Small cause, large effect

The Mre11 complex is a multisubunit nuclease that contains three components — Mre11, Rad50 and Nbs1 (known as Xrs2 in yeast). It acts as a sensor of double-stranded DNA breaks, but also has roles in checkpoint signalling and DNA replication. Research into the individual components has been hampered by the fact that null mutants are not viable, but John Petrini and colleagues now present their studies of a functionally impaired Rad50 mutant. And the results underline the importance of the Mre11 complex in the homeostasis of proliferative tissues.

Reporting in Genes and Development, Petrini and co-workers describe the generation of a group of hypomorphic Rad50 mutants, one of which — Rad50^K22M — was used to derive so-called Rad50^S/S-mutant mice. Rad50^S/S mouse-embryo fibroblasts contained wild-type levels of Mre11, Nbs1 and Rad50^K22M, and these components could assemble functional complexes. However, the Rad50^S/S mice showed partial embryonic lethality, and those that survived were only 60% of the weight of their wild-type littermates. By 4–8 weeks of age, the mice showed signs of anaemia, and most of them died by 4 months. Of those that survived up to 7 months, 20% died from a variety of tumours.

As the premature death was associated with severe anaemia, the authors first looked more closely at haematopoietic cells in the Rad50^S/S mice. At 2 weeks there was no difference between wild-type and Rad50^S/S mice, but by 4 weeks of age, lymphocytes, macrophages, red blood cells and platelets were severely depleted in the mutant mice. Further experiments indicated that the progressive depletion was due to the failure of haematopoietic stem cells. Moreover, a similar depletion of cells was observed in the spermatogenic lineages of Rad50^S/S mice.

Given the age-dependent cellular depletion in the bone marrow and testes, and the fact that those mice that survived longest developed tumours, Petrini and co-workers wondered whether the Rad50^K22M mutation might cause genotoxic stress. If this were the case, mutation of p53 — which is involved in the response to genotoxic stress — could be expected to lessen the severity of the observed defects. And this is just what the authors saw. For example, macrophages were increased threefold and T cells 3–20-fold in p53^−/− Rad50^S/S mice compared with Rad50^S/S mice. Moreover, p53^−/− Rad50^S/S mice developed tumours and died much earlier than p53^−/− mice alone, again supporting the idea that the Rad50^K22M mutation causes genotoxic stress.

A final confirmation came when the authors analysed various indices of genotoxic stress. Levels of phosphorylated histone H2AX — which correlate with levels of DNA damage — were the same in unirradiated Rad50^S/S cells as in irradiated wild-type cells. And karyotype analyses of Rad50^S/S thymic lymphoma cells revealed increased chromosome breaks and rearrangements (including telomeric short-arm fusions) compared with wild-type cells. So, as the authors conclude, “the data clearly indicate that the Mre11 complex exerts a profound influence on homeostasis in mammalian tissues, even when its checkpoint and DNA recombination functions are not overtly impaired”.