ABSTRACT
The human type IIA topoisomerases (Top2) are essential enzymes that regulate DNA topology and chromosome organization. The Topo IIα isoform is a prime target for antineoplastic compounds used in cancer therapy that form ternary cleavage complexes with the DNA. Despite extensive studies, structural information on this large dimeric assembly is limited to the catalytic domains, hindering the exploration of allosteric mechanism governing the enzyme activities and the contribution of its non-conserved C-terminal domain (CTD). Herein we present cryo-EM structures of the entire human Topo IIα nucleoprotein complex in different conformations solved at subnanometer resolutions (3.6-7.4 Å). Our data unveils the molecular determinants that fine tune the allosteric connections between the ATPase domain and the DNA binding/cleavage domain. Strikingly, the reconstruction of the DNA-binding/cleavage domain uncovers a linker leading to the CTD, which plays a critical role in modulating the enzyme's activities and opens perspective for the analysis of post-translational modifications.
Subject(s)
DNA Topoisomerases, Type II/ultrastructure , Poly-ADP-Ribose Binding Proteins/ultrastructure , Allosteric Regulation , Animals , Catalytic Domain , Cell Line , Cryoelectron Microscopy , DNA/metabolism , DNA/ultrastructure , DNA Topoisomerases, Type II/genetics , DNA Topoisomerases, Type II/isolation & purification , DNA Topoisomerases, Type II/metabolism , Humans , Mesocricetus , Models, Molecular , Nucleoproteins , Poly-ADP-Ribose Binding Proteins/genetics , Poly-ADP-Ribose Binding Proteins/isolation & purification , Poly-ADP-Ribose Binding Proteins/metabolism , Protein Processing, Post-Translational , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Recombinant Proteins/ultrastructureABSTRACT
Liquid-liquid phase separation (LLPS) is a mechanism of intracellular organization that underlies the assembly of a variety of RNP granules. Fundamental biophysical principles governing LLPS during granule assembly have been revealed by simple in vitro systems, but these systems have limitations when studying the biology of complex, multicomponent RNP granules. Visualization of RNP granules in cells has validated key principles revealed by simple in vitro systems, but this approach presents difficulties for interrogating biophysical features of RNP granules and provides limited ability to manipulate protein, nucleic acid, or small molecule concentrations. Here, we introduce a system that builds upon recent insights into the mechanisms underlying RNP granule assembly and permits high-fidelity reconstitution of stress granules and the granular component of nucleoli in mammalian cellular lysate. This system fills the gap between simple in vitro systems and live cells and allows for a variety of studies of membraneless organelles, including the development of therapeutics that modify properties of specific condensates.
