SummaryThe Mre11–Rad50–Nbs1 (MRN) complex tethers, processes and signals DNA double strand breaks, promoting genomic stability. To understand the functional architecture of MRN, we determined the crystal structures of the Schizosaccharomyces pombe Mre11 dimeric catalytic domain alone and in complex with a fragment of Nbs1. Two Nbs1 subunits stretch around the outside of Mre11’s nuclease domains, with one subunit additionally bridging and locking the Mre11 dimer via a highly conserved asymmetrical binding motif. Our results reveal that Mre11 forms a flexible dimer and suggest that Nbs1 is not only a checkpoint adaptor, but also functionally impacts on Mre11-Rad50. Clinical mutations in Mre11 are located along the Nbs1 interaction sites and weaken the Mre11–Nbs1 interaction. However, they differentially affect DNA repair and telomere maintenance in Saccharomyces cerevisiae, potentially providing insight into their different human disease pathologies.

The Saccharomyces cerevisiae Ku heterodimer comprising Yku70p and Yku80p is involved in telomere maintenance and DNA repair by the pathway of nonhomologous end joining. It is also a key regulator of transcriptional silencing of genes placed in close proximity to telomeres. Here, we describe the identification of separation-of-function mutants of Yku80p that exhibit defects in silencing but not DNA repair and show that these mutations map to an evolutionarily conserved domain within Yku80p. Furthermore, we reveal that Yku80p interacts with the silent information regulator protein Sir4p and that this interaction is mediated by the N-terminal 200 amino acid residues of Sir4p. Notably, this interaction also requires the region of Yku80p that contains the sites of the silencing defective mutations. Finally, we show that these mutations impair the Yku80p-Sir4p interaction and recruitment of Sir3p to telomeric regions in vivo. Taken together with other data, these findings indicate that the Yku80p-Sir4p interaction plays a vital role in the assembly of telomeric heterochromatin.

Pur-alpha (Purα) plays an important role in a variety of cellular processes including transcriptional regulation, cell proliferation and oncogenic transformation. To better understand the role of Purα in the developing and mature brain, we generated Purα-deficient mice, which we were able to raise to the age of six months. Purα(-/-) mice were born with no obvious pathological condition. We obtained convincing evidence that lack of Purα prolongs the postnatal proliferation of neuronal precursor cells both in the hippocampus and in the cerebellum, however, without affecting the overall number of postmitotic neurons. Independent of these findings, we observed alterations in the expression and distribution of the dendritic protein MAP2, the translation of which has been proposed previously to be Purα-dependent. At the age of 2 weeks, Purα(-/-) mice generated a continuous tremor which persisted throughout lifetime. Finally, adult Purα(-/-) mice displayed a megalencephaly and histopathological findings including axonal swellings and hyperphosphorylation of neurofilaments. Our studies underline the importance of Purα in the proliferation of neuronal precursor cells during postnatal brain development and suggest a role for Purα in the regulation of the expression and cellular distribution of dendritic and axonal proteins. Since recent studies implicate a link between Purα and the fragile X tremor/ataxia syndrome, our Purα(-/-) mouse model will provide new opportunities for understanding the mechanisms of neurodegeneration.

The Mre11-Rad50 nuclease-ATPase is an evolutionarily conserved multifunctional DNA double-strand break (DSB) repair factor. Mre11-Rad50's mechanism in the processing, tethering, and signaling of DSBs is unclear, in part because we lack a structural framework for its interaction with DNA in different functional states. We determined the crystal structure of Thermotoga maritima Rad50 NBD (nucleotide-binding domain) in complex with Mre11 HLH (helix-loop-helix domain), AMPPNP, and double-stranded DNA. DNA binds between both coiled-coil domains of the Rad50 dimer with main interactions to a strand-loop-helix motif on the NBD. Our analysis suggests that this motif on Rad50 does not directly recognize DNA ends and binds internal sites on DNA. Functional studies reveal that DNA binding to Rad50 is not critical for DNA double-strand break repair but is important for telomere maintenance. In summary, we provide a structural framework for DNA binding to Rad50 in the ATP-bound state.

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