The Molecular Mechanism Of Cell Cycle Regulation In Budd

Diabetes, Digestive, Kidney Diseases

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Abstract

During cell division daughter cells must receive one and only one copy of each and every chromosome. To achieve this all chromosomes must be duplicated and the duplicated products must be recognized as being equivalent, i.e. sister chromatids, so that they can be segregated to the two daughter cells. The identity of the sister chromatids is maintained by the presence of sister chromatid cohesion, which is dissolved only after all duplicated chromosomes have formed bipolar spindle attachments. A key regulator of this process in budding yeast is the protein Pds1. Functional homologues of Pds1, known as securins, are found in many organisms including human, where securin over expression is linked to genomic instability. Pds1 inhibits anaphase initiation by inactivating a conserved protease known as separase (Esp1 in budding yeast) that dissolves cohesion between sister chromatids. Esp1 becomes active only after Pds1 is degraded in a process that involves a ubiquitin ligase called the anaphase promoting complex/cyclosome (APC/C). During Pds1 degradation the APC/C acts in conjunction with an associated subunit, the Cdc20 protein. We have previously shown that Cdc20 acts as a substrate recognition subunit of the APC/C and that it binds Pds1 directly. Pds1 function becomes crucial in the presence of DNA or spindle damage, when it inhibits mitotic progression until the damage is repaired. In the presence of DNA damage Pds1 is stabilized and we have recently determined the molecular mechanism for this stabilization (section 1). In addition to its role as an inhibitor of Esp1, Pds1 is also required for proper Esp1 function. In the past year we carried out two genetic screens that exploited these properties of Pds1 and that have lead to the identification of proteins involved in spindle function, DNA integrity and Esp1 activation (section 2). Finally, we are searching for proteins that are needed for nuclear structure and integrity. Thus far we have focused on two proteins, Mlp1 and Mlp2, and found that they are needed for proper DNA replication (section 3). 1. The Pds1-Cdc20 interaction in the presence of DNA damage. In the presence of DNA damage budding yeast cells arrest in metaphase due to the activation of a conserved regulatory mechanism known as the DNA damage checkpoint pathway. Two checkpoint kinases are necessary for the arrest: Chk1 and Rad53. Chk1 phosphorylates Pds1 whereas the target of Rad53 is unknown. We have previously demonstrated that the APC/C-dependent ubiqutination of Pds1 requires an association between Pds1 and Cdc20. Thus we wished to examine whether the presence of DNA damage affects this association. We found that in the presence of DNA damage Pds1 no longer binds Cdc20, and that the inhibition of this interaction was Chk1-independent but Rad53 dependent, thus establishing a previously unknown downstream target of Rad53 in mediating the checkpoint arrest. To investigate the role of the Chk1-dependent Pds1 phosphorylation we examined whether it is sufficient to block ubiquitination by the APC/C in vitro. In collaboration with Dr. Hongtao Yu (University of Texas Southwestern Medical Center, Dallas TX) we found that to be the case, suggesting that Chk1 phosphorylation stabilizes Pds1 by preventing its ubiquitination. Taken together our results suggest that the mitotic arrest in response to DNA damage is obtained by two distinct mechanisms, both of which lead to Pds1 stabilization: the Rad53-dependent inhibition of the Pds1-Cdc20 interaction, and the Chk1-dependent inhibition of APC/C-mediated ubiquitination. A manuscript describing these results has been accepted for publication in J. Biol.Chem. 2. The role of Pds1 as a mitotic regulator. Pds1 is not essential for viability but there are several conditions under which cells lacking Pds1 function cannot survive. These include conditions that lead to spindle defects or induce DNA damage. In addition, we hypothesize that Pds1-independent defects in in the activation of Esp1 or nuclear localization will also render Pds1 essential because under these conditions cells will have to depend on the ability of Pds1 to promote Esp1 activation. To identify proteins required for spindle function, DNA integrity or Esp1 activation we carried out two genetic screens for mutants whose viability depends on Pds1 (known as a yeast synthetic lethality screen). The first screen was based on a colony-sectoring assay in which cells that are mutated at the PDS1 locus and at a second, randomly induced site, cannot survive in the absence of a plasmid carrying the PDS1 gene. So far we isolated 14 complementation groups that show synthetic lethality with a pds1 deletion. Of these, several mutants have been identified and were shown to require Pds1 for viability due to spindle defects. Of particular interest was our finding that a protein required for mitotic cyclin degradation, Cdh1, is also synthetically lethal with the deletion of PDS1. We found that the cdh1 mutant strain requires the spindle checkpoint, and Pds1 in particular, to prevent chromosome mis-segregation. These and additional results suggest that an untimely increase in cyclin-Cdk activity can lead to genomic instability. A manuscript describing these results has been accepted for publication in Genetics. We also conducted a genetic screen in collaboration with Dr. Mike Tyers (Samuel Lunenfeld Research Institute, Toronto Canada), in which a deletion of Pds1 was combined, individually, with a collection of strains each carrying a deletion of one out of the 4600 non-essential genes in budding yeast. This screen has yielded over twenty mutants that require Pds1 for viability, many of which were not previously known to be involved in cell cycle regulation. These genes were analyzed for possible involvement in spindle function, DNA replication/damage and Esp1 inactivation. As expected, several of the mutants were indeed defective in spindle function, one required the DNA damage checkpoint for viability and two were found to be involved in DNA replication. Interestingly, in the course of this study we discovered two heat shock/chaperon proteins needed for Esp1 activation. A manuscript describing these findings is currently in preparation. 3. Exploring nuclear structure and integrity. Many nuclear processes, including transcription, DNA replication and gene silencing, are functionally linked to nuclear structure. In an attempt to uncover proteins that contribute to nuclear structure and integrity, we have taken two experimental approaches: a genetic screen to identify mutants whose nuclear structure is compromised, and a gene candidate approach in which we investigated the function of two proteins suspected to be involved in maintaining nuclear structure. The genetic screen makes use of a phenomenon in which in rare cases individual chromosomes can move from one nucleus to another in binucleated cells that cannot undergo nuclear fusion. We hypothesize that in this system mutants defective in nuclear integrity will have an altered rate of chromosome transfer, which can be monitored genetically. In the gene candidate approach we focused on the Mlp1 and Mlp2 proteins that were reported to be required for proper intranuclear chromosome localization. We found that the absence of these proteins leads to the activation of the DNA damage checkpoint pathway and elongated telomeres. We also have evidence that these phenotypes are due to defects in DNA synthesis. A manuscript describing these results is in preparation.