Nup60 directly induce membrane curvature by amphipathic helix insertion into the lipid bilayer. In a cell-free system, both Nup1 and Nup60 transform spherical liposomes into highly curved membrane structures. In vivo, high levels of the Nup1/Nup60 amphipathic helices cause deformation of the yeast nuclear membrane, whereas adjacent helical regions contribute to anchoring the basket to the NPC core. Basket amphipathic helices are functionally linked to distinct transmembrane nucleoporins of the NPC core, suggesting a key contribution to the membrane remodeling events that underlie NPC assembly.

Nuclear actin and lamins in viral infections.

Lamins are the best characterized cytoskeletal components of the cell nucleus that help to maintain the nuclear shape and participate in diverse nuclear processes including replication or transcription. Nuclear actin is now widely accepted to be another cytoskeletal protein present in the nucleus that fulfills important functions in the gene expression. Some viruses replicating in the nucleus evolved the ability to interact with and probably utilize nuclear actin for their replication, e.g., for the assembly and transport of capsids or mRNA export. On the other hand, lamins play a role in the propagation of other viruses since nuclear lamina may represent a barrier for virions entering or escaping the nucleus. This review will summarize the current knowledge about the roles of nuclear actin and lamins in viral infections.