Abstract. We have recently cloned and characterized a human member (BM28) of the MCM2-3-5 family of putative replication factors (Todorov, I. T., R. Pepperkok, R. N. Philipova, S. Kearsey, W. Ansorge, and D.Werner. 1994. J. Cell Sci. 107:253-265). While this protein is located in the nucleus throughout interphase, we report here a dramatic alteration in its nuclear binding during the cell cycle. BM28 is retained in the nucleus after Triton X-100 extraction in G1 and early S phase cells, but is progressively lost as S phase proceeds, and little BM28 is retained in detergent-extracted G2 nuclei. BM28 that is resistant to extraction in G1 nuclei is removed by DNase I digestion, suggesting that the protein is chromatin associated.In addition, we present evidence for variations in the electrophoretic mobility of BM28 that may reflect posttranslational modifications of BM28 during the cell cycle. During mitosis, BM28 is present as a fast-migrating form, but on entry into G1, the protein is converted into a slow-migrating form. With the onset of S phase, the slow-migrating form is progressively converted into the fast form. BM28 is phosphorylated at all stages of the cell cycle, but during interphase the fast form is hyperphosphorylated compared with the slow form. These apparent changes in modification may reflect or effect changes in the nuclear binding of BM28.The behavior of BM28 is not dissimilar to related proteins in Saccharomyces cerevisiae, such as Mcm2p, which are excluded from the nucleus after DNA replication. We speculate that BM28 may be involved in the control that limits eukaryotic DNA replication to one round per cell cycle. STUDIES of chromosomal replication have shown that, in many systems, an initiation event at a replication origin is followed by a latent period, during which that origin is inactive. This is demonstrated clearly in eukaryotic cells, where initiation events, which occur throughout S phase, only take place on unreplicated DNA. Replicated chromosomes usually have to pass through mitosis before further initiation events can occur, and thus one round of replication per cell cycle is ensured (for review see Coverley and Laskey, 1994; Diffiey, 1994;Stillman, 1994). The nature of the control is not clear but appears to be cis-acting at the level of the template, as cell fusion experiments have indicated that a G1, but not a G2, nucleus can replicate when fused to an S phase cell (Rao and Johnson, 1970). Studies on the effects of nuclear permeabilization on DNA replication in Xenopus egg extracts have suggested that G1 but not 1988; for review see Coverley and Laskey, 1994). In this model, a hypothetical licensing factor binds to chromatin during mitosis and permits DNA replication during the following S phase. The licensing factor is inactivated by the passage of the replication fork, excluded from the nucleus, and its access to the chromatin is blocked by the nuclear membrane until the following mitosis, thus preventing overreplication of DNA during a single cell cycle.Evidence for a lice...

Timelapse video microscopy has been used to record the motility and dynamic interactions between an H-2D b -restricted murine cytotoxic T lymphocyte clone (

We have used time-lapse video microscopy to study cytotoxic T lymphocyte (CTL)-mediated apoptosis of LDb fibroblast target cells at different phases of the cell cycle. When aphidicolin-synchronized target cells were exposed to the CTL clone F5, apoptosis occurred with similar morphology during G1, S/G2 and M phase, showing that apoptosis and mitosis are not mutually exclusive cellular events. Interestingly, following normal mitosis of target cells that had been previously contacted by CTL, pairs of daughter cells would occasionally undergo apoptosis within minutes of each other. Such synchronous post-mitotic apoptosis was also observed when using mitotically unsynchronized target cells, and also when using d11S T cell hybridomas as alternative Fas- (CD95-) based effector cells, even if these effectors were physically washed away after an initial period of co-incubation with the target cells. Our observations show that cytotoxic cells can induce a condemned state in pre-mitotic target cells, which can be inherited by both daughter cells, leading to their synchronous apoptosis after mitosis.