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Regulation of Chromosome Segregation
Summary: Sandra Holloway studies the regulation of chromosome segregation and mitotic exit in the yeast Saccharomyces cerevisiae and in Xenopus extracts. She and her colleagues would like to know how cellular damage can regulate these transitions.
The eukaryotic cell cycle is defined as the time during which a cell is born, replicates its genetic material, and segregates this material into two identical daughter cells. The two daughter cells are identical only if, despite challenges such as the presence of DNA-damaging agents, each step in the cell cycle is accurate. To ensure this accuracy and maintain genomic integrity following DNA damage, eukaryotic cells activate the DNA damage checkpoint pathway that temporarily arrests the cell cycle and allows time for the repair of DNA lesions. This protective mechanism is defective in many cancer cells, suggesting that one step during the progression of a normal cell to the cancerous state is the loss of checkpoint controls, which results in genomic instability that alters the cell's growth properties. This model is well supported. For example, humans who have mutations in the ATM protein that is critical for the DNA damage checkpoint pathway lack this protective mechanism and have a predisposition to cancer. An understanding of checkpoint pathways and their regulation is thus critical to an understanding of the treatment and prevention of cancer.
The DNA damage checkpoint pathway is well conserved from yeast to humans. When the yeast Saccharomyces cerevisiae is treated with DNA-damaging agents it responds (like human cells) in a multifaceted fashion. It will delay the cell cycle before DNA synthesis (G1), during DNA synthesis (early S phase), or after DNA synthesis but just prior to chromosome segregation (metaphase of mitosis). We have shown that mutants lacking the S-phase cyclins Clb5 and Clb6 cannot stop at metaphase in response to DNA damage and are therefore hypersensitive to this damage. After DNA damage, these strains undergo an apparently normal S-phase delay and, like wild-type cells, eventually complete DNA replication and arrest with a fully replicated genome. Unlike wild-type cells, however, this arrest is not at metaphase of mitosis with an undivided nucleus. Instead, in the presence of the DNA-damaging agent methyl methanesulfonate (MMS), more than 50 percent of clb5 clb6 mutants bypass the normal metaphase checkpoint and arrest instead with divided (but damaged) nuclei. A defect of this type could be the consequence of a defective DNA damage checkpoint pathway or a defect in the targets of this pathway—the proteins that ensure that nuclear division does not occur until the damage is repaired.
To distinguish between these two possibilities, we tested the ability of clb5 clb6 mutants to prevent nuclear division after spindle damage. In the presence of the spindle-damaging agent nocodazole, more than 50 percent of clb5 clb6 mutants bypass the metaphase checkpoint and arrest instead with divided nuclei. Since clb5 clb6 mutants are unable to prevent nuclear division in response to two separate checkpoint pathways, we concluded that clb5 clb6 mutants must have a defect in the proteins that regulate the timing of nuclear division, and not in the checkpoint pathways themselves.
During normal nuclear division the cohesion between duplicated DNA molecules (sister chromatids) is dissolved and the sister chromatids segregate to the two daughter cells. Sister chromatid cohesion is established and maintained by the cohesin complex that contains Scc1/Mcd1. The cohesin complex normally holds sister chromatids together until Scc1/Mcd1 is cleaved by the Esp1 protease at the metaphase-to-anaphase transition, allowing sister chromatid separation. Sister chromatid cohesion can be prevented during damage by the stabilization of Pds1, which sequesters Esp1 and keeps it away from Scc1/Mcd1. This situation ends once the signal is received to resume sister chromatid separation once, for example, the damage is repaired. At this point Pds1 is degraded by the anaphase-promoting complex/cyclosome (APC/C), Esp1 is released, Scc1 is cleaved, and sister chromatid separation occurs.
We have tested the response of clb5 clb6 mutants to the presence of nondegradable forms of Pds1 and Scc1. Like wild-type cells, clb5 clb6 mutants arrest at metaphase in response to the presence of nondegradable forms of Scc1/Mcd1. Unlike wild-type cells, however, clb5 clb6 mutants undergo nuclear division despite the presence of nuclear nondegradable Pds1. Our results suggest a novel role for the S-phase cyclins Clb5 and Clb6 in maintaining sister chromatid cohesion during DNA damage–induced arrest, perhaps by regulating Pds1 activity. Inactivation of these cyclins causes dramatic chromosome missegregation and cell death only after damage (for example, DNA damage). This is significant because (by analogy to the yeast) it predicts that drugs that inactivate the human orthologs cyclin E and cyclin A might enhance the effectiveness of DNA-damaging cancer drugs without many side effects on nonirradiated tissues.
Once the genetic material is duplicated during the S phase of the cell cycle, it is segregated physically between two daughter cells by the spindle apparatus. Due to its importance, the proper function of this apparatus is closely monitored by the spindle damage checkpoint pathway. If spindle damage is detected, this pathway triggers events that arrest the cell cycle prior to nuclear division. The main event that is triggered by this pathway is the binding of Mad2 to Cdc20, which sequesters it away from the APC/C. Since Cdc20 is required to target the APC/C to Pds1, this prevents Pds1 degradation and enables cells with spindle damage to arrest at metaphase of mitosis, prior to chromosome segregation. The cell is thus provided with time during which it can repair the spindle damage.
Once the damage is repaired or if the damage is not significant enough to maintain a prolonged arrest, the cell cycle resumes and chromosomes segregate. During cell cycle resumption, Cdc20 rebinds the APC/C and the Cdc20/APC/C ubiquitinates Pds1, leading to its degradation, sister chromatid separation, exit from mitosis, and the start of a new cell cycle. Very little is known about the changes that allow the re-formation of the Cdc20-APC/C complex during resumption of the cell cycle.
We have generated a mutant version of the APC/C that affects cell recovery after spindle damage. This version bears a mutation in the CDC16 subunit of the APC/C, and the allele is called cdc16-183. Cells bearing cdc16-183 grow at 30°C but are supersensitive to spindle-damaging agents at this temperature. Unlike spindle checkpoint mutants (such as the mad and bub mutants) that cannot arrest in response to spindle damage, cdc16-183 mutants arrest normally in response to spindle damage but die because they are unable to resume cell cycling properly. Consistent with this, cdc16-183 mutants are unable to degrade Pds1 properly (relative to wild-type cells) after exposure to spindle-damaging agents. Our results suggest that the mutation in cdc16-183 may affect the rebinding of Cdc20 to the APC/C after spindle damage and gives us excellent tools to now dissect out the pathways that regulate Cdc20 rebinding to the APC/C after spindle damage. Yeast bearing cdc16-183 are not supersensitive to DNA-damaging agents, suggesting that distinct mechanisms are used to reactivate the APC/C after DNA damage or spindle damage.
These new insights into cell cycle regulation during and after DNA or spindle damage suggest that the S-phase cyclins may be good candidates for cancer chemotherapy, and our results with cdc16-183 add to the information required to design new drugs to treat cancer.
Last updated May 24, 2001
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