Rmi1 is a conserved oligonucleotide and oligosaccharide binding-fold protein that is associated with RecQ DNA helicase complexes from humans (BLM-TOP3␣) and yeast (Sgs1-Top3). Although human RMI1 stimulates the dissolution activity of BLM-TOP3␣, its biochemical function is unknown. Here we examined the role of Rmi1 in the yeast complex. Consistent with the similarity of top3⌬ and rmi1⌬ phenotypes, we find that a stable Top3⅐Rmi1 complex can be isolated from yeast cells overexpressing these two subunits. Compared with Top3 alone, this complex displays increased superhelical relaxation activity. The isolated Rmi1 subunit also stimulates Top3 activity in reconstitution experiments. In both cases elevated temperatures are required for optimal relaxation unless the substrate contains a single-strand DNA (ssDNA) bubble. Interestingly, Rmi1 binds only weakly to ssDNA on its own, but it stimulates the ssDNA binding activity of Top3 5-fold. Top3 and Rmi1 also cooperate to bind the Sgs1 N terminus and promote its interaction with ssDNA. These results demonstrate that Top3-Rmi1 functions as a complex and suggest that Rmi1 stimulates Top3 by promoting its interaction with ssDNA.Mutations in BLM result in Bloom syndrome, a rare autosomal disease characterized by a variety of symptoms including a predisposition to cancer (1). Cells from Bloom syndrome patients display genomic instability characterized by elevated rates of sister chromatid exchange (2). BLM is a RecQ-family DNA helicase that forms a complex with DNA topoisomerase III␣ (TOP3␣) 2 (1,3,4). This complex is conserved throughout eukaryotes where it acts to suppress recombination, especially in response to DNA-damaging agents such as interstrand cross-linkers (e.g. mitomycin C) or the alkylating agent methylmethanesulfonate (5-10). The fact that the lesions created by these agents are known to impede replication forks has led to the notion that the hypersister chromatid exchange phenotype of Bloom syndrome cells is a consequence of an alternative repair pathway for replication-induced DNA damage.Although the molecular function of BLM-TOP3␣ is not completely understood, the ability of the helicase-topoisomerase complex to "dissolve" double Holliday junction (HJ) substrates in vitro has suggested a compelling mechanism by which it could suppress crossing over during recombinational repair (11,12). Studies of orthologous RecQ complexes from model systems such as budding yeast (Sgs1-Top3), fission yeast (Rqh1-Top3), and Drosophila have provided genetic insight into the function of these proteins as well as support for the HJ dissolution model (12)(13)(14)(15)(16)(17)(18)(19).Rmi1/BLAP75 is a conserved protein that was recently identified based on its association with BLM, Sgs1, and Rqh1. In human cells, RMI1 co-purifies with a complex of proteins including BLM and TOP3␣ (20). This complex has not been purified to homogeneity from yeast, but Rmi1 co-fractionates with a complex containing Sgs1 and Top3 (21,22). Human RMI1 is a 625-amino acid protein that contains a predict...

Protein sumoylation plays an important but poorly understood role in controlling genome integrity. In Saccharomyces cerevisiae, the Slx5-Slx8 SUMO-targeted Ub ligase appears to be needed to ubiquitinate sumoylated proteins that arise in the absence of the Sgs1 DNA helicase. WSS1, a high-copy-number suppressor of a mutant SUMO, was implicated in this pathway because it shares phenotypes with SLX5-SLX8 mutants, including a wss1⌬ sgs1⌬ synthetic-fitness defect. Here we show that Wss1, a putative metalloprotease, physically binds SUMO and displays in vitro isopeptidase activity on poly-SUMO chains. Like that of SLX5, overexpression of WSS1 suppresses sgs1⌬ slx5⌬ lethality and the ulp1ts growth defect. Interestingly, although Wss1 is relatively inactive on ubiquitinated substrates and poly-Ub chains, it efficiently deubiquitinates a Ub-SUMO isopeptide conjugate and a Ub-SUMO fusion protein. Wss1 was further implicated in Ub metabolism on the basis of its physical association with proteasomal subunits. The results suggest that Wss1 is a SUMO-dependent isopeptidase that acts on sumoylated substrates as they undergo proteasomal degradation.Protein modification by SUMO is implicated in a wide variety of cellular processes (19,20). Well known for its ability to control subcellular localization (29, 57), sumoylation has been shown to compete with ubiquitination (15), to mediate proteinprotein interactions (38, 39), and to affect protein turnover through SUMO-targeted ubiquitin (Ub) ligases (23,40,47,48,51,53,54). Indeed, modification by SUMO is so common that the number of proteins targeted for sumoylation in Saccharomyces cerevisiae has been estimated to be over 500 (28). Genetic studies of budding yeast have revealed a role for sumoylation in recombination-mediated DNA repair, although the role of SUMO in this process is not completely understood (4,5,16,56).Similar to ubiquitination, the conjugation of SUMO (Smt3 in budding yeast) to substrate proteins requires an ATP-dependent E1-activating enzyme (Aos1/Uba2), an E2-conjugating enzyme (Ubc9), and one of several E3 ligases (e.g., Siz1 or Siz2). The product of this reaction is an isopeptide linkage between the C-terminal glycine residue of mature SUMO and the ε-amino group of lysine residues in target proteins. In vivo, this bond is unstable, as it is subject to hydrolysis by a family of SUMO-specific cysteine proteases known as Ulps in yeast (Ulp1 and Ulp2) and SENPs (six members) in mammals (24,26,33).SUMO-specific proteases regulate SUMO metabolism at multiple steps. In yeast, Ulp1 carries out the essential role of processing the C terminus of the Smt3 precursor [Smt3(Y101)] into its mature form [Smt3(G98)] by removing its 3 terminal amino acids (aa) (24). Both Ulp1 and Ulp2 are responsible for deconjugating SUMO from target proteins, while Ulp2 and its human homologs, SENP6/7, suppress the accumulation of poly-SUMO chains (6, 27, 33). Ulp1 and Ulp2 are primarily located in the nucleus, although Ulp1, which concentrates at nuclear pores, has cytoplasmic targets (22,25...

