Cell cycle checkpoints are surveillance systems which delay or arrest cell division when conditions are inappropriate or inopportune. During mitosis, the replicated chromatin fibers are condensed and compacted into sister chromatids and then the sister chromatids are segregated to the daughter cells. Several checkpoint systems regulate the onset of mitosis in mammalian cells. The DNA damage G2 checkpoint delays mitosis by inhibiting the protein kinase that promotes mitotic entry (mitosis-promoting-factor, MPF) and excluding MPF from the nucleus so that it cannot interact with nuclear substrates. Ataxia telangiectasia-mutated (ATM) and BRCA1 are required for effective DNA damage G2 checkpoint function. DNA topoisomerase II (topoll) is an essential enzyme that is required to separate topologically intertwined daughter chromatids after DNA replication and prior to mitosis. G2 human cells actively delay the onset of mitosis when chromatid decatenation is blocked using the topoll catalytic inhibitor, ICRF-193. Override of the ICRF-193-induced G2 delay caused the appearance of constrictions between chromatid arms in metaphase spreads, suggesting the presence of linkages or catenations due to inhibition of topoll. The active arrest of G2 cells when topoll was inhibited did not require ATM and was not associated with activation of Chkl or significant inhibition of MPF. The topoll-dependent G2 delay that is induced by ICRF- 193 does not, therefore, appear to be a DNA damage G2 checkpoint response. The topoll-dependent G2 delay did require the activity of the AT- and rad3-related (ATR) checkpoint kinase and expression of BRCA11 establishing the G2 delay in ICRF-193-treated cells as an active, checkpoint response. ATR and BRCA1 appear to enforce the topoll-dependent G2 checkpoint through inhibition of polo-like kinase I (Plkl). Plkl phosphorylates cyclin B1 on ser 147, and the Crml nuclear exporter binds at this site to actively transport cyclin B1 out of the nucleus. Plkl-dependent phosphorylation of ser147 blocks Crml interaction with cyclin B1 and causes accumulation of MPF within the interphase nucleus where it can interact with its various substrates to initiate mitosis. ATR- and BRCAl-dependent inhibition of Plkl should cause MPF to be excluded from the nucleus, thereby blocking the onset of mitosis. Studies in this project will test several questions concerning the ICRF-193-induced G2 checkpoint response. Does the ICRF-193-induced G2 delay reflect the activity of a checkpoint that monitors the status of chromatid decatenation following DNA replication? Does ATR sends a signal through BRCA1 to inhibit Plkl when chromatid catenations are sensed? Is defective decatenation checkpoint function associated with genetic instability in cancer?
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