Structure chromosome (SMC) proteins organize the core of cohesin, condensin and Smc5-Smc6 complexes. The Smc5-Smc6 complex is required for DNA repair, as well as having another essential but enigmatic function. Here, we generated conditional mutants of SMC5 and SMC6 in budding yeast, in which the essential function was affected. We show that mutant smc5-6 and smc6-9 cells undergo an aberrant mitosis in which chromosome segregation of repetitive regions is impaired; this leads to DNA damage and RAD9-dependent activation of the Rad53 protein kinase. Consistent with a requirement for the segregation of repetitive regions, Smc5 and Smc6 proteins are enriched at ribosomal DNA (rDNA) and at some telomeres. We show that, following Smc5-Smc6 inactivation, metaphase-arrested cells show increased levels of X-shaped DNA (Holliday junctions) at the rDNA locus. Furthermore, deletion of RAD52 partially suppresses the temperature sensitivity of smc5-6 and smc6-9 mutants. We also present evidence showing that the rDNA segregation defects of smc5/smc6 mutants are mechanistically different from those previously observed for condensin mutants. These results point towards a role for the Smc5-Smc6 complex in preventing the formation of sister chromatid junctions, thereby ensuring the correct partitioning of chromosomes during anaphase.

The NIFL regulatory protein controls transcriptional activation of nitrogen fixation (nif) genes in Azotobacter vinelandii The high energetic requirements for nitrogen fixation and the extreme oxygen sensitivity of the nitrogenase enzyme impose physiological constraints on diazotrophy, which necessitate stringent control of nitrogen fixation (nif) gene expression at the transcriptional level (1). In both Azotobacter vinelandii and Klebsiella pneumoniae, the NIFL protein regulates nif gene transcription in response to environmental oxygen and fixed nitrogen (2, 3). This control by NIFL is achieved through modulation of the activity of the transcriptional activator NIFA, an enhancer binding protein that catalyzes the formation of open promoter complexes by the alternative holoenzyme form of RNA polymerase containing the sigma factor eN (Eo4N) (4). Stimulation of open promoter complex formation by NIFA requires nucleoside triphosphate hydrolysis catalyzed by the central domain of this activator (5).Sequence analysis of NIFL indicates that this protein is composed of two domains separated by a glutamine-rich flexible linker. The amino-terminal domain shows homology to the bat gene product from Halobacterium halobium, which potentially has an oxygen-sensing function and also to the rhizobial FixL family of heme-based oxygen sensors, although the significance of these homologies is at present unknown (2). The carboxyl-terminal domain of NIFL shares characteristic features with the histidine protein kinase family of twocomponent regulatory proteins, and in the case of the A. vinelandii protein possesses all five of the conserved regions found in other transmitter domains. However, although A.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.vinelandii NIFL contains a conserved histidine residue known to be the site of autophosphorylation in other members of this family, a number of substitutions of this residue do not impair function, implying that sensory transduction by NIFL does not involve phosphorylation of this residue (6). Moreover, neither autophosphorylation of NIFL nor phosphotransfer to NIFA has so far been detected in vitro (7,8). Inhibition of NIFA activity by NIFL apparently requires stoichiometric amounts of the two proteins, implying direct protein-protein interaction rather than catalytic modification of NIFA activity. Since the nucleoside triphosphatase activity of A. vinelandii NIFA decreases when the inhibitory complex between NIFL and NIFA is formed, NIFL may block NIFA activity by inhibiting its catalytic function. Moreover, inhibition by A. vinelandii NIFL is stimulated by the presence of adenosine nucleotides, particularly ADP, suggesting that formation of the inhibitory complex might be regulated by the ATP/ADP ratio (9).When NIFL is overexpressed aerobically in nitrogen-rich medium and purified under aerobic conditions, it is c...

SummaryAzotobacter vinelandii NIFL is a nitrogen fixationspecific regulatory flavoprotein that modulates the activity of the transcriptional activator NIFA in response to oxygen and fixed nitrogen in vivo. NIFL is also responsive to ADP in vitro. Limited proteolysis of NIFL indicates that it comprises a relatively stable N-terminal domain and a C-terminal domain that is protected from trypsin digestion in the presence of adenosine nucleotides. ATP protects the protein from cleavage in the vicinity of potential nucleotide-binding sites in the C-terminus, whereas ADP protects the entire C-terminal domain. NIFL has an apparent K d of 130 M for ATP and 16 M for ADP. The purified N-terminal domain has an identical UV/visible absorption spectrum to the wild-type protein and is reduced by sodium dithionite, demonstrating that it is a flavinbinding domain. The isolated N-terminal domain does not inhibit NIFA activity. A subdomain fragment containing 160 residues of the C-terminal region, including the nucleotide-binding sites, is also not competent to inhibit NIFA. Removal of the first 146 residues of NIFL, which includes a conserved S-motif (PAS-like domain), found in a large family of sensory proteins from eubacteria, archea and eukarya eliminates the redox response. However, this truncated protein remains competent to inhibit NIFA activity in response to ADP in vitro and to the level of fixed nitrogen in vivo. The redox and nitrogen-sensing functions of A. vinelandii NIFL are therefore separable and are discrete functions of the protein.

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