Three Bo beta fruct cDNAs encoding acid invertases were cloned from shoots of the green bamboo Bambusa oldhamii. On the basis of the amino acid sequences of their products and phylogenetic analyses, Bo beta fruct1 and Bo beta fruct2 were determined to encode cell wall invertases, whereas Bo beta fruct3encodes a vacuolar invertase. The recombinant proteins encoded by Bo beta fruct2 and Bo beta fruct3 were produced in Pichia pastoris and purified to near homogeneity using ammonium sulfate fractionation and immobilized metal affinity chromatography. The pH optima, pI values, and substrate specificities of the isolated enzymes were consistent with those of plant cell wall or vacuolar invertases. The growth-dependent expression of Bo beta fruct1 and Bo beta fruct2 in the base regions of shoots underscores their roles in sucrose unloading and providing substrates for shoot growth. Its high sucrose affinity suggests that the Bo beta fruct2-encoded enzyme is important for maintaining the sucrose gradient between source and sink organs, while the predominant expression of Bo beta fruct3 in regions of active cell differentiation and expansion suggests functions in osmoregulation and cell enlargement.

The gene mutated in Bloom's syndrome, BLM, encodes a member of the RecQ family of DNA helicases that is needed to suppress genome instability and cancer predisposition. BLM is highly conserved and all BLM orthologs, including budding yeast Sgs1, have a large N-terminus that binds Top3-Rmi1 but has no known catalytic activity. In this study, we describe a sub-domain of the Sgs1 N-terminus that shows in vitro single-strand DNA (ssDNA) binding, ssDNA annealing and strand-exchange (SE) activities. These activities are conserved in the human and Drosophila orthologs. SE between duplex DNA and homologous ssDNA requires no cofactors and is inhibited by a single mismatched base pair. The SE domain of Sgs1 is required in vivo for the suppression of hyper-recombination, suppression of synthetic lethality and heteroduplex rejection. The top3D slow-growth phenotype is also SE dependent. Surprisingly, the highly divergent human SE domain functions in yeast. This work identifies SE as a new molecular function of BLM/Sgs1, and we propose that at least one role of SE is to mediate the strand-passage events catalysed by Top3-Rmi1.

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A revised Tai-Ta centrifugal impeller pump was designed to study the interaction of the left ventricular assist device (LVAD) with the cardiovascular system in a canine model. Six healthy dogs weighing 12-16 kg were used. Blood flows in the aortic arch, the pulmonary artery (PA), and the LVAD outlet were measured simultaneously with the arterial blood pressure (ABP), the pump outflow pressure (POP), and the electrocardiograph (ECG). Normally, the blood flows in the aorta and the PA started at the S-wave of the ECG. When the LVAD was operated at a higher rotational speed (increased from 2900 to 5400 rpm), the ABP, POP, the pump flow, and the maximum rate of change of PA flow increased. However, the fluctuating amplitudes of ABP, POP, and the pump flow decreased significantly. The cardiovascular hemodynamics change with the pump speeds. For a typical 1.1-1.5 L/min cardiac output in canine, the revised LVAD was able to deliver a flow bypass ratio from 15% up to 100%. The LVAD outflow appeared to be pulsatile and matched the cardiac cycle, showing that the centrifugal impeller pump could be used as a pediatric assist device when cardiac function was impeded.

Mutations in BLM result in Bloom's syndrome, a rare autosomal disease characterized by a variety of symptoms including a predisposition to cancer. BLM is conserved in the budding yeast Saccharomyces cerevisiae as Sgs1. BLM/Sgs1 is a 3′‐5′ DNA helicase with a large N‐terminal domain of unknown function. To determine the role of the N‐terminus, we assayed purified recombinant sub‐domains using biochemical methods. We found that an internal domain of the Sgs1 N‐terminus (amino acids #103–322, named the SA domain) can bind single‐stranded (ss) DNA using gel‐shift assay. Moreover, this ssDNA‐binding activity is conserved in the N‐termini of human and Drosophila BLM. Further studies demonstrate that the SA domain exhibits strand‐annealing and strand‐exchange activities in vitro. To determine the biological function of this domain, we tested the sgs1Δ103–322 allele for a variety of sgs1Δ phenotypes including MMS sensitivity, top3Δ slow‐growth and sgs1Δ slxΔ synthetic‐lethality. The results show that sgs1Δ103–322 complements MMS sensitivity but is defective for top3Δ slow‐growth promotion and sgs1Δslx4Δ and sgs1Δslx5Δ synthetic lethality. While further defining its modular structure, these results identify an essential pro‐recombinogentic function for BLM/Sgs1 and provide a framework to analyze the coordinated activities of Top3, Rmi1 and strand annealing.This work is supported by NIH‐R01 GM071268

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