Genome stability requires a set of RecQ-Top3 DNA helicase-topoisomerase complexes whose sole budding yeast homolog is encoded by SGS1-TOP3. RMI1/NCE4 was identified as a potential intermediate in the SGS1-TOP3 pathway, based on the observation that strains lacking any one of these genes require MUS81 and MMS4 for viability. This idea was tested by confirming that sgs1 and rmi1 mutants display the same spectrum of synthetic lethal interactions, including the requirements for SLX1, SLX4, SLX5, and SLX8, and by demonstrating that rmi1 mus81 synthetic lethality is dependent on homologous recombination. On their own, mutations in RMI1 result in phenotypes that mimic those of sgs1 or top3 strains including slow growth, hyperrecombination, DNA damage sensitivity, and reduced sporulation. And like top3 strains, most rmi1 phenotypes are suppressed by mutations in SGS1. We show that Rmi1 forms a heteromeric complex with Sgs1-Top3 in yeast and that these proteins interact directly in a recombinant system. The Rmi1-Top3 complex is stable in the absence of the Sgs1 helicase, but the loss of either Rmi1 or Top3 in yeast compromises its partner's interaction with Sgs1. Biochemical studies demonstrate that recombinant Rmi1 is a structurespecific DNA binding protein with a preference for cruciform structures. We propose that the DNA binding specificity of Rmi1 plays a role in targeting Sgs1-Top3 to appropriate substrates.

In the presence of emetine, an inhibitor of protein synthesis, nascent DNA on forward arms of replication forks in hamster cell lines containing either single or amplified copies of the DHFR gene region was enriched 5‐ to 7‐fold over nascent DNA on retrograde arms. This forward arm bias was observed on both sides of the specific origin of bidirectional DNA replication located 17 kb downstream of the hamster DHFR gene (OBR‐1), consistent with at least 85% of replication forks within this region emanating from OBR‐1. However, the replication fork asymmetry induced by emetine does not result from conservative nucleosome segregation, as previously believed, but from preferentially inhibiting Okazaki fragment synthesis on retrograde arms of forks to produce ‘imbalanced DNA synthesis’. Three lines of evidence support this conclusion. First, the bias existed in long nascent DNA strands prior to nuclease digestion of non‐nucleosomal DNA. Second, the fraction of RNA‐primed Okazaki fragments was rapidly diminished. Third, electron microscopic analysis of SV40 DNA replicating in the presence of emetine revealed forks with single‐stranded DNA on one arm, and nucleosomes randomly distributed to both arms. Thus, as with cycloheximide, nucleosome segregation in the presence of emetine was distributive.

One of the most common chromosomal abnormalities in acute leukemia is a reciprocal translocation involving the HRX gene at chromosome locus 11q23, resulting in HRX fusion proteins. Using the yeast two-hybrid system, in vitro binding studies, and human cell culture coimmunoprecipitation experiments, we show here that a region of the HRX protein that is consistently retained in HRX leukemic fusion proteins interacts directly with SET, another protein implicated in leukemia. We have identified the binding sites on HRX for SET and show that these sequences are clustered near the A⅐T hooks that have been shown to bind DNA. We also show that carboxyl-terminal SET sequences, possibly the acidic tail of SET, bind to HRX. We have also found serine/ threonine-specific protein phosphatase activity in anti-HRX coimmunoprecipitates. Using the phosphatase inhibitor okadaic acid and Western blotting, the phosphatase was identified as protein phosphatase 2A (PP2A). Mutation of a single amino acid in one of the SET binding sites of HRX resulted in lower amounts of both coimmunoprecipitated SET protein and coimmunoprecipitated PP2A. These results suggest that the leukemogenic effects of HRX fusion proteins may be related to interactions with SET and PP2A.

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In the presence of oligomeric chondroitin 4-sulfate (C4-S), cathepsin K (catK) forms a specific complex that was shown to be the source of the major collagenolytic activity in bone osteoclasts. C4-S forms multiple contacts with amino acid residues on the backside of the catK molecule that help to facilitate complex formation. As cathepsin L does not exhibit a significant collagenase activity in the presence or in the absence of C4-S, we substituted the C4-S interacting residues in catK with those of cathepsin L. Variants revealed altered collagenolytic activities with the largest inhibitory effect shown by the hexavariant M5. None of the variants showed a reduction in their gelatinolytic and peptidolytic activities when compared with wild-type catK, indicating no structural alteration within their active sites. However, the crystal structure of the M5 variant in the presence of oligomeric C4-S revealed a different binding of chondroitin 4-sulfate. C4-S is not continuously ordered as it is in the wildtype catK⅐C4-S complex. The orientation and the direction of the hexasaccharide on the catK surface have changed, so that the hexasaccharide is positioned between two symmetry-related molecules. Only one M5 variant molecule of the dimer that is present in the asymmetric unit interacts with C4-S. These substitutions have changed the mode of catK binding to C4-S and, as a result, have likely affected the collagenolytic potential of the variant. The data presented here support our hypothesis that distinct catK/C4-S interactions are necessary for the collagenolytic activity of the enzyme.Collagen degradation is a natural process observed in bone remodeling, wound healing, and organ development. However, excessive collagen degradation is implicated in many serious diseases such as osteoporosis, different forms of arthritis, and some vascular disorders. Triple-helical collagens are the major organic components of bone matrix (type I collagen) and of cartilage (type II collagen). These collagens are highly resistant to general proteolysis, and it requires specific peptidases for their degradation. Cathepsin K (catK), 7 a member of the papain family of lysosomal cysteine peptidases, is the predominant peptidase of bone-degrading osteoclasts (1, 2). It has the ability, unique among mammalian proteinases, of cleaving triple-helical collagen at multiple sites (3, 4). Analysis of catK activities revealed that the collagen-degrading activity is not proportional to the general peptidase activity suggesting some other factors affect the specificity of catK. It was shown (5, 6) that the ability to cleave collagen is highly dependent on the formation of an oligomeric complex of catK with glycosaminoglycans, in particular, with chondroitin 4-sulfate (C4-S). Glycosaminoglycans are naturally present in bone and cartilage in sufficient quantities to form complexes with catK. Complex formation with glycosaminoglycans is unique for catK among other papain-like proteases (6, 7). It is remarkable that monomeric catK has no significant collag...