Topoisomerase I (TOP1) relaxes superhelical DNA through a breakage/rejoining reaction in which the active site tyrosine links covalently to a 3 phosphate at the break site as a transient intermediate. The antitumor drug camptothecin (CPT) and its analogs inhibit the rejoining step of the breakage/rejoining reaction, which traps the enzyme in covalent linkage with DNA (the cleavable complex). Little is known about the fate of cellular TOP1 trapped in the cleavable complex. We have analyzed TOP1 in mammalian cell lines treated with CPT. When CPT-treated cells were lysed with either SDS or alkali and analyzed by Western blotting, greater than 90% of the TOP1 was linked to DNA. Nuclease treatment of the cell lysate to remove the covalently linked DNA from TOP1 revealed a distinct ladder of higher molecular weight bands having properties indicative of multi-ubiquitin (Ub) conjugates of TOP1. Approximately 5-10% of TOP1 was present as these conjugates within minutes of CPT treatment. Consistent with ubiquitination, TOP1 was not modified in ts85 cells at the restrictive temperature for its thermolabile ubiquitin-activating enzyme (E1). Because conjugation with ubiquitin can mark proteins for destruction by the 26S proteasome, we analyzed TOP1 protein levels during prolonged CPT treatment. TOP1 protein levels were reduced to about 25% during CPT treatments of 2-4 h resulting from increased destruction, with the half-life dropping from 10 -16 h down to 1-2 h. The destruction of TOP1, like the formation of Ub-TOP1 conjugates, was not observed in ts85 cells at the restrictive temperature. The destruction of TOP1 was also prevented in cells treated with MG-132 and lactacystin, specific inhibitors of the 26S proteasome. Finally, the multi-Ub conjugates of TOP1 were observed whether or not aphidicolin was included in cotreatment with CPT, indicating that replication fork activity was not involved in making TOP1 a substrate for ubiquitination. These results demonstrate that independent of DNA replication, the TOP1 cleavable complex is ubiquitinated and destroyed in cells treated with antitumor drugs that block the religation step of the TOP1 reaction. DNA topoisomerase I (TOP1)1 is a vital DNA metabolic enzyme as well as a molecular target of antitumor drugs. TOP1 relaxes DNA supercoils by cleaving a single strand of duplex DNA and passing the complimentary DNA strand though the cleaved strand before religation (1). In a transient reaction intermediate, termed the cleavable complex, the enzyme links covalently to DNA through tyrosine 723 (human TOP1) leaving a DNA break having a free 5Ј hydroxyl end. Stopping the TOP1 reaction with strong protein denaturants produces a very low yield of TOP1 linked covalently to DNA. The rarity of this product is a reflection of the transient nature of the "cleaved" reaction intermediate. Antitumor drugs that act on TOP1 inhibit the DNA religation step through a reversible mechanism. The inhibition of religation can be observed as an increase in the yield of the enzyme-DNA covalent complex pro...

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Objective. To determine the sensitivity and specificity of anti-Jo-1 in systemic sclerosis (SSc) patients with and without myositis.Methods. Immunoblots on HeLa nuclei were used to screen sera from 554 consecutive connective tissue disease patients. Those who had 45-55-kd bands, all patients with polymyositis/derrnatomyositis (PM/DM), and a random selection of SSc, Raynaud's disease, systemic lupus erythematosus, and rheumatoid arthritis patients were also studied by anti-Jo-1 enzyme-linked immunosorbent assay and by immunoblots on rabbit pooled aminoacyl-transfer RNA synthetase.Results. Anti-Jo-1 was present only in 8 of the 40 PM/DM patients.Conclusion. Anti-Jo-1 is specific for PM/DM.Anti-Jo-1 antibodies are found in polymyositis/ dermatomyositis (PM/DM) patients, especially in those with lung involvement (1). Anti-Jo-1 is directed at histidyl-transfer RNA (tRNA) synthetase (Jo-1 autoantigen), which is a dimer of 50-kd subunits found in the cytoplasm (1,2). Anti-Jo-1 may be detected by double immunodiffusion, immunoblotting, and enzyme-linked immunosorbent assay (ELISA) (3) using an antigen containing Jo-1 and standard positive and negative control sera. The presence of anti-Jo-1 in systemic sclerosis (SSc) patients has not been examined in large-scale studies. SSc patients frequently present with lung involvement, and, in some cases, with myositis. Therefore, in the present study, we decided to screen our large series of consecutive SSc patients for the Submitted for publication July 25, 1995; accepted in revised form September 29, 1995. presence of anti-Jo-1 and compare them with other connective tissue disease patients with and without myositis.Our results show that anti-Jo-1 is present in 20.0% of PM/DM patients (8 of 40), and is absent from SSc patients' sera, including those with PM/DM overlap and those with elevated creatine phosphokinase (CPK) levels and muscle pain, but who do not fulfill criteria for PM/DM, with or without interstitial lung fibrosis. PATIENTS AND METHODSPatients and controls. All patients and controls were seen in the Rheumatology Clinics at