Spindle assembly checkpoint and chromosome stability in Caenorhabditis elegans

In many species, proper chromosome segregation is accomplished with the aid of a surveillance mechanism, the spindle assembly checkpoint. To identify the mechanisms involved in this process, the mutants that suppress or enhance the mdf-1(gk2)/MAD1 checkpoint lethality were characterized. The suppressors of mdf-1 (gk2) fall into two classes. The major class of suppressors compensates for the loss of the checkpoint by delaying mitotic progression. This class includes two known suppressors and anaphase promoting complex/cyclosome (APC/C) components, emb-30/APC4 and fzy-1/CDC2O, and four new such (suppressors of spindle checkpoint defect) genes. One of the new such genes was found to be an APC5-like gene not previously identified as a component of the APC/C in C. elegans. This analysis revealed that APC5 and APCJO genes have paralogs in the C. elegans genome. Furthermore, a class of suppressors was identified that does not delay mitotic progression. In mouse cells, mutations that result in defective apoptosis rescued the lethality associated with deletion of the Mad2 gene. The suppressor mutants were analyzed for the apoptotic response and the such-7(h1985) suppressor with normal anaphase onset was found to abrogate the DNA damage-induced apoptosis. Despite the defective apoptotic response in this suppressor, loss of apoptosis alone could not rescue the mdf-1 lethality in C. elegans, indicating that other processes affected by the such-7 mutation, could account for the rescue of mdf-1(gk2) lethality. The upstream components required for the genome stability would be recognized as enhancers of the mdf-1 (gk2) lethality. In yeast, 79 genes were found to result in synthetic lethal phenotype with MAD1. Of the 21 non-essential putative C. elegans orthologs assayed, nine enhanced mdf-1(gk2) lethality. The enhancers have a specific effect on the SAC, since six of them enhanced the mdf-2(tm2910)/MAD2 lethality, three also enhanced the san-1 (ok1580) lethality and none enhanced lethality in the kinetochore mutant him-1O(e15l1ts)/NUF2. In addition two interactions, hcp-1 and bub-3, were identified in C. elegans that are not conserved in yeast. This analysis also showed that HCP-1 and HCP-2, the two CENP-F-related proteins, have a non redundant role, since only the hcp-1(RNAi) enhances lethality of the SAC mutants.Medicine, Faculty ofMedical Genetics, Department ofGraduat