Accumulation of DNA damage leading to adult stem cell exhaustion has been proposed to be a principal mechanism of ageing. Here we address this question by taking advantage of the highly specific role of DNA ligase IV in the repair of DNA double-strand breaks by non-homologous end-joining, and by the discovery of a unique mouse strain with a hypomorphic Lig4(Y288C) mutation. The Lig4(Y288C) mouse, identified by means of a mutagenesis screening programme, is a mouse model for human LIG4 syndrome, showing immunodeficiency and growth retardation. Diminished DNA double-strand break repair in the Lig4(Y288C) strain causes a progressive loss of haematopoietic stem cells and bone marrow cellularity during ageing, and severely impairs stem cell function in tissue culture and transplantation. The sensitivity of haematopoietic stem cells to non-homologous end-joining deficiency is therefore a key determinant of their ability to maintain themselves against physiological stress over time and to withstand culture and transplantation.

The signalosome is implicated in regulating cullin-dependent ubiquitin ligases. We find that two signalosome subunits, Csn1 and Csn2, are required to regulate ribonucleotide reductase (RNR) through the degradation of a small protein, Spd1, that acts to anchor the small RNR subunit in the nucleus. Spd1 destruction correlates with the nuclear export of the small RNR subunit, which, in turn, correlates with a requirement for RNR in replication and repair. Spd1 degradation is promoted by two separate CSN-dependent mechanisms. During unperturbed S phase, Spd1 degradation is independent of checkpoint proteins. In irradiated G2 cells, Spd1 degradation requires the DNA damage checkpoint. The signalosome copurifies with Pcu4 (cullin 4). Pcu4, Csn1, and Csn2 promote the degradation of Spd1, identifying a new function for the signalosome as a regulator of Pcu4-containing E3 ubiquitin ligase. The COP9 signalosome (CSN) complex was originally identified as a negative regulator of photomorphogenesis in plants (for review, see Schwechheimer and Deng 2001). Subsequently, it was purified from human cell extracts during attempts to isolate the 19S regulatory lid complex of the proteosome (Seeger et al. 1998). The human signalosome consists of eight core subunits, each sharing significant homology with a corresponding subunit in the regulatory 19S lid complex of the proteosome . The purified CSN complex can cleave the ubiquitin-like Nedd8 protein from cullins Wee et al. 2002). Csn5 contains a putative metalloprotease motif that is presumed to mediate deneddylation activity (Cope et al. 2002). Cullins are subunits of E3 ubiquitin ligases (Feldman et al. 1997;Skowyra et al. 1997), and deneddylation of cullins decreases SCF E3 ubiquitin ligase activity (Osaka et al. 2000). SCF E3 complexes typically consist of a cullin, the Rbx1 RING domain protein that binds an E2 enzyme Skowyra et al. 1999), and an adapter protein, Skp1, that binds an F-box protein that determines the substrate specificity (Skowyra et al. 1997).In Arabidopsis, the signalosome is involved in the degradation of the two bZIP transcription factors (Hy5, HyH) that lie at the top of a transcriptional cascade required to induce ∼ 30% of Arabidopsis genes during photomorphogenesis (Holm et al. 2002). An E2-like protein, Cop10, and an E3 RING protein, Cop1, are also required to degrade Hy5 and HyH, which occurs when seedlings are germinated in the dark (Osterlund et al. 2000;Holm et al. 2002;Suzuki et al. 2002). The biochemical role of the signalosome is unknown, although a correlation with Cop1 nuclear localization (von Arnim et al. 1997) and the associations between the signalosome and E3 ubiquitin ligases Schwechheimer et al. 2002) suggest a regulatory role in ubiquitination that may be linked to subcellular localization (Chamovitz et al. 1996;Hellmann and Estelle 2002).A highly conserved signalosome complex was identified in the fission yeast Schizosaccharomyces pombe (Mundt et al. 1999) and subsequently shown to be required to remove the Nedd8 ubiquitin-like protein fr...

Vps4p (End13p) is an AAA-family ATPase that functions in membrane transport through endosomes, sorting of soluble vacuolar proteins to the vacuole, and multivesicular body (MVB) sorting of membrane proteins to the vacuole lumen. In a yeast two-hybrid screen with Vps4p as bait we isolated VPS20 (YMR077c) and the novel open reading frame YLR181c, for which the name VTA1 has recently been assigned (Saccharomyces Genome Database). Vps4p directly binds Vps20p and Vta1p in vitro and binding is not dependent on ATP - conversely, Vps4p binding to Vps20p is partially sensitive to ATP hydrolysis. Both ATP binding [Vps4p-(K179A)] and ATP hydrolysis [Vps4p-(E233Q)] mutant proteins exhibit enhanced binding to Vps20p and Vta1p in vitro. The Vps4p-Vps20p interaction involves the coiled-coil domain of each protein, whereas the Vps4p-Vta1p interaction involves the (non-coiled-coil) C-terminus of each protein. Deletion of either VPS20 (vps20Δ) or VTA1 (vta1Δ) leads to similar class E Vps- phenotypes resembling those of vps4Δ, including carboxypeptidase Y (CPY) secretion, a block in ubiquitin-dependent MVB sorting, and a delay in both post-internalisation endocytic transport and biosynthetic transport to the vacuole. The vacuole resident membrane protein Sna3p (whose MVB sorting is ubiquitin-independent) does not appear to exit the class E compartment or reach the vacuole in cells lacking Vps20p, Vta1p or Vps4p, in contrast to other proteins whose delivery to the vacuole is only delayed. We propose that Vps20p and Vta1p regulate Vps4p function in vivo.

The COP9/signalosome complex is highly conserved in evolution and possesses significant structural similarity to the 19S regulatory lid complex of the proteasome. It also shares limited similarity to the translation initiation factor eIF3. The signalosome interacts with multiple cullins in mammalian cells. In the fission yeast Schizosaccharomyces pombe, the Csn1 subunit is required for the removal of covalently attached Nedd8 from Pcu1, one of three S. pombe cullins. It remains unclear whether this activity is required for all the functions ascribed to the signalosome. We previously identified Csn1 and Csn2 as signalosome subunits in S. pombe. csn1 and csn2 null mutants are DNA damage sensitive and exhibit slow DNA replication. Two further putative subunits, Csn4 and Csn5, were identified from the S. pombe genome database. Herein, we characterize null mutations of csn4 and csn5 and demonstrate that both genes are required for removal of Nedd8 from the S. pombe cullin Pcu1 and that their protein products associate with Csn1 and Csn2. However, neither csn4 nor csn5 null mutants share the csn1 and csn2 mutant phenotypes. Our data suggest that the subunits of the signalosome cannot be considered as a distinct functional unit and imply that different subunits of the signalosome mediate distinct functions.