Molecular Determinants of Faithful Chromosome Transmission and Cell Cycle Checkpoint Regulation.A fundamental requirement of the cell division cycle is the maintenance, replication, and segregation of chromosomal DNA. Failure of complex mechanisms involved in maintaining genome integrity has been implicated in cancer, aging, and congenital birth defects. Research in our laboratory focuses on the molecular mechanisms of high fidelity chromosome transmission, the organization of chromatin structure, and the checkpoint regulatory mechanisms that ensure the proper execution of the cell cycle in Saccharomyces cerevisiae and the study of its human homologs. We are also interested in the application of genome technologies such as serial analysis of gene expression (SAGE) and DNA microarrays for analysis of transcription profiles and the identification of small non-annotated open reading frames (NORFs). We use a combination of genetic, biochemical, cell biology and whole genome approaches for analysis of gene function. The latter is made feasible through the application of a colony picking robot and whole genome arrays that we have in our laboratory.Molecular Determinants of Faithful Chromosome Transmission. CEN DNA sequences and the trans-acting kinetochore components (centromere-specific DNA binding proteins) are required for high fidelity chromosome transmission. Additionally, a higher order chromatin structure provides a framework for interactions of histones, CEN DNA, and the kinetochore. We have characterized genes that are important for the structure/function of the kinetochore and studied the corresponding human homolog. We initiated our studies using a large reference set of chromosome transmission fidelity mutants, the ctf mutants of S. cerevisiae previously isolated by Spencer et al., using a colony color assay for chromosome loss. Using genetic screens we determined that mutations or deletions in S. cerevisiae SPT4 and NUP170 lead to defects in chromosome transmission fidelity and kinetochore integrity. In the first studies we established that Spt4p is a novel component of centromeric and heterochromatic chromatin, which is required for kinetochore function and gene silencing in S. cerevisiae. In cross-species approach we have also shown that the spt4, ctf, and silencing phenotypes of yeast are functionally complemented by a human homolog of SPT4. In the second study we determined that the evolutionarily conserved Nup170p is a specialized component of the nucleopore complex (NPC) with roles in chromosome segregation and kinetochore function. These studies represent one of the first reports describing a functional relationship between the NPC and chromosome segregation and they have since been followed by similar observations in several other systems, including humans. In an extension of these results, we established an important functional and physical association between members of the Nup170p complex and the spindle checkpoint proteins Mad1p and Mad2p in S. cerevisiae. Prior to our analysis using live cell imaging no reports had been published on the localization of mitotic spindle checkpoint proteins in S. cerevisiae. We recently defined a minimal domain of Mad1p that is required for chromosome transmission and checkpoint functions. Our novel findings that S. cerevisiae Mad1p and Mad2p are localized to the NPC prompted us to investigate the localization of another spindle checkpoint protein Bub3p. We designed a novel genetically engineered reporter strain and showed preferential enrichment of Bub3p at defective kinetochores. This finding was an important observation because enrichment of a spindle checkpoint protein at kinetochores upon checkpoint activation had not previously reported in S. cerevisiae.
