The Rad50 zinc-hook is a structure joining Mre11 complexes in DNA recombination and repair

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.

The Rad50 hook domain regulates DNA damage signaling and tumorigenesis

“…Mutations that target the invariant cysteines of the Rad50 hook domain (Hopfner et al 2002;Wiltzius et al 2005;Hohl et al 2011;He et al 2012) globally disrupt Mre11 complex functions and thus phenocopy the null Rad50 mutation. Hence, the specific contribution of the Rad50 hook to the repair and signaling functions of the Mre11 complex has not been examined in isolation from the other domains of the complex.…”

“…Having previously shown that alteration of the cysteine residues of the Rad50 hook domain was tantamount to complete elimination of the protein (Hopfner et al 2002), we reasoned that alteration of adjacent residues might result in a partial loss of function that would provide insight regarding the functional importance of the Rad50 hook. Using S. cerevisiae, we identified two mutants, designated rad50-46 and rad50-47, that behaved as hook domain hypomorphs and have in common the presence of charged residues in the vicinity of the cysteines that coordinate zinc (Fig.…”

“…A large coiled-coil domain of Rad50 extends out from the globular domain and folds back on itself in an anti-parallel manner, with the Rad50 hook domain at the apex. The hook domain is a highly conserved interface that mediates homotypic dimerization via the coordination of a Zn 2+ atom by two invariant cysteines from each of two Rad50 protomers (Hopfner et al 2002).…”

ATM and the Mre11 complex combine to recognize and signal DNA double-strand breaks

“…This is arranged as a globular domain with Rad50 Walker A and B motifs (ATPase domains) and the bridging of DNA molecules is achieved through CXXC sequences in the middle of Rad50 (Figure 1). These sequences are displayed at the ends of coiled-coil regions and appear to dimerize by the coordination of a Zn 2 þ ion (Hopfner et al, 2002). Upon binding to DNA, the dynamic architecture of the MRN complex is altered to give rise to parallel orientation of the coiled-coils of Rad50, preventing intracomplex interaction and favouring intercomplex association (Moreno-Herrero et al, 2005).…”

Non-homologous end-joining, a sticky affair

“…An important clue for the MRN function in DSB repair may come from the peculiar structure of this protein complex. The RAD50 component of this complex has two very long, flexible coiled-coil arms that contain a Zinc hook at the tip (Hopfner et al, 2002). In the presence of Zn 2 þ , this structure forms a bridge between two RAD50 arms, suggesting that the MRN complex may be involved in keeping two DNA ends in close proximity.…”