Interdependence of the Rad50 Hook and Globular Domain Functions

Abstract

SUMMARY Rad50 contains a conserved Zn2+ coordination domain (the Rad50 hook) that functions as a homodimerization interface. Hook ablation phenocopies Rad50 deficiency in all respects. Here we focused on rad50 mutations flanking the Zn2+-coordinating hook cysteines. These mutants impaired hook-mediated dimerization, but recombination between sister chromatids was largely unaffected. This may reflect that cohesin-mediated sister chromatid interactions are sufficient for double strand break repair. However, Mre11 complex functions specified by the globular domain, including Tel1 (ATM) activation, nonhomologous end-joining, and DNA double strand break end resection were affected, suggesting that dimerization exerts a broad influence on Mre11 complex function. These phenotypes were suppressed by mutations within the coiled coil and globular ATPase domain, suggesting a model in which conformational changes in the hook and globular domains are transmitted via the extended coils of Rad50. We propose that transmission of spatial information in this manner underlies the regulation of Mre11 complex functions.

Functional Analysis of the Bacteriophage T4 Rad50 Homolog (gp46) Coiled-coil Domain

“…Previous in vivo experiments using the yeast model system indicate that the presence of the zinc-hook and a full-length coiled-coil are critical to the proper functioning of the MR complex (22,40,46). Here, we show that this is also true in the T4 system, as mutation of the zinc-hook cysteine residues is lethal, indicating that the ability to form a complex capable of DNA tethering is necessary for phage survival.…”

“…The effects of altering the zinc-hook and coiled-coil in vivo have most often been interpreted under the assumption that the enzymatic properties of the MR complex are unaffected, although recent work has suggested communication between the coiled-coil/zinc-hook and the globular domain (30,40). The data presented here directly demonstrate this functional crosstalk.…”

“…These results suggest that the combined binding of Mre11 and DNA alter the position or reduce the flexibility of the coiledcoil, and these changes are transmitted allosterically to the ATP active site. As suggested by Petrini and co-workers (40), the coupling of the coiled-coil to the ATPase active site likely enables tethering-dependent regulation of ATP-dependent nuclease activity.…”

“…The function of the Rad50 coiled-coil has been the subject of intense research (22,24,25,40,43). Previous in vivo experiments using the yeast model system indicate that the presence of the zinc-hook and a full-length coiled-coil are critical to the proper functioning of the MR complex (22,40,46).…”

“…The Rad50 2CS mutation was created to eliminate Zn 2ϩ binding by the zinc-hook and corresponds to the SPTS construct used in the phage infections described above. The two cysteine residues that are mutated to serine in the Rad50 2CS protein are absolutely conserved in all Rad50 proteins and were previously known to affect the in vivo properties of the MR complex in other systems (22,39,40).…”