BackgroundThe SMC5-6 protein complex is involved in the cellular response to DNA damage. It is composed of 6–8 polypeptides, of which Nse1, Nse3 and Nse4 form a tight sub-complex. MAGEG1, the mammalian ortholog of Nse3, is the founding member of the MAGE (melanoma-associated antigen) protein family and Nse4 is related to the EID (E1A-like inhibitor of differentiation) family of transcriptional repressors.Methodology/Principal FindingsUsing site-directed mutagenesis, protein-protein interaction analyses and molecular modelling, we have identified a conserved hydrophobic surface on the C-terminal domain of Nse3 that interacts with Nse4 and identified residues in its N-terminal domain that are essential for interaction with Nse1. We show that these interactions are conserved in the human orthologs. Furthermore, interaction of MAGEG1, the mammalian ortholog of Nse3, with NSE4b, one of the mammalian orthologs of Nse4, results in transcriptional co-activation of the nuclear receptor, steroidogenic factor 1 (SF1). In an examination of the evolutionary conservation of the Nse3-Nse4 interactions, we find that several MAGE proteins can interact with at least one of the NSE4/EID proteins.Conclusions/SignificanceWe have found that, despite the evolutionary diversification of the MAGE family, the characteristic hydrophobic surface shared by all MAGE proteins from yeast to humans mediates its binding to NSE4/EID proteins. Our work provides new insights into the interactions, evolution and functions of the enigmatic MAGE proteins.

The SMC protein complexes play important roles in chromosome dynamics. The function of the SMC5-6 complex remains unclear, though it is involved in resolution of different DNA structures by recombination. We have now identified and characterized the four non-SMC components of the human complex and in particular demonstrated that the MAGEG1 protein is part of this complex. MAGE proteins play important but as yet undefined roles in carcinogenesis, apoptosis, and brain development. We show that, with the exception of the SUMO ligase hMMS21/ hNSE2, depletion of any of the components results in degradation of all the other components. Depletion also confers sensitivity to methyl methanesulfonate. Several of the components are modified by sumoylation and ubiquitination.SMC (structural maintenance of chromosomes) proteins form the cores of three essential protein complexes involved in important aspects of chromosome dynamics (see reference 15 for a review). Cohesin, containing Smc1 and Smc3, is the molecular glue that holds sister chromatids together from the time that they are established during DNA replication until they separate in anaphase. Smc2 and Smc4 form the core of condensin, which plays a crucial role in compaction of chromosomes at the beginning of mitosis. The function of the SMC5-6 complex is less clear, but it has an important role in a variety of different DNA repair processes and in resolving recombination structures.The SMC proteins are conserved throughout eukaryotes, and all have similar structures. Globular domains at the N and C termini are separated by two long coiled-coil domains linked together by a hinge. Each SMC protein is doubled back at the hinge region such that the coiled-coil domains interact with each other in an antiparallel manner and the globular domains are brought together. In this way a Walker A motif in the N-terminal domain and a Walker B motif in the C-terminal domain are juxtaposed to generate a globular ATP binding and hydrolysis "head" domain. The two Smc proteins in each complex interact with each other via the hinge. In cohesin the head domains of Smc1 and Smc3 are also brought together with the non-Smc components to form a ring, which is thought to encircle the two sister chromatids. It is not known if the other Smc complexes also form such a ring structure.The rad18 gene of Schizosaccharomyces pombe was isolated as a mutant sensitive to both UV and ionizing radiation. It was subsequently found to encode an SMC protein (12) that was redesignated Smc6 as part of the SMC5-6 complex. Further genetic and biochemical studies revealed a role in the repair of double-strand breaks as well as in other repair processes involving repair by homologous recombination (12, 29). The precise function remains unclear. The SMC5-6 complex is essential for cell proliferation, but cells are still able to go through several rounds of division in the absence of Smc6 (9). This suggests that death might result from an accumulation of unrepaired damage. Recent work has shown an accumulation of SMC...

Breaking and sealing one strand of DNA is an inherent feature of chromosome metabolism to overcome torsional barriers. Failure to reseal broken DNA strands results in protein-linked DNA breaks, causing neurodegeneration in humans. This is typified by defects in tyrosyl DNA phosphodiesterase 1 (TDP1), which removes stalled topoisomerase 1 peptides from DNA termini. Here we show that TDP1 is a substrate for modification by the small ubiquitin-like modifier SUMO. We purify SUMOylated TDP1 from mammalian cells and identify the SUMOylation site as lysine 111. While SUMOylation exhibits no impact on TDP1 catalytic activity, it promotes its accumulation at sites of DNA damage. A TDP1 SUMOylation-deficient mutant displays a reduced rate of repair of chromosomal single-strand breaks arising from transcription-associated topoisomerase 1 activity or oxidative stress. These data identify a role for SUMO during single-strand break repair, and suggest a mechanism for protecting the nervous system from genotoxic stress.

DNA double-strand breaks (DSBs) can be repaired by either homologous recombination (HR) or nonhomologous end-joining (NHEJ). In vertebrates, the first step in NHEJ is recruitment of the DNA-dependent protein kinase (DNA-PK) to DNA termini. DNA-PK consists of a catalytic subunit (DNA-PKcs) that is recruited to DNA ends by the Ku70/Ku80 heterodimer. Although Ku has been identified in a wide variety of organisms, to date DNA-PKcs has only been identified experimentally in vertebrates. Here, we report the identification of DNA-PK in the nonvertebrate Dictyostelium. Dictyostelium Ku80 contains a conserved domain previously implicated in recruiting DNA-PKcs to DNA and consistent with this observation, we have identified DNA-PKcs in the Dictyostelium genome. Disruption of the gene encoding Dictyostelium DNA-PKcs results in sensitivity to DNA DSBs and defective H2AX phosphorylation in response to this form of DNA damage. However, these phenotypes are only apparent when DNA damage is administered in G(1) phase of the cell cycle. These data illustrate a cell cycle-dependent requirement for Dictyostelium DNA-PK in signaling and combating DNA DSBs and represent the first experimental verification of DNA-PKcs in a nonvertebrate organism.

SummaryGenomic damage can feature DNA-protein crosslinks whereby their acute accumulation is utilized to treat cancer and progressive accumulation causes neurodegeneration. This is typified by tyrosyl DNA phosphodiesterase 1 (TDP1), which repairs topoisomerase-mediated chromosomal breaks. Although TDP1 levels vary in multiple clinical settings, the mechanism underpinning this variation is unknown. We reveal that TDP1 is controlled by ubiquitylation and identify UCHL3 as the deubiquitylase that controls TDP1 proteostasis. Depletion of UCHL3 increases TDP1 ubiquitylation and turnover rate and sensitizes cells to TOP1 poisons. Overexpression of UCHL3, but not a catalytically inactive mutant, suppresses TDP1 ubiquitylation and turnover rate. TDP1 overexpression in the topoisomerase therapy-resistant rhabdomyosarcoma is driven by UCHL3 overexpression. In contrast, UCHL3 is downregulated in spinocerebellar ataxia with axonal neuropathy (SCAN1), causing elevated levels of TDP1 ubiquitylation and faster turnover rate. These data establish UCHL3 as a regulator of TDP1 proteostasis and, consequently, a fine-tuner of protein-linked DNA break repair.