The enzyme DNA topoisomerase II, which removes the catenations formed between the DNA molecules of sister chromatids during replication and is a structural component of chromosome cores, is needed for chromosome condensation in yeast and in Xenopus extracts. Inhibitors of topoisomerase II arrest mammalian cells before mitosis in the G2 phase of the cell cycle, but also produce DNA damage, which causes arrest through established checkpoint controls. It is open to question whether cells need topoisomerase II to leave G2, or control late-cycle progression in response to its activity. Bisdioxopiperazines are topoisomerase II inhibitors that act without producing direct DNA damage; the most potent, ICRF-193, blocks mammalian entry into but not exit from mitosis. Here we show that checkpoint-evading agents such as caffeine override this block to produce abortively condensed chromosomes, indicating that topoisomerase II is needed for complete condensation. We find that exit from G2 is regulated by a catenation-sensitive checkpoint mechanism which is distinct from the G2-damage checkpoint.

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Molecular analysis revealed a diverse ocular surface bacterial population. In addition to the normal flora, various potentially pathogenic bacteria were identified. The detection of known pathogens in both normal and dry eyes, with minimal signs of infection, presents a diagnostic dilemma. It remains unknown whether their presence is associated with inflammation and reduced goblet cell density or whether they adversely affect the ocular surface predisposing it to abnormal microbial colonization. In the absence of overt clinical infection, it is unknown whether such results should prompt intervention with therapy.

The mitotic state is associated with a generalized repression of transcription. We show that mitotic repression of RNA polymerase III transcription can be reproduced by using extracts of synchronized HeLa cells. We have used this system to investigate the molecular basis of transcriptional repression during mitosis. We find a specific decrease in the activity of the TATA-binding-protein (TBP)-containing complex TFIIIB. TBP itself is hyperphosphorylated at mitosis, but this does not appear to account for the loss of TFIIIB activity. Instead, one or more TBP-associated components appear to be regulated. The data suggest that changes in the activity of TBP-associated components contribute to the coordinate repression of gene expression that occurs at mitosis.Nuclear transcription is repressed during mitosis (4,7,9,31,45,46). In mammalian cells, all RNA synthesis stops by midprophase before any disintegration of the nuclear membrane is apparent, and it does not resume again until late in telophase (31). This repression may be necessary to allow chromosomal condensation and division to occur without interference from the transcriptional apparatus. However, very little is known about the molecular mechanisms responsible for transcriptional inhibition at mitosis. An important step toward characterizing this process came with the recent demonstration that mitotic repression can be mimicked in vitro by using Xenopus egg extracts (15). Hartl et al. (15) showed that extracts shifted to a mitotic state by the addition of recombinant cyclin B exhibit a marked decrease in ability to transcribe tRNA or 5S RNA genes relative to untreated interphase extracts. This happens even if active transcription complexes are preassembled on the promoters of these genes (15). The in vitro inhibition does not require nucleosome deposition on the template and occurs in the presence of the topoisomerase II inhibitor VM-26, which prevents the complete assembly of chromosomes into metaphase structures. It appears to involve phosphorylation-dependent inactivation of one or more components of the transcription machinery that are required for tRNA and 5S RNA synthesis (15).tRNA and 5S RNA genes are transcribed by RNA polymerase III (Pol III). The proteins involved in this process have been studied extensively (reviewed in references 51 and 59). Recruitment of Pol III to specific promoter sites requires a factor called TFIIIB, which is a multisubunit complex containing the TATA-binding protein (TBP) and associated polypeptides (reviewed in references 16 and 34). Since most Pol III promoters contain no TATA sequence and cannot be recognized directly by TBP (19,22,53), TFIIIB is normally recruited via protein-protein interactions with an assembly factor called TFIIIC that binds downstream of the initiation site (reviewed in references 16, 51, and 59).We have investigated how the human Pol III transcription apparatus behaves at mitosis by comparing extracts made from asynchronous HeLa cells with those of cells synchronized in M phase of the active gro...

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Abstract. Metaphase chromatids are believed to consist of loops of chromatin anchored to a central scaffold, of which a major component is the decatenatory enzyme DNA topoisomerase II. Silver impregnation selectively stains an axial element of metaphase and anaphase chromatids; but we find that in earlier stages of mitosis, silver staining reveals an initially single, folded midline structure, which separates at prometaphase to form two chromatid axes. Inhibition of topoisomerase II prevents this separation, and also prevents the contraction of chromatids that occurs when metaphase is arrested. Immunolocalization of topoisomerase IItx reveals chromatid cores analogous to those seen with silver staining. We conclude that the chromatid cores in early mitosis form a single structure, constrained by DNA catenations, which must separate before metaphase chromatids can be resolved.

DNA topoisomerase II (EC 5.99.1.3) is necessary for chromosome condensation and disjunction in yeast but not for other functions. In mammalian cells, it has been reported to be necessary for progression toward mitosis but not for transit through mitosis. We have found, on the contrary, that specific inhibition of topoisomerase II (but not of topoisomerase I) interferes with mammalian mitotic progression. Metaphase is prolonged, and anaphase separation of chromatids is completely inhibited, in cells given high concentrations of topoisomerase II inhibitors; nevertheless these cells attempt cleavage, sometimes generating nucleate and anucleate daughters. Lower concentrations of inhibitors interfere with anaphase and produce abnormalities of segregation. DNA topoisomerase II activity is therefore necessary for mammalian chromatid separation, but it is not tightly coupled to the control of other mitotic events.In eukaryotic cells DNA topoisomerase II (EC 5.99.1.3) is a major component of the mitotic chromosome core (1), with sites of action adjacent to the attachment points of supercoiled DNA loops (2). This structural role is conserved in all known cases.In budding and in fission yeast, temperature-sensitive top2 mutants show no defect in normal interphase cell functions, including cell cycle progression, at the restrictive temperature; but they are arrested in mitosis (3, 4). In top2 mutants of the fission yeast Schizosaccharomyces pombe, chromosome condensation is partly achieved at the restrictive temperature, but it produces long entangled prophase-like structures, which become fully condensed when the topoisomerase II activity is restored. If topoisomerase II is inactivated after chromosome condensation, the cells remain blocked at metaphase, unable to achieve chromatid separation (4). After release from such arrest, yeast cells show chromosome nondisjunction (5); meiotic nondisjunction can be similarly produced (6). This requirement for topoisomerase II in mitotic condensation and in separation of sister chromatids may be due to the need to separate sister strands of DNA, which inevitably become concatenated during replication but are not decatenated until later (7). Other cellular DNA topoisomerases appear to be adequate for other necessary manipulations of DNA topology but cannot perform this decatenation activity; in top2 mutants, concatenated plasmids accumulate at the restrictive temperature (3,8).Though the topoisomerase II function is necessary for chromatid segregation in fission yeast, other aspects of yeast mitosis continue without it. Temperature-sensitive top2 cells at the restrictive temperature divide into two portions, joined by chromatid fibers that prevent total cytokinesis; in top2 cdcll double mutants, in which septum formation is also blocked, cells reform single active nuclei after an abortive mitosis and enter a new round of replication (8).In mammalian cells the role of topoisomerase II in cycle progression has been reported to be rather different from its role in yeast. No tempe...