A topoisomerase II-dependent G2 cycle checkpoint in mammalian cells

Abstract

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.

The decatenation checkpoint

“…The decatenation checkpoint therefore monitors the approach to completion, not the full completion, of chromosome disentanglement (Downes et al, 1994). A similar threshold basis for checkpoint activation has been documented for other cell cycle checkpoints, notably the DNA replication checkpoint in which activation requires a certain quantity of single-stranded DNA (Branzei and Foiani, 2005).…”

“…Gain and loss of whole chromosomes and chromosome fragments result when cells complete mitosis in the presence of entangled chromosomes ( Figure 1A). The G 2 phase decatenation checkpoint delays entry into mitosis until chromosomes have been decatenated (disentangled) by topoisomerase II (topo II) (Downes et al, 1994). The decatenation checkpoint is distinct from the DNA damage checkpoint, the chromatin deacetylation checkpoint, the spindle assembly checkpoint and other G 2 /M checkpoints.…”

“…Studies with these topo II poisons precluded the discovery of the decatenation checkpoint. In contrast, the catalytic inhibitors, which include ICRF-193 and related bisdioxopiperazines, inhibit topo II before DSB formation and activate the decatenation checkpoint, but not the DNA damage checkpoint (Downes et al, 1994;Roca et al, 1994). The decatenation checkpoint is typically studied with the bisdioxopiperazine topo II inhibitors.…”

“…The original report documented a functional checkpoint in the Indian muntjac cells, PtK2 rat kangaroo cells, Chinese hamster ovary cells, SV-MRC5-transformed human fibroblasts, MSU1.1 v-myc-immortalised human fibroblasts, and HeLa human cervical carcinoma cell lines (Downes et al, 1994). Subsequently, the checkpoint was observed in human lymphoblastoid cell lines (Deming et al, 2001), mouse embryonic fibroblasts (Damelin et al, 2005), and primary cultures and strains of human uroepithelial cells (Doherty et al, 2003) and human fibroblasts (Kaufmann and Kies, 1998;Deming et al, 2001;Franchitto et al, 2003;Damelin et al, 2005).…”

“…The checkpoint response does not require ATM kinase (Deming et al, 2001). Caffeine, which inhibits certain signalling molecules including ATR, was shown to override the G 2 delay (Downes et al, 1994). ATR mediates the delay by inhibition of Plk1 (Polo-like kinase 1); Plk1 activity was reduced following ICRF-193 treatment, and conversely the checkpoint response was attenuated when active Plk1 was overexpressed (Deming et al, 2002).…”