No abstract

The Mre11 complex (Mre11 Rad50 Nbs1) is central to chromosomal maintenance and functions in homologous recombination, telomere maintenance and sister chromatid association. These functions all imply that the linked binding of two DNA substrates occurs, although the molecular basis for this process remains unknown. Here we present a 2.2 A crystal structure of the Rad50 coiled-coil region that reveals an unexpected dimer interface at the apex of the coiled coils in which pairs of conserved Cys-X-X-Cys motifs form interlocking hooks that bind one Zn(2+) ion. Biochemical, X-ray and electron microscopy data indicate that these hooks can join oppositely protruding Rad50 coiled-coil domains to form a flexible bridge of up to 1,200 A. This suggests a function for the long insertion in the Rad50 ABC-ATPase domain. The Rad50 hook is functional, because mutations in this motif confer radiation sensitivity in yeast and disrupt binding at the distant Mre11 nuclease interface. These data support an architectural role for the Rad50 coiled coils in forming metal-mediated bridging complexes between two DNA-binding heads. The resulting assemblies have appropriate lengths and conformational properties to link sister chromatids in homologous recombination and DNA ends in non-homologous end-joining.

To clarify functions of the Mre11/Rad50 (MR) complex in DNA double-strand break repair, we report Pyrococcus furiosus Mre11 crystal structures, revealing a protein phosphatase-like, dimanganese binding domain capped by a unique domain controlling active site access. These structures unify Mre11's multiple nuclease activities in a single endo/exonuclease mechanism and reveal eukaryotic macromolecular interaction sites by mapping human and yeast Mre11 mutations. Furthermore, the structure of the P. furiosus Rad50 ABC-ATPase with its adjacent coiled-coil defines a compact Mre11/Rad50-ATPase complex and suggests that Rad50-ATP-driven conformational switching directly controls the Mre11 exonuclease. Electron microscopy, small angle X-ray scattering, and ultracentrifugation data of human and P. furiosus MR reveal a dual functional complex consisting of a (Mre11)2/(Rad50)2 heterotetrameric DNA processing head and a double coiled-coil linker.

Type IV pili (T4P) are long, thin, flexible filaments on bacteria that undergo assembly-disassembly from inner membrane pilin subunits and exhibit astonishing multifunctionality. Neisseria gonorrhoeae (gonococcal or GC) T4P are prototypic virulence factors and immune targets for increasingly antibiotic-resistant human pathogens, yet detailed structures are unavailable for any T4P. Here, we determined a detailed experimental GC-T4P structure by quantitative fitting of a 2.3 A full-length pilin crystal structure into a 12.5 A resolution native GC-T4P reconstruction solved by cryo-electron microscopy (cryo-EM) and iterative helical real space reconstruction. Spiraling three-helix bundles form the filament core, anchor the globular heads, and provide strength and flexibility. Protruding hypervariable loops and posttranslational modifications in the globular head shield conserved functional residues in pronounced grooves, creating a surprisingly corrugated pilus surface. These results clarify T4P multifunctionality and assembly-disassembly while suggesting unified assembly mechanisms for T4P, archaeal flagella, and type II secretion system filaments.

To clarify the key role of Rad50 in DNA double-strand break repair (DSBR), we biochemically and structurally characterized ATP-bound and ATP-free Rad50 catalytic domain (Rad50cd) from Pyrococcus furiosus. Rad50cd displays ATPase activity plus ATP-controlled dimerization and DNA binding activities. Rad50cd crystal structures identify probable protein and DNA interfaces and reveal an ABC-ATPase fold, linking Rad50 molecular mechanisms to ABC transporters, including P glycoprotein and cystic fibrosis transmembrane conductance regulator. Binding of ATP gamma-phosphates to conserved signature motifs in two opposing Rad50cd molecules promotes dimerization that likely couples ATP hydrolysis to dimer dissociation and DNA release. These results, validated by mutations, suggest unified molecular mechanisms for ATP-driven cooperativity and allosteric control of ABC-ATPases in DSBR, membrane transport, and chromosome condensation by SMC proteins